13.4: The Chemistry and Biochemistry of Dioxygen - Biology

13.4: The Chemistry and Biochemistry of Dioxygen - Biology

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The History of Oxygen

Oxygen may be considered one of the most important element in chemistry. Why is oxygen so special?

Oxygen reacts with atoms of all elements except the Noble gases to form molecules. One of the most important molecules of course, from a biological sense is water. It :

  • provides a perfect solvent for biomolecules
  • moderates the earths climate
  • is the source of almost all the dioxygen in the air

From a chemical point of view, water is a(n):

  • nucleophile and electrophile
  • acid and base
  • oxidizing agent and reducing agent
  • a protic solvent that can form H-bonds

The formation of earth and the development of life:

The gaseous and dusty environment from which earth formed contained metals and water, which as you remember from introductory chemistry, can react to form hydrogen gas. H2 reacts with nonmetals (under various conditions of temperature and pressure) to form H2S, HCl, CH4, and NH3 which contributed to the reducing nature of the early atmosphere. This kept the transition metals in their lowest oxidation states. Many metals, including the coinage metals (Cu, Ag, and Au) and the platinum group (Ru, Rh, Pd, Pt) were stable in elemental form.

Then, around 2.7-2.8 billion years ago, photosynthetic organisms (blue/green algae- also called cyanobacteria) developed which could oxidize water to form dioxygen. Oxygen was generally unavailable for redox chemistry before then as photosynthesis, the process that would evolve to oxidize water to produce dioxygen, was unavailable. Remember that to oxidize water to dioxygen, itself a strong oxidizing agent, requires a stronger oxidizing agent than dioxygen and lots of energy. Fossilized remains of cyanobacteria are found in stromatolites. Using knowledge of how atmospheric oxygen can alter the chemistry of different sulfur isotopes of SO2, it has been shown that O2 did not exist in the atmosphere as a whole above 1 ppm earlier than 2.4 billion years ago, although there might have been isolated pockets with higher concentrations. After that it rose, presumably as a result of cyanobacteria. Before this time, bacteria oxidized a similar molecule, H2S to form elemental sulfur. It could do this through photosynthetic reduction of CO2 by H2S. It is probable that volcanic gases like H2 might have kept oxygen levels from rising between 2.7 billion year ago and 2.4 billion years ago, when its build-up started. Hydrogen in the form of H2 and methane, probably decreased around 2.4 billion years ago as methane with its hydrogen atoms escaped to the upper atmosphere and space. Methane levels would also be decreased by its easy reaction with dioxygen in the presence of UV light to form CO2. This would paradoxically lead to a cooling of the earth and pronounced glaciation as a more potent greenhouse gas, methane, was replaced with a less potent one, carbon dioxide.

Over the next billion years, dioxygen rose to perhaps 0.2 - 2% (compared to the present levels of 20%) Why? Because the early atmosphere was reducing, the added oxygen combined with a large "sink" of reduced metals (like elemental Cu and Fe) or nonmetals (like C and ammonia), preventing a large buildup. Only after these reduced substances were "titrated" did dioxygen build up to present levels. In addition, the oxygen might have increased weathering (by oxidation) of sulfur deposits which can lead to sulfides entering the ocean, where they could precipitate ocean iron ions that are necessary for cyanobacterial chemistry. This would place constraints on cyanobacterial growth until dioxygen levels in the atmosphere increased enough so sulfides were converted to sulfates. This first increase in atmospheric oxygen is often called the Great Oxidation Event as it correlated and presumably caused one of the greatest mass extinctions (of anerobic organisms) of all time.

  • The continuing puzzle of the great oxidation event

Around 2.3 billion years ago, as trace dioxygen had accumulated in the atmosphere, redox chemistry changed, although isotope evidence suggest that little dioxgen was found in water. Around 1.8 - 1.5 billion years ago, the earth's atmosphere became somewhat oxygenated, which was also coincident with the development of eukaryotic organisms. Until then, life was restricted to the oceans since there was no ozone to absorb dangerous UV radiation. The buildup of dioxygen in the air must have led to another extinction of anaerobic organisms, since as we shall see, products of oxygen metabolism are very toxic. Some evolved to use dioxygen. Ozone developed, and life could then migrate from the sea to the land. It wasn't until around 600 million years ago that animals arose, however. Was this event associated with the development of a fully oxygenated (20%) atmosphere? Recent evidence, which shows that substantial oxygen wasn't available in the deep sea until about 600 million years, seems to suggest that. Based on analysis of iron compounds in waters in Newfoundland, it appears that oxygen was very low in the sea 580 million years ago, during the Gaskiers glaciation period. Immediately after that it rose to levels consistent with atmospheric dioxygen levels of 15%, levels necessary for large animals. Similar trends in carbon and sulfur isotopes in marine rocks in Oman also suggest large increases in oxygen at the end of the Gaskiers glaciation period. What caused this second great oxygenation event? One possibility is that organic matter was sequestered from reaction with atmospheric dioxygen, as clays bound organic molecules in the ocean and lichens and zooplankton facilitated weather and production of insoluble organic material in the oceans.

Dioxygen is obviously critically important for higher organisms, so an understanding of its chemistry becomes important. This chapter will show that dioxygen is a ground state diradical that has low solubility in aqueous solution, reacts in a kinetically sluggish fashion in oxidation reaction, and forms toxic byproducts as it gets reduced. Life forms hence evolved ways to deal with these problems, including ways to increase its solubility (with dioxygen binding and transport proteins), and enzymes (that could activate it kinetically and also detoxify oxygen by-products). Dioxygen is toxic to many cells. Obligate aerobes die in an oxygen environment as many of their cellular components get oxidized by this excellent oxidizing agent. Several strains of bacteria actually swim away from high levels of dioxygen. A graph showing log of survival vs log pO2 is linear with a negative slope for a variety of organisms, including mice, fish, rats, rabbits, and insects. Pure oxygen can induce chest soreness, coughs, and sore throats in people. Premature infants put in pure dioxygen environments often developed blindness due to retrolental fibroplasia (a build-up of fibrous tissue behind the lens). The trade off for this toxicity is clear. Energy is derived from organic molecule through oxidation. Before dioxygen became available to power aerobic catabolism of reduced molecules like fatty acids and the less reduced sugars, such molecules were only partially oxidized. The glycolytic pathway, found in most organisms, oxidizes glucose (6 Cs) to two molecules of pyruvate (3 Cs). It was only with the availability of dioxygen did pathways evolve (Kreb Cycle, mitochondrial electron transport/oxidative phosphorylation) that allowed pyruvate to be fully oxidized to carbon dioxide, with the release of much more energy.

The Properties of Dioxygen

It is important to understand the properties of dioxygen since oxidation reactions using it power not only our bodies but our entire civilization. We will obviously concentrate on biological reactions, but even these show the same characteristics as non-biological ones.

  • oxidation of organic molecules by oxygen is thermodynamically favored but kinetically slow.
  • pure oxygen environments are toxic to cells and organisms.

First we will try to understand these properties of oxygen, and then we will see how organisms overcome these problem to use dioxygen.

We can understand both of these properties by looking at the molecular orbitals of oxygen and its reduction products as shown in the diagrams below. Ground state oxygen is a diradical, which explains the paramagnetic behavior of oxygen. The two unpaired oxygens each have a spin state of 1/2 for a total resultant spin S of 1, making ground state oxygen a triplet (2S+1) = 3. Organic molecules typically undergo 2 electron oxidation steps. Consider the stepwise oxidation of methane below. The oxidation number of C in methane is -4, -2 in methanol, 0 in formaldehyde, +2 in formic acid, and finally +4 in carbon dioxide, indicating two electron losses in each step.

The two electrons lost by the organic substrate are added to oxygen, but since the two lost electrons are spin paired, a spin flip must occur to allow the electrons to enter the unfilled oxygen orbitals. Alternatively, energy can be put into ground state dioxygen to produce excited state singlet oxygen (S=0, 2S+1 = 1). The source of the large activation energy required (about 25 kcal or 105 kJ/mol) to flip the electron spin accounts for the kinetic sluggishness of reactions of dioxygen with organic reactants.

A traditional Lewis structure for ground state dioxygen can not be easily written since the electrons are added in pairs, and dioxygen is a diradical. There are 6 electrons in the sigma molecular orbitals from second shell electrons (two each in σ2s, σ2s*, and σ2p,) and 6 electrons in the pi molecular orbitals from second shell electrons (two each in two different π2p orbitals, and one electron each in two different π2p*), so the net number of electrons in bonding orbitals is 4, giving a bond order (or number of 2). In contrast it is easy to write the Lewis structure of singlet, excited state oxygen, since all electrons can be viewed as paired, with two net bonds (1 sigma, 1 pi) connecting the atoms of oxygen. This Lewis structure will be used to represent singlet, excited oxygen, which should react more quickly with organic molecules. The excited state single on the far right (below) is unstable and decays to the middle singlet state. The middle state is approximately 94.3 kJ/mol higher in energy than the ground state triplet (on the left). In quantum mechanical parlance, the transition from the ground state triplet to the singlet state is forbidden for a number of reasons, making it unlikely that absorption of a photon will induce the transition.

The Reductions of Dioxygen

When oxygen oxidizes organic molecules, it itself is reduced. By adding electrons one at a time to the molecular orbitals of ground state dioxygen we produce the step-wise reduction products of oxygen. On the addition of one electron, superoxide is formed. A second electron produces peroxide. Two more produces 2 separated oxides since no bonds connect the atoms (the number of electrons in antibonding and bonding orbitals are identical). Each of these species can react with protons to produce species such as HO2, H2O2 (hydrogen peroxide) and H2O. It is the first two reactive reduction products of dioxygen that make it potentially toxic.

How are the potential problems in oxygen chemistry dealt with biologically?

Kinetic sluggishness: Enzymes that utilize dioxgen must activate it in some way, which decreases the activation energy. Enzymes that use dioxygen typically are metalloenzymes, and often heme-containing proteins. Since metals such as Fe2+ and Cu2+ are themselves free radicals (i.e. they have unpaired electons), they react readily with ground state oxygen which itself is a radical. The molecular orbitals of the metal and oxygen combine to produce new orbitals which for oxygen are more singlet-like in nature. Likewise, dioxygen reacts more readily with organic molecules which can themselves form reasonably stable free radicals, such as flavin adenine dinucleotide (FAD), as we shall see later.

Dioxygen toxicity: Since toxicity arises from the reduction products of oxygen, enzymes that use oxygen have evolved to bind oxygen and its reduction products tightly (through metal-oxygen bonds) so they are not released into the cells where they can cause damage. In addition, enzymes which detoxify free dioxygen reduction products are widely found in nature. For example:

  • superoxide dismutase catalyzes the dismutation (self-redox) of 2 superoxides into dioxygen and hydrogen peroxide;
  • catalase converts hydrogen peroxide into water and oxygen;
  • peroxidase catalyzes the reaction of hydrogen peroxide with an alcohol to form water and an aldehyde
  • peroxiredoxins react with peroxides and thioredoxin (a small electron donor) to form water and oxidized thioredoxin .

Finally free radical scavengers such as vitamins A, C, E, and selenium can react with reactive free radicals to produce more stable free radical derivatives of the vitamins and Se. More on this later.

The Reactions of Dioxygen and its Reduction Products

1. Triplet O2 - Ground State:

Figure: Triplet O2 - Ground State

  • Metals ions - Metal ions are radicals themselves, so can react with dioxygen. Ex:
    Fe2+ + O2 <--> [ Fe2+-- O2 <--> Fe3+-- O2-.] <--> Fe3+ + O2-. (superoxide)
  • Autoxidation of organic molecules to produce peroxides. In this free radical reaction, several reactions occur, including
    RH ---> R. (Initiation)
    R. + O2 ---> ROO. (Propagation)
    ROO. + RH ---> R. + ROOH (Propagation)
    R. + R. ---> R--R (Termination)
    ROO. + ROO. ---> ROOR + O2 (Termination)
    ROO. ---> ROOR (Termination)

    The initiation step above occurs mostly at C atoms which can produce the most stable free radicals (allylic, benzylic position, and 3o > 2o >> 10 carbons). Hence unsaturated fatty acids are extra reactive at the methylene C that separate the double bonds.

Figure: unsaturated fatty acids are extra reactive at the methylene C that separate the double bonds

2. Single O2 - Excited State.

Figure: Single O2 - Excited State

It can be made from triplet oxygen by photoexcitation. Alternatively, it can be made from triplet oxygen through collision with an excited molecule which relaxes to the ground state after a radiationless transfer of energy to triplet oxygen to form reactive singlet oxygen. (This later process accounts for photobleaching of colored clothes when the conjugated dye molecules absorb UV and Vis light, relax by transferring energy to triplet oxygen to form singlet oxygen, which then chemically reacts with the conjugated double bonds in the dye. )

  • Alkenes react with oxygen to form hydroperoxides, potentially through a epoxide intermediate
  • Dienes reacts with oxygen in a Diels-Alder like reaction to form endoperoxides

3. Superoxide

Figure: Superoxide

  • Dismuation: O2-. + O2-. ---> H2O2 + O2
  • Acid/Base: HO2. ----> O2-. + H+ (pKa = 4.8)
  • With metal ions: Fe3+ (as in heme) + O2-. ---> O 2 + Fe2+

4. Peroxide

Figure: Peroxide

In contrast to dioxygen which contains multiple bonds between the O atoms, peroxide has only one bond. In fact, it is quite weak and requires only 38 kcal/mol to break it. Remember, bonds can be broken in a heterolytic way (both electrons in a bond go to one of the atoms, or in a homolytic fashion, in which the one electron goes to each atom.

  • Acid/Base: H2O2 ---> HO2- ---> O22- (pKa1 = 11.8; pKa2 > 14)
  • Reaction with Fe2+ - The Fenton Reaction: (similar to reaction of triplet O2 with Fe2+ above)
    Fe2+ + OOH- <--> Fe2+-- OOH <--> Fe2+-- O + OH- <--> Fe3+-- O . <--> Fe3+ + OH. (last step a proton is added). In this reaction, a homolytic cleavage of the O--O bond occurs generating OH- and the hydroxy free radical, OH., which will react with any molecule it encounters.
  • thermal or photochemical homolytic cleavage of peroxide. This forms alkoxide free radicals which reacts like the hydroxy free radical.
  • Reactions with alkyl groups in the presence of metal ions such as Cu, Co, or Mn:
    RH + R'OOH ----> ROOR'

5. Hydroxy free radical:

Figure: Hydroxy free radical

As mentioned above this species is extremely reactive. It will react with any molecule it encounters and does so immediately. It can abstract a H atom leaving another free radical. For example, the hydroxy free radical could extract a hydrogen atom from a polyunsaturated fatty acid to from a carbon-centered radical. A particularly nasty reaction is the insertion of the hydroxy radical into bases in DNA, as shown in the diagram.

Oxidative Modification of Proteins

Oxidative Modification of Proteins:

Figure: Oxidative Modification of Proteins

Oxidized levels of proteins (as evidenced by increased levels of aldehydes) increase dramatically with age (especially after age 40 ). The reactions seem to be catalyzed by metals and may proceed by generation of hydroxy free radicals. Diseases associated with premature aging (Werner's Syndrome, another link to Werner's Syndrome, Progeria) show very high levels of oxidized proteins at an early age. Fibroblasts from 10 yr. old children with progeria have levels of oxidized proteins usually not seen until the age of 70. Beta amyloid protein deposits (found in Alzheimer's and Down's Syndrome) cause neurotoxicity and death, partly by increasing superoxide production by endothelial cells, causing vasoconstriction/dilation, and ultimately disease progression. Beta amyloid aggreagates appear to increase H2O2 levels, in a process facilitated by Fe2+ and Cu+. Free radical scavengers (antioxidants) help to prevent this damage. Recent studies suggest that vitamin E may delay the symptoms of Alzheimers.

Lou Gehrigs Disease (Amyotrophic Lateral Schlerosis) is a disease of progressive motor neuron degeneration, which affects 1/100,000 people, and is 10-15% familial. Of the familial cases, about 25% have a mutation in superoxide dismutase I, a copper-zinc enzyme. About 2-3% of ALS patients carry 1 of 60 different dominant mutations in this enzyme. Mutations often decrease the stability of the protein which decreases Zn2+ affinity 5-50 fold. The A4V mutation (valine at amino acid 4 substituted for Ala) has the weakest Zn affinity and causes rapid disease progression. In the absence of Zn2+, the apoprotein somehow seems to induce cell death in neurons. This superoxide dismutase also expresses a second activity. It also acts as a peroxidase which takes ROH + H2O2 to form an RHO (an aldehyde) plus water. In some cases, the enzyme retains normal activity against superoxide but altered peroxidase activity.

Is your hair going white?: Wood et al have shown that millimolar concentrations of hydrogren peroxide build up in hairs that have grayed and whitened. This was associated with a decrease in catalase and in increases in Met oxidation (to Met-sulfoxide) in proteins, also associated with a decrease in the repair enzyme Met-sulfoxide reductase, Met 374 in the active site of tyrosinase, an enzyme required for production of melanin in hair follicles, is also damaged, leading to lack of melanin, a pigment necessary for hair coloration and "senile hair graying".

ROS and Protein Folding

As discussed in Chapter 2D: Protein Folding in Vitro and in Vivo, the cytoplasm has sufficient concentrations of "β-mercaptoethanol"-like molecules (used to reduce disulfide bonds in proteins in vitro) such as glutathione (γ-Glu-Cys-Gly) and reduced thioredoxin (with an active site Cys) to prevent disulfide bond formation in cytoplasmic proteins. Disulfide bonds in proteins are typically found in extracellular proteins, where they serve to keep multisubunit proteins together as they become diluted in the extracellular milieu. These proteins destined for secretion are cotranslationally inserted into the endoplasmic reticulum (see below) which presents an oxidizing environment to the folding protein and where sugars are covalently attached to the folding protein and disulfide bonds are formed (see Chapter 3D: Glycoproteins - Biosynthesis and Function). Protein enzymes involved in disulfide bond formation contain free Cys which form mixed disulfides with their target substrate proteins. The enzymes (thiol-disulfide oxidoreductases, protein disulfide isomerases) have a Cys-XY-Cys motif and can promote disulfide bond formation or their reduction to free sulfhydryls. They are especially redox sensitive since their Cys side chains must cycle between and free disulfide forms.

Reactive oxygen species (ROS) can significantly affect redox chemistry, and if present in excess can place the cell in a condition of "oxidative" stress. ROS can indiscriminately oxidize lipids, nucleic acids, and proteins, but more specifically, they may also oxidize proteins involved in creating and maintaining the normal disulfide bond formation in proteins. As the concentration of ROS increase, the concentration of cytoplasmic proteins with incorrect disulfides should increase. Using a two dimension PAGE system (first dimension run under nonreducing and the second reducing conditions) of neural cell proteins derived from cells exposed to normal and differing oxidative conditions (hydrogen peroxide or decreased intracellular glutathione levels, Cumming et al showed that oxidizing stress increased the levels of disulfide bonds in redox sensitive enzymes and, unexpectedly, among other cytoplasmic proteins involved in many aspects of life, effecting the activity of many cellular processes, suggesting that disulfide bond formation may have not only a structural but regulatory role.

Oxidative Modification of Lipids:

Figure: Oxidative Modification of Lipids

The initial stages of cardiovascular disease appear to involve the development of fatty acid streaks under the artery walls. Macrophages, an immune cell, have receptors which appear to recognize oxidized lipoproteins in the blood, which they take-up. The cells then become fat-containing foam cells which form the streaks. Oxidation of fatty acids in lipoproteins (possibly by ozone) could produce lipid peroxides and to protein oxidation in lipoproteins. Cortical neurons from fetal Down's Syndrome patients show 3-4 times levels of intracellular reactive O2 species and increased levels of lipid peroxidation compared to control neurons. This damage is prevented by treatment of the neurons in culture with free radical scavengers or catalase.

Figure: oxidized LDL uptake

A recent study of peroxiredoxins by Neumann et al showed the importance of these gene products in mice. Peroxidredoxins (which catalyze the conversion of a peroxides and thioredoxin into water and oxidized thioredoxin) are small proteins with an active site cysteine and are found in most organisms. Transcription of the mammalian peroxiredoxin 1 gene is activated by oxidative stress. They inactivated the gene which produced a mouse that could reproduce and appeared vital, but which had a shortened lifespan. These mice developed severe hemolytic anemia and several types of cancers. High levels of reactive oxygen species and resulting increased levels of oxidized proteins were found in red blood cells of the knockout mice with anemia. High levels of 8-oxoguanine, resulting from oxidative damage to DNA, were found in tumor cells.

Oxidative Modification of DNA

Significant evidence suggests oxygen free radicals are linked to aging and diseases. Mutations caused by hydroxylation reactions (presumably from the generation of hydroxyl free radicals as shown above) can potentially lead to cancer. Recently it has been shown that mitochondrial DNA is more susceptible to oxidation that is nuclear DNA. Human mitochondria has its own small genome (16.5 Kb compared to the nuclear genome of 3 Gb) which code 13 protein subunits involved in respiration, 22 tRNAs and two ribosomal RNAs. (The mitochondria presumably are vestiges of a bacteria which invaded an early cell and established a symbiotic relationship with the cell). A recent study has shown that there is an inverse correlation of oxidized mitochondrial DNA [8-oxoG] with maximal life span of an organism, but this correlation is not seen with nuclear DNA. Presumably the nuclear DNA is somewhat protected from oxidative damage since it is bound to histone proteins (which form nucleosome core particles with DNA) and by DNA repair enzymes. DNA repair enzymes that are encoded in the nucleus are found in the mitochondria and mitochondrial DNA is package with mitochondrial transcription factor A (TFAM). Examination of human bladder, head and neck and lung primary tumors reveals a high frequency of mitochondrial DNA mutations. In addition most dioxygen use by the cell occurs in the mitochondria. Hence this organelle probably faces the highest concentration of toxic oxygen reduction products. Recently, the crystal structure of an enzyme, adenine DNA glycosylase (MutY), that repairs 8-oxyG modified DNA has been determined in complex with the oxidatively damaged DNA. If not repaired, the 8-oxyG base pairs with adenine instead of cytosine, causing a GC to AT mutation on DNA replication.

Jmol: Updated Adenine DNA glycosylase:8-oxyG DNA complex Jmol14 (Java) | JSMol (HTML5)

Although oxidative damage in mitochondria clearly can promote premature aging, other independent mechanisms may also. In a recent study, Kujoth et al. developed a mouse model that expressed a mutant form of mitochondrial DNA polymerase that was defective in the proofreading activity of the enzyme. These mice displayed premature aging but showed no increased levels of oxidized mitochondrial lipids or hydroxylated G residues in mitochondrial DNA. They did show significant activation of a cytosolic enzyme called caspase-3, which when active lead to the programmed death of cells (a process called apoptosis). This calcium-activated aspartic acid proteases (with an active site Asp) is activated by binding mitochondrial cytochrome C that has "leaked" into the cytoplasm from its normal location in the intermembrane space in mitochondria. The process is usually associated with DNA damage (mutations, fragmentation) that would arise if the proofreading function of DNA polymerase was defective. This was indeed found in these mice.

Oxidative damage to biomolecules might not initiate aging and disease processes, but rather might be a secondary effect of other initiating events. Reversing or preventing oxidative damage might slow the progression of aging and disease. Aging is a complex feature of organisms and would be expected to have complex causes and biological effects. At the organismal level, aging has been studied in the round worm C. elegans which lives for only a few weeks. Genetic analyses can be easily used to find gene alterations associated with premature aging. One hormonal system that has recently been associated with aging in eukaryotes (and in C. elegans) involves the signaling pathways for insulin and insulin growth factor I (IGF-1), which regulate carbohydrate, lipid, and reproductive pathways in C. elegans. Mutations that decrease signaling from this pathway increase C. elegans life span. These mutations lead to increased activity of the DAF16 transcription factor, which upregulates the expression of many genes. In contrast, wild type organisms, when exposed to insulin or IGF-1, decrease the activity of DAF16. Using DNA microarrays, investigators determined which DAF16-controlled genes were upregulated in mutant worms in the mid-life point of the organism. These genes included, among others, peroxisomal and cytosolic catalase, Mn-superoxide dismutase, cytochrome P450s, metallothionein-related Cd-binding protein, and heat shock proteins. We will investigate the function of several of these gene products in the next section, but needless to say, they are all involved in cellular responses to stress, often involving dioxygen metabolites. Over expression of mitochondrial catalase in mice increased their lifespan by 20%. It has also been showed that decreased levels of insulin-like growth factor also promote longevity in mice, indicating again that mechanisms in addition to oxidative damage by ROS are involved in aging.

13.4: The Chemistry and Biochemistry of Dioxygen - Biology

Oxygen-Activating Enzymes, Chemistry of

Corinna R. Hess*, Richard W. D. Welford* and Judith P. Klinman**, Departments of Chemistry and Molecular Cell Biology, University of California, Berkeley, California

Aerobic organisms derive the energy required for cellular processes from the conversion of dioxygen to water, which highlights the importance of dioxygen chemistry in biologic systems. The biochemistry of dioxygen is far from simple and has been the subject of intense study in a range of chemical and biologic disciplines. The reduction of dioxygen is energetically favorable however, dioxygen is a ground state triplet, kinetically unreactive with singlet organic molecules. Nature has developed a diverse array of catalysts to overcome this kinetic barrier. These dioxygen-activating enzymes are divided into two classes: oxygenases and oxidases. Oxygenases incorporate directly at least one atom from dioxygen into the organic products of their reaction. Oxidases couple the reduction of dioxygen with the oxidation of substrate. Typically, enzymes that react with dioxygen contain transition metal ions and/or conjugated organic molecules as cofactors. The reaction with dioxygen is initiated by electron transfer from the cofactor to O2. The subsequent chemistry varies depending on both the nature of the cofactor and the protein scaffold. Here we review the fascinating chemistry of the dioxygen-activating enzymes and identify some of the common strategies and themes that have emerged from over half a century of research.

* These authors contributed equally.

Biological Background

The introduction of an oxygen-rich atmosphere led to the evolution of enzymes capable of exploiting this diatomic gas for a variety of different chemistries. Viewing life today, we can conclude that the use of dioxygen ultimately turned out to be advantageous. Dioxygen is required by all aerobic forms of life for a variety of chemical transformations and biological processes, which are conducted by a rich and diverse set of protein catalysts (1). In addition to its crucial function in the respiratory chain, dioxygen also is involved in biosynthesis, signaling, xenobiotic metabolism, DNA repair, and biodegradation. However, a darker side exists to dioxygen biochemistry. The reactive oxygen species (ROS) formed by reduction of dioxygen species have been linked to several detrimental processes such as aging and cancer (2). These ROS can damage proteins, cell walls, and DNA. One key area of interest is how dioxygen-activating enzymes use ROS without damaging their own peptide backbone in the process.

The full four-electron, four-proton reduction of O2 to two molecules of H2O is strongly exothermic (Table 1). However, most redox processes involve sequential one or two-electron transfer pathways. The first electron transfer to dioxygen is the most difficult step (Table 1) because of the loss in O-O bond strength associated with formation of superoxide (3). Most organic molecules do not possess enough reducing power to facilitate this initial reduction, and as a consequence, their one-electron oxidation by dioxygen is thermodynamically unfavorable. Moreover, dioxygen is a triplet in its ground state, whereas most organic molecules possess singlet ground states. Consequently, the direct reaction of dioxygen with organic molecules necessitates a spin conversion to the singlet excited state of O2, which has 22.5 kcal/mol more energy.

Enzymes have evolved to couple efficiently the reduction of dioxygen to the oxidation of biological compounds. Difficult C-H abstraction and O-atom insertion reactions are catalyzed by oxygen-activating enzymes under mild conditions and with high specificity.

Table 1. Reduction potentials of dioxygen and reduced oxygen species in water (3)

Redox reactions of O2, pH 7, 25° C

Cofactors used in Dioxygen Activation

Practically all known oxygen-activating enzymes employ a cofactor in the form of a metal center, a highly conjugated organic molecule, or both. Ultimately, the reactions involve the reduction of O2by two or four electrons to generate hydrogen peroxide or water. Metallocofactors and flavin molecules are powerful reductants, able to overcome the large potential associated with the formation of superoxide (Table 1), and with comparatively stable one-electron oxidized forms. The activated dioxygen products are highly reactive and readily accept additional electrons to yield the final enzymatic products.

Dioxygen activation by metalloenzymes is dominated by Fe and Cu, although a few examples of other transition metal active sites, such as Mn, have been documented. Metallocofactors bind dioxygen and its many reduced forms. The resultant metal-oxygen bonds compensate partly for the loss of bond strength upon reduction of O2. The rich spectroscopy associated with metal sites has advanced our understanding of the chemistry of dioxygen-activating enzymes. Methods such as electron paramagnetic resonance spectroscopy (EPR), X-ray absorbtion spectroscopy (XAS), and Mossbauer have been used to obtain structural information regarding the metallocofactors at various stages in the catalytic mechanisms (4).

Dioxygen-activating enzymes can be categorized according to the type of cofactor(s) employed. Although similar types of cofactors can catalyze different reactions, common structural features and reaction intermediates often exist for a given type of cofactor, as discussed below.

Iron-containing motifs for dioxygen activation

Iron-containing proteins are classified as heme, mononuclear non-heme, and binuclear non-heme enzymes. The Fe center in heme proteins is ligated by four nitrogens of a porphyrin molecule, in addition to one or two axial ligands (Fig. 1a). The reaction of Fe II with O2 generates an Fe III -superoxide, in which the reduced O2 is coordinated at an axial position of the heme unit in a bent, end-on fashion.

Mononuclear non-heme iron centers with several different types of coordination environments have been identified (5-6-7). The most prevalent coordination is an octahedral or square pyramidal site with a 2His-1carboxylate ligand set coordinated to one face of an Fe II center (Fig. 1b). In the resting state, the other positions are occupied by water molecules, which can be readily substituted during the reaction cycle by the substrate, the co-substrate, or O2. Another group of mononuclear non-heme iron enzymes activate their organic substrate for direct reaction with dioxygen. These enzymes contain an Fe III , in which the coordination environment differs by subfamily. For example, intradiol dioxygenases contain a trigonal bipyrimidal Fe III ligated by 2His, 2Tyr, and a hydroxide in the resting state (Fig. 1c).

The active sites of binuclear non-heme enzymes consist of two Fe atoms, separated by 3-4 A and bridged by O-atoms derived from hydroxide or carboxylate residues (Fig. 1d) (8). The iron centers can adopt 4-, 5-, or 6-coordinate geometries, with the bridging ligands bound via one or two O-atoms. The remaining coordination sites are occupied by His and Asp/Glu residues.

The reactions of iron-containing enzymes with O2 often involve high oxidation states of the metal. Generally, the initial reaction of dioxygen with both heme and mononuclear non-heme ferrous enzymes results in the formation of Fe III -superoxide intermediates. Highly reactive Fe IV =O intermediates often are employed often for C-H activation. The mechanism of substrate oxidation by binuclear non-heme enzymes involves high valent, oxo-bridged species, with Fe in the +3 or +4 oxidation state.

Figure 1. X-ray crystal structures of representative motifs used in reactions with dioxygen. Amino acids and the heme cofactor are shown as sticks. Iron, copper, and water are shown as green, aquamarine, and red spheres, respectively. PDB codes are given in parentheses. (a) P450, the red stick represents a bound O2 molecule (1DO9) (b), non-heme iron, 2His-1 carboxylate (1RXF) (c) non-heme iron, intradiol dioxygenase (1DLM) (d) di-iron, methane monooxygenase, including a bound molecule of acetic acid (1MMO) (e) type 2 copper, copper amine oxidase (1A2V) (f) coupled binuclear copper, tyrosinase (1WX3) (g) reduced flavin.

Copper-containing motifs for dioxygen activation

The copper-containing enzymes can be classified as mononuclear, binuclear, or multicopper proteins (9). Unlike iron, copper cannot readily access high oxidation states and the formation of Cu III in biological systems remains controversial. Generally, Cu I centers undergo one electron oxidations to activate dioxygen. Thus, the Cu-containing enzymes tend to employ multiple copper atoms or an additional cofactor for the final two or four-electron oxidation of their substrates.

Mononuclear copper enzymes capable of dioxygen activation contain a Type 2 copper center (Fig. 1e). Generally, Type 2 copper sites have a tetragonal geometry in the Cu II state and can be identified by their EPR spectra (10). Reduction of the Cu site is accompanied by a loss of water molecules and a change in coordination geometry trigonal or tetrahedral geometries are common for Cu I (11). Dioxygen is thought to bind to the reduced copper site in an end-on or side-on fashion, to yield a Cu II -superoxide intermediate.

A Type 3 active site, which consists of two antiferromagnetically coupled Cu atoms,

3 A apart, is found in binuclear copper enzymes (10). Each copper is coordinated by three histidine residues and by one or two water molecules that serve as η 2 bridging ligands (Fig. 1f). These enzymes all bind to O2 in an η 2 fashion, where each O atom is bound by the two coppers. Each copper atom transfers one electron to dioxygen to yield a Cu II 2-peroxide intermediate (for example, “oxy state” in Fig. 3g). Proteins with Type 3 copper centers include dioxygen transport and dioxygen-activating enzymes.

The multicopper enzymes contain a trinuclear metal cluster that consists of a Type 2 copper site and a binuclear, Type 3 copper site (10). The Type 3 site of multicopper enzymes is distinct from the active site of the coupled binuclear copper enzymes described above. Although the Type 3 Cu centers are antiferromagnetically coupled, the centers are separated by

5 A and are bridged by a hydroxide ligand. The multicopper oxidases also contain at least one mononuclear, Type 1, blue copper site in addition to the trinuclear Cu cluster. The Type 1 Cu site is ligated by two histidines, a cysteine, and often a weakly coordinating axial 4th ligand. Blue copper sites also are found commonly in electron transfer proteins and are defined by their intense Cys-to-metal chargetransfer transition at

Dioxygen activation by flavins

A large group of enzymes react with O2 by using an organic flavin cofactor (Fig. 1g) (12, 13). The reactions can be divided into two half-reactions. First the flavin is reduced by substrate, and then the reduced flavin reacts with an electron acceptor, such as dioxygen. When O2 is the electron acceptor, the first step in the oxidative half-reaction is the rate-limiting electron transfer, which leads to the formation of a caged radical pair of superoxide anion and flavin semiquinone. The fate of the radical pair depends on whether the enzyme is an oxidase or an oxygenase and will be discussed below. The second-order reaction of a reduced free flavin with O2 in solution proceeds at a rate of 2.5 x 10 2 M -1 s -1 . For an enzyme-bound flavin, the rate can vary between 2 M -1 s -1 and 10 6 M -1 s -1 . It is not fully understood how enzymes with extremely similar active sites can access such a range of rates for reaction with O2.

The following sections describe specific examples of oxygenase and oxidase chemistry catalyzed by the cofactors described above. It is by no means an extensive list, but it should offer the reader a flavor of the diverse mechanisms of dioxygen activation by enzymes.

The enzymes in this section can incorporate either one or two atoms from dioxygen into the organic product(s) of their reaction. It is of note that many oxygenases also can catalyze oxidase like reactions, such as desaturation and ring closure. Figure 2 shows illustrative examples of the reactions catalyzed by some of these enzymes, whereas Fig. 3 shows partial reaction mechanisms, including key intermediates.

Heme-Fe: The cytochromes P450

The cytochromes P450 are some of the most well-studied oxygenase enzymes their oxidation reactions, typically hydroxylations, are important for xenobiotic metabolism and biosynthesis (18, 19). Several notable differences exist between P450s, which catalyze oxygenations, and the O2 transport globins, which simply bind O2 reversibly. P450s have an axial cysteine thiolate iron ligand on the proximal side of the heme, whereas the transport globins have a histidine residue (20). Additionally, P450s have a conserved GX(E/D)T sequence motif on the distal side of the heme, which is thought to be involved in the proton donation required for cleavage of O2. In the resting state, the P450s contain a heme-ligated Fe III . Typically, the electrons for the reduction of O2 are supplied by NAD(P)H via protein redox partners. Extensive spectroscopic work has led to the observation of several of the peroxy intermediates involved in the conversion of the Fe-oxygen complex, Fe m -O-O•, to the compound I + H2O (Fig. 3a). Most recently, elegant one-electron cryo reduction and EPR spectroscopy allowed the detection of the Fe m -O-O 2- and Fe m -O-OH 1- intermediates. The loss of water from the latter generates the highly oxidizing compound I, which is believed to be an Fe IV =O species with radical character localized on either the porphyrin ring or thiol ligand. Compound I can abstract a hydrogen atom from an organic substrate. The substrate radical combines rapidly with the iron-ligated hydroxyl in a “radical rebound” mechanism (21). Several facets of P450 catalysis are underlying themes for some other oxygenase enzymes. The reduction of the ferric heme and the reaction with dioxygen occur only after binding of the organic substrate molecule. This “substrate-triggering” mechanism is of primary importance, as it prevents the formation of potentially damaging ROS in the absence of a suitable oxidizable substrate. Additionally, the substrate selectivity can be either broad or narrow, depending on the enzyme’s function.

Mononuclear non-heme iron oxygenases

The mononuclear, non-heme iron oxygenases can be classified into two groups: those that activate substrate for reaction with O2 and those in which the iron activates O2 directly (5, 7). Examples of enzymes that employ substrate activation mechanisms are the intradiol cleaving dioxygenases and lipoxygenases, which catalyze ring opening and hydroperoxidation, respectively (Figs. 2 and 3).

Intradiol dioxygenases prime their substrate for direct reaction with O2 by inducing radical character in the organic molecule. The iron is Fe III in the resting state, and spectroscopic studies have shown that its oxidation state does not change detectably throughout the catalytic cycle (Fig. 3b). The substrate-bound form is believed to have some semiquinone radical character and is thought to combine directly with O2. The transient peroxy substrate complex then breaks down by a Criegee-type alkyl migration to yield the products.

The Fe III center in lipoxygenase is coordinated by 3His/Asn, the protein’s C-terminal carboxylate and a hydroxide (Fig. 3c). The Fe III -OH abstracts a hydrogen atom from the substrate yielding a five-carbon delocalized radical intermediate (designated as R•) that combines directly with O2 to yield the peroxy product. Entry of O2 to the enzyme active site is believed to be controlled tightly, such that it can access only a single carbon of the substrate radical, giving strict regio- and stereospecificity (22).

The 2His-1carboxylate mononuclear, non-heme iron enzymes can be divided into subfamilies, which differ in the cosubstrate that provides the electrons for the reduction of O2 and the relative positions of the 2His-1carboxylate residues in the protein sequence (7). Even among members of the same subfamily, the only conserved residues are the 2His-1carboxylate, which indicates the use of this motif for activation of O2. Despite the conserved ligand set, the chemistry can vary greatly. In the reaction cycle of the α-ketogluturate (α-KG) dependent subfamily, it is the α-KG co-substrate that ligates the Fe, whereas the prime substrate occupies a second sphere position. The counter is observed for the extradiol catechol dioxygenases where the “prime” substrate ligates directly to the iron.

The largest subfamily in the 2His-1carboxylate group of non-heme Fe II -dependent enzymes depends on α-KG as a cosubstrate (6). The substrate binding is ordered. First α-KG ligates the Fe II , then the prime substrate binds adjacent to the metal site, and finally O2 binds to the metal (Fig. 3d). The ternary complex reacts intramolecularly to yield the oxidizing species. This was characterized recently as an Fe IV =O by Mossbauer, Raman, and EXAFS spectroscopy, which is the first enzymatic example of a non-heme Fe IV (14). As with P450s, the Fe IV =O is thought to be the oxidizing intermediate in a wide variety of reactions, including hydroxylation, desaturation, and oxidative ring closure (6). The multifaceted, yet highly controlled, nature of this chemistry is exemplified brilliantly by the enzyme clavaminate acid synthase, which uses a single active site to catalyze a hydroxylation, a desaturation, and a ring closure at different stages in a single biosynthetic pathway. The α-KG-dependent enzymes, like other oxygenases, can hydroxylate aromatic amino acid side chains in the vicinity of the active site under certain conditions (7). This illustrates the enzymes’ need to control the oxidizing intermediate to minimize such deleterious side reactions. In fact, mammals are thought to require vitamin C for the reduction of Fe III , generated as a by-product of unproductive reaction cycles of α-KG enzymes.

The extradiol dioxygenases, similar to their intradiol counter parts, cleave aromatic compounds, but the position of ring opening differs (Fig. 2) (7). In the resting state, the extradiol dioxygenases contain a 2His-1carboxylate, five-coordinate Fe II . Bidentate binding of the catechol to the Fe activates the metal for O2 binding. This process is analogous to the α-KG enzymes, except an additional substrate is not required to make the center five-coordinate. The Fe III superoxide formed on dioxygen binding can induce radical character in the catechol that leads eventually to ring opening.

Another subgroup of the 2His-1carboxylate family is dependent on a reduced pterin cofactor (5). They catalyze hydroxylations at the aromatic positions of amino acids in phenylalanine catabolism and hormone biosynthesis (Fig. 2). Unlike the α-KG-dependent enzymes, the pterin co-substrate does not ligate to the iron directly. In the reaction cycle, the pterin cosubstrate supplies two electrons for the heterolysis of O2 to give a yet to be characterized iron-oxygen hydroxylating species.

X-ray crystallography and magnetic circular dichroism spectroscopy have shown that, for the α-KG- and pterin-dependent mononuclear non-heme iron enzymes, the binding of the penultimate substrate promotes a change to a five-coordinate iron center. As with the P450s, this finding implies that dioxygen will not bind in the absence of an organic substrate, which prevents the build up of potentially damaging intermediates (23).

Figure 2. Some common reactions carried out by oxygenase enzymes. A * represents an oxygen atom derived from O2. The reactions listed are those thought to be the enzyme's biologically relevant reaction. Where appropriate the name of the enzyme catalyzing the example reaction is given.

Binuclear non-heme iron oxygenases

Soluble methane monooxygenase (sMMO) is the best studied binuclear non-heme iron oxygenase enzyme, largely due to its remarkable ability to hydroxylate the stable C-H (440 kJ /mol) of methane (15). sMMO is a three-component enzyme system, which consists of the di-iron hydroxylating protein, a flavin-Fe2S2 protein, and a third regulatory protein that does not contain a cofactor. The role of the flavin-Fe2S2 protein is to provide two electrons from NADPH to form the active Fe II -Fe Ii form of the di-iron hydroxylating protein. Although incompletely understood, the regulatory protein seems to coordinate the interaction of the other two components such that uncoupled reaction cycles do not occur. The intermediates in the sMMO reaction cycle accumulate in the absence of methane, which is a feature that has allowed their spectroscopic characterization (14). Studies on the Mossbauer suggest that O2 binds initially to the binuclear Fe site forming a μ-1,2-peroxo-Fe III 2 intermediate, which then decays to form the highly oxidizing intermediate termed Q (Fig. 3e). EXAFS and Mossbauer have indicated collectively that Q is a bis-(μ-oxo)-Fe IV 2 with a short Fe-Fe separation of 2.46 A. A strong consensus on how this intermediate compound hydroxylates methane has not yet been reached despite a wealth of studies using isotopically labeled and radical clock substrates (15). Currently, a radical rebound mechanism or a nonsynchronous concerted rearrangement remains possible.

Figure 3. Illustration of possible partial reaction cycles of some oxygenase enzymes. Water molecules and protein ligands have sometimes been omitted for clarity. (a) P450 (18) (b) intradiol dioxygenase (7) (c) lipoxygenase (7) (d) α-KG-dependent non-heme iron enzymes (14) (e) soluble methane monooxygenase (15) (f) uncoupled binuclear copper (16) (g) coupled binuclear copper (h) flavin monooxygenases (17).

Copper-containing oxygenases are less common than their iron counterparts, perhaps because of the greater difficulty in obtaining more reactive, higher oxidation states. Examples include dopamine β-monooxygenase (DβM), tyramine β-monooxygenase (TβM), peptidylglycine α-hydroxylating enzyme (PHM), tyrosinase, and a membrane bound form of MMO (16, 17, 24). The membrane bound form of MMO contains both a Type 3 binuclear Cu site and a mononuclear Type 2 Cu site (25). At the moment, its mechanism has not been studied in detail and will not be discussed additionally here.

Uncoupled binuclear copper oxygenases

DβM, TβM, and PHM all employ two uncoupled, Type 2 copper sites in the biosynthesis of neurotransmitters and of hormones (16). The reaction entails the hydroxylation of unactivated carbon centers, similar to the chemistry of the P450s and α-KG-dependent non-heme iron enzymes. The two metal sites of the uncoupled binuclear Cu monooxygenases are separated by more than 11 zA, which is a feature that distinguishes this family of enzymes from the binuclear Type 3 copper proteins. Each Cu site has a discrete coordination environment and serves a unique function in the hydroxylation reaction. The CuM site functions as the dioxygen activation site, whereas CuH serves as the electron transfer site, providing the additional electron required for substrate oxidation. Evidence suggests that the reaction of dioxygen with the reduced Cu I M generates a Cu II M -superoxide intermediate (Fig. 3f), which subsequently abstracts a hydrogen atom from the substrate (26, 27). A methionine ligand to CuM is believed to stabilize the reduced enzyme form, such that oxygen activation is coupled strongly to the ensuing C-H activation step. The steps following C-H activation are still unresolved. A Cu II -oxyl radical (Cu II —O•) is a postulated, short-lived intermediate along the reaction pathway. This high energy intermediate is unprecedented in copper chemistry, but it could provide the driving force for the requisite electron transfer from Cu I H to Cu II M.

Coupled binuclear copper oxygenases

A characteristic Type 3, binuclear Cu site is found in tyrosinase (10). Tyrosinase catalyzes the conversion of monophenols to orthoquinones, the formation of which is coupled to the two electron reduction of O2 and proceeds in two steps. The first step occurs via electrophilic attack on the phenol ring of the substrate and is followed by the oxidation of the di-hydroxybenzene to yield the quinone product. Thus, tyrosinase functions as both an oxygenase and an oxidase. Much of the structural information pertaining to the tyrosinase active site and various Cu-dioxygen intermediates was derived by comparison to several spectroscopic studies on model complexes (10). The resting form of tyrosinase assumes two forms: 15% of the enzyme exists in the oxy state (Cu II 2O2 2— ), whereas 85% is present in the met state (both Cu sites are oxidized, but O2 is not bound). The reaction is initiated by the binding of phenolic substrate to one of the Cu atoms (CuA) in the oxy form (Fig. 3g). Substrate hydroxylation converts the active site to the met state, which binds diphenol in a bidentate fashion (as for catechol oxidase, Fig. 5c). The ensuing oxidation to yield the o -quinone generates the reduced, deoxy state (Cu I 2) of the enzyme. The oxy form is regenerated by the reaction with dioxygen and the catalytic cycle resumes. Alternative mechanisms, invoking the formation of Cu III intermediates or radicals, also have been postulated.

Several types of flavoprotein monooxygenases exist. One group catalyzes electrophilic aromatic substitution or heteroatom oxidation reactions, whereas the other group catalyzes Baeyer-Villiger-type oxidations of ketones (Fig. 2) (13, 17). In the well-studied, single-component flavoprotein monooxygenases, the flavin is reduced by a hydride delivered from a single molecule of NAD(P)H (Fig. 3h). The reduced flavin then reacts with O2 producing a peroxyflavin intermediate, which is in equilibrium with the hydroperoxyflavin form. For electrophilic substrates, as in the Baeyer-Villiger-type oxidation, it is the peroxyflavin intermediate that reacts with substrate. For hydroxylation of aromatic rings, the more electrophilic hydroperoxy intermediate is the reactive species. In the final step, the flavin is dehydrated to regenerate the resting state.

Single-component flavin monooxygenases employ different strategies to prevent the buildup of ROS. Extensive work on the p-hydroxybenzoate hydroxylase has demonstrated how protein dynamics compartmentalize different parts of the reaction (17). Reduction of the flavin and reaction with dioxygen occur in different conformations. Flavin reduction by NADPH only occurs in the presence of substrate, leading to the dioxygen reactive conformation. The Baeyer-Villiger monooxygenases and mammalian flavin monooxygenases use an alternative strategy. The flavin is reduced readily by NADPH in the absence of substrate, but the (hydro)peroxyflavin intermediate is stabilized by the bound NADP until a suitable substrate binds.

Members of the single-component flavin monooxygenase family have been shown to have wide substrate and reaction selectivities. In particular, one Baeyer-Villiger monooxygenase is known to react with over a 100 different substrates and is finding use as a tool for green biocatalysis in synthetic organic chemistry (28).

Oxidases couple the oxidation of an organic substrate to the two- or four-electron reduction of O2, producing H2O2 or two molecules of H2O, respectively. Oxygen atoms from dioxygen are not incorporated into the product, unlike reactions catalyzed by oxygenases. Oxidase reactions may proceed via inner-sphere or outer-sphere mechanisms.

Non-heme mononuclear iron oxidases

As mentioned, many non-heme iron enzymes also catalyze oxidase-type reactions, such as desaturation, in biological systems (7). Similar to the non-heme iron oxygenases, the reactions are thought to proceed through an Fe IV =O intermediate. Two examples of enzymes that catalyze biologically interesting oxidase reactions are isopenicillin N-synthase (IPNS) and 1-aminocyclopropane-1-carboxylate oxidase (ACCO).

IPNS catalyzes a double ring closure in the formation of isopenicillin concomitant with the four-electron reduction of dioxygen to two molecules of water, without the use of any cofactors or cosubstrates other than the Fe (Fig. 4a) (7). Ligation of the substrate thiolate activates the Fe for reaction with dioxygen by converting the active site from a six-coordinate to a five-coordinate metal center. The first ring closure is believed to occur with heterolysis of Fe III -O-OH forming Fe IV =O, which then closes the second ring.

ACCO breaks down 1-aminocyclopropane-1-carboxylate (ACC) in plants to form the growth hormone ethylene, hydrogen cyanide, and CO2 (Fig. 4b) (7). In the initial stages of catalysis, ACC ligates the iron, priming the system for reaction with dioxygen. Electrons for the reduction of O2 are derived from ascorbate, leading to formation of the reactive iron oxygen intermediate, possibly an Fe IV =O. Although the later steps of the reaction are poorly characterized, it is thought that an Fe IV =O may remove a hydrogen atom from ACC, generating a substrate radical, which breaks down to generate the gaseous product molecules.

Figure 4. Illustration of possible partial reaction cycles of some iron oxidase enzymes. (a) isopenicillin N-synthase (7) (b) 1-aminocyclopropane-1-carboxylate oxidase (7) (c) ribonucleotide reductase R2 (14).

Binuclear non-heme iron oxidases: Class I ribonucleotide reductas

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and can be found in all organisms. RNRs are categorized into three classes and employ different cofactors for this process. The Class I RNRs contain a binuclear iron cofactor (29). The iron active site is located in the R2 subunit, one of two homodimeric subunits that compose the Class I RNRs, and is very similar structurally to the active site of sMMO (Fig. 1d). The binuclear iron site reacts with dioxygen, ultimately generating a tyrosyl radical, which transfers an electron across 35 A and oxidizes a cysteine residue in the R1 subunit. The resulting Cys radical abstracts a hydrogen atom from the nucleotide to initiate deoxynucleotide synthesis. The di-iron center is required by the enzyme for the initial activation of the tyrosine residue but is not necessary for catalytic activity, as electrons are shuttled back and forth between the Tyr• in R2 and the Cys• in R1. Spectroscopic methods such as EPR, XAS, and Mossbauer have been employed to characterize many of the short-lived di-iron intermediates formed on reaction of the metal site with dioxygen (14). The initial activation of dioxygen yields a peroxide-bound binuclear Fe 111 center (Fig. 4c). This intermediate molecule may require protonation prior to reduction. An additional electron is transferred to the Fe III 2-peroxide adduct, generating an oxo-bridged Fe III /Fe IV center. The nature of the electron donor was revealed to be a tryptophan residue, which is oxidized to a cation radical in the electron transfer process. The ultimate formation of the tyrosyl radical by the Trp•/ + species has been shown to occur by several pathways. The iron active site in RNR-R2 shares several features and intermediates with sMMO. However, formation of an Fe IV /Fe IV species is unique to the latter enzyme and may dictate its monooxygenase ability.

Mononuclear copper oxidases

Most copper-containing oxygenases and oxidases use multiple metal centers to conduct their biotransformations. Copper amine oxidases (CAO) and galactose oxidase (GalO), instead, employ posttranslationally derived amino acid side chains as cofactors to supply additional electrons (24).

CAOs use a quinone cofactor to catalyze the oxidation of primary amines to aldehydes (Fig. 5a) (30). The metal cofactor of CAO is a square pyramidal Cu II center ligated by three histidines and by two water molecules (Fig. 1e). A distance of

3 A separates the copper site and the organic cofactor. Although its catalytic function is as an oxidase, CAO also functions as an oxygenase in the self-processing mechanism of quinone cofactor biogenesis (31). The biogenesis reaction requires two equivalents of molecular dioxygen for the six-electron oxidation of an active site tyrosine to 2,4,5-trihydroxyphenylquinone (TPQ). The catalytic oxidase reaction of the CAOs can be separated into two half-reactions. In the reductive half-reaction, the amine substrate is oxidized to the corresponding aldehyde. Concurrently, TPQ is converted to the reduced aminoquinol form. Dioxygen subsequently reacts with the reduced aminoquinol, generating TPQ and one equivalent of hydrogen peroxide. Reduction of the copper center has not proven essential for oxidase activity. In selected CAOs dioxygen has been shown to be activated via direct electron transfer from the reduced TPQ (30). Kinetic studies suggest that the metal center in the oxidase reaction contributes primarily to charge stabilization of the activated dioxygen (Fig. 5a).

GalO contains a unique cofactor, which is composed of a cysteine-crosslinked tyrosine ligated to a copper center, derived from posttranslational crosslinking of the two amino acids (Fig. 5b) (32, 33). A second Tyr, two His residues, and water molecule comprise the remaining ligands of the square pyramidal Cu II site. Cofactor formation is catalyzed by the enzyme itself, in a dioxygen and Cu I -dependent reaction. The Tyr-Cys residue is oxidized during the posttranslational process to yield the active form of GalO, which is a Cu II -Tyr-Cys• cation radical. The reaction catalyzed by GalO is the oxidation of primary alcohols to their corresponding aldehydes. The oxidation of substrate is coupled to the two-electron reduction of dioxygen to H2O2. Kinetic studies support the oxidation of alcohols via a ping-pong mechanism. In the reductive half-reaction, a hydrogen atom is abstracted from the C-a position of the metal bound alcohol to produce Cu I and the reduced cysteinyl-tyrosine. In the oxidative half-reaction (Fig. 5b), dioxygen is believed to bind directly to the reduced Cu site, displacing the water molecule. The Tyr-Cys ligand supplies the additional electron that is required for the two-electron reduction of dioxygen to peroxide, and for the regeneration of the cofactor radical.

Figure 5. Illustration of possible partial reaction cycles of some copper- and flavin-dependent oxidase enzymes. (a) Copper amine oxidase 30, 31 (b) galactose oxidase (32) (c) catechol oxidase (10) (d) multicopper oxidases (10) (e) flavin oxidases (30) (f) cytochrome c oxidase (38).

Binuclear copper oxidases

The active site of the catechol oxidases is virtually identical to the Type 3 binuclear Cu site found in tyrosinases (vide ultra) (10, 34). In contrast to the tyrosinases, catechol oxidases do not exhibit monooxygenase activity and are capable only of the second reaction, the oxidation of diphenols to quinones. The resting form of catechol oxidases lies exclusively in the met state. According to the accepted mechanism, two molecules of catechol are oxidized on binding to either the reduced, deoxy form or the oxy form. Dioxygen reacts with the reduced form after product release to yield the Cu II 2-peroxide adduct and allow binding of the next substrate molecule (Fig. 5c). The difference in reactivity toward O2 for the Type 3 copper centers in tyrosinases, catechol oxidases, and hemocyanins has been attributed to the partial or the complete occlusion of the substrate binding site in the latter two enzyme families. The degree of flexibility around the copper active site also has been cited as a possible factor (35).

The multicopper oxidases couple the one-electron or two-electron oxidation of their substrates to the four-electron reduction of dioxygen to water (36). The reaction with substrate can proceed via an outer-sphere or an inner-sphere mechanism, and as a result, the substrate specificity varies substantially among the enzymes. The best-characterized enzymes are laccase, ascorbate oxidase, and ceruloplasmin. Radical phenol and amine species formed by laccase and ascorbate oxidase react further via polymerization reactions with other organic molecules, or disproportionate to generate the final biological products. The substrate for ceruloplasmin is Fe II , which is oxidized to Fe III . Four substrate molecules transfer electrons sequentially to the Type 1 Cu site, which shuttles three electrons to the trinuclear cluster to generate the fully reduced enzyme form. Dioxygen is activated by the trinuclear copper cluster, which obtains two electrons from the Type 3 site (Fig. 5d). The Type 2 center is required for dioxygen activation but remains reduced at this stage in the reaction. The hydroperoxide is bound to the trinuclear cluster near the Type 3 binuclear Cu center, but both the Type 2 and the binuclear copper centers contribute significant electron density to the reduced oxygen molecule (37). The bound peroxide is reduced further by two electrons, one from the Type 2 Cu center and one from the distant Type 1 Cu site, forming the native intermediate. The native intermediate is believed to contain an oxide coordinated by the three Cu atoms of the trinuclear cluster. This species reacts with the substrate in the catalytic cycle.

Flavin Dependent Oxidases

Flavoprotein oxidases can conduct a variety of oxidation reactions, such as the conversion of alcohols and amines to aldehydes (12). Typically, the organic substrate provides an equivalent of hydride to reduce the flavin. As mentioned previously, the rate-limiting step in the reaction of the reduced flavin with dioxygen is the initial electron transfer to form a superioxide anion (30). Unlike the flavin oxygenases, a hydroperoxyflavin intermediate has never been detected for the flavin oxidases during catalysis. Instead, the mechanism is thought to proceed by two sequential one-electron transfers forming H2O2 (Fig. 5e). The lack of a solvent deuterium isotope effect in kcat/Km(O2) for glucose oxidase has provided evidence that proton transfer is not rate-limiting for the reduction of dioxygen.

Cytochrome c oxidase is a vital enzyme in the respiratory pathway of most aerobic organisms (38, 39). The enzyme couples the four-electron reduction of dioxygen to the generation of a proton gradient and the resulting synthesis of ATP, which is the primary source of energy for all cellular processes. Cytochrome c oxidase contains two iron-porphyrin units (heme a and heme a3) and two mononuclear Cu sites (CuA and CuB). Heme a and CuA serve as electron transfer sites, whereas heme a 3 and CuB form a binuclear metal site that activates dioxygen. CuB is coordinated by three His residues and is located 5 A from the Fe center of heme a3. A tyrosine residue is bound covalently to one CuB His ligand and is believed to be critical to the four-electron reduction of O2. The first intermediate generated upon the reaction of dioxygen with the reduced Cu I B-Fe II a3 center has been identified, based on resonance Raman spectroscopic studies, as an Fe nII a3-O2, although a peroxide bridged Fe III a3-O2 2- -Cu II B species has not been ruled out entirely (Fig. 5f) (38). The heme-bound dioxygen molecule is reduced rapidly by four electrons, two of which are obtained from the iron center and one each obtained from CuB and the active site tyrosine. The resultant intermediate Pm consists of an Fe IV =O, Cu II , and a tyrosyl radical, as deduced from Raman and EPR spectroscopy (40, 41). A series of proton and electron transfer events regenerates the resting, fully oxidized form of cytochrome c oxidase. The electrons for this process are derived from cytochrome c and shuttled through the CuA and heme a sites, during which protons are pumped across the cell membrane. Thus, cytochrome c oxidase functions as an electron-coupled proton pump.

The mechanisms of the oxygenases and oxidases detailed here represent some of the numerous strategies employed by enzymes to overcome the kinetic barrier for reaction of organic molecules with dioxygen. This list is far from exhaustive new reactions are discovered continually, and many intermediates in more established systems have not yet been characterized. A key feature of the dioxygen-activating enzymes is their ability to form highly ROS using carefully tuned cofactors. The enzymes prevent the release of ROS at a stage in the catalytic cycle when damage to the protein or the wider cellular environment could occur as a consequence. Efforts to mimic nature’s dioxygen chemistry with synthetic inorganic complexes often are only partially successful (7, 42). Many biomimetic complexes will react with dioxygen, but the resultant M-O bonds are either too stable to catalyze the subsequent chemistry or the products are too unstable and lead to undesirable side reactions.

Although great strides have been made to understand dioxygen activation by enzymes, many questions remain. The relationship between protein structure and enzyme catalysis is not well understood. Changes in the active site, beyond the primary coordination sphere, lead to altered cofactor redox properties. An identical cofactor often is employed by different enzymes to carry out dissimilar chemistry. The protein fold tunes the reactivity of these sites through electrostatic effects, control of solvent and substrate access, and by carefully organizing the orientation of substrates around the active site. The role of protein dynamics in tuning enzyme reactivity has become the focal point of recent studies, as well. These aspects of enzyme catalysis present the primary difficulties in the design of synthetic complexes that function as protein mimics.

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15. Merkx M, Kopp DA, Sazinsky MH, Blazyk JL, Muller J, Lippard SJ. Dioxygen activation and methane hydroxylation by soluble methane monooxygenase: a tale of two irons and three proteins. Angew. Chem. Int. Ed. 2001 40:2782-2807.

16. Klinman JP. The copper-enzyme family of dopamine β-mono-oxygenase and peptidylglycine α-hydroxylating monooxygenase: resolving the chemical pathway for substrate hydroxylation. J. Biol. Chem. 2006 281:3013-3016.

17. Ballou DP, Entsch B, Cole LJ. Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases. Biochem. Biophys. Res. Comm. 2005 338:590-598.

18. Denisov IG, Makris TM, Sligar SG, Schlichting I. Structure and chemistry of cytochrome P450. Chem. Rev. 2005 105:2253-2277.

19. Sligar SG, Makris TM, Denisov IG. Thirty years of microbial P450 monooxygenase research: Peroxo-heme intermediates - the central bus station in heme oxygenase catalysis. Biochem. Biophys. Res. Comm. 2005 338:346-354.

20. Green MT, Dawson JH, Gray HB. Oxoiron(IV) in chloroper-oxidase compound II is basic: Implications for P450 chemistry. Science 2004 304:1653-1656.

21. Groves JT. High-valent iron in chemical and biological oxidations. J. Inorg. Biochem. 2006 100:434-447.

22. Knapp MJ, Seebeck FP, Klinman JP. Steric control of oxygenation regiochemistry in soybean lipoxygenase-1. J. Am. Chem. Soc. 2001 123:2931-2932.

23. Solomon EI, Decker A, Lehnert N. Non-heme iron enzymes: contrasts to heme catalysis. Proc. Natl. Acad. Sci. 2003 100:3589-3594.

24. Klinman JP. Mechanisms whereby mononuclear copper proteins functionalize organic substrates. Chem. Rev. 1996 96:2541-2561.

25. Lieberman RL, Rosenzweig AC. Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature 2005 434:177-182.

26. Chen P, Solomon EI. Oxygen activation by the noncoupled binuclear copper site in peptidylglycine α-hydroxylating monooxygenase. Reaction mechanism and role of the noncoupled nature of the active site. J. Am. Chem. Soc. 2004 126:4991-5000.

27. Evans JP, Ahn K, Klinman JP. Evidence that dioxygen and substrate activation are tightly coupled in dopamine β-mono-oxygenase. Implications for the reactive oxygen species J. Biol. Chem. 2003 278:49691-49698.

28. Kamerbeek NM, Janssen DB, van Berkel WJH, Fraaije MW. Baeyer-Villiger monooxygenases, an emerging family of flavin-dependent biocatalysts. Ad. Syn. Cat. 2003 345:667-678.

29. Stubbe J, Nocera DG, Yee CS, Chang MCY. Radical initiation in the class I ribonucleotide reductase: long-range proton-coupled electron transfer. Chem. Rev. 2003 103:2167-2201.

30. Mure M, Mills SA, Klinman JP. Catalytic mechanism of the topa quinone containing copper amine oxidases. Biochemistry 2002 41:9269-9278.

31. DuBois JL, Klinman JP. Mechanism of post-translational quinone formation in copper amine oxidases and its relationship to the catalytic turnover. Arch. Biochem. Biophys. 2005 433:255-265.

32. Whittaker JW. The radical chemistry of galactose oxidase. Arch. Biochem. Biophys. 2005 433:227-239.

33. Firbank SJ, Rogers M, Hurtado-Guerrero R, Dooley DM, Halcrow MA, Phillips SEV, Knowles PF, McPherson MJ. Cofactor processing in galactose oxidase. Biochem. Soc. Trans. 2003 31:506-509.

34. Gerdemann C, Eicken C, Krebs B. The crystal structure of catechol oxidase: New insight into the function of type-3 copper proteins. Acc. Chem. Res. 2002 35:183-191.

35. Matoba Y, Kumagai T, Yamamoto A, Yoshitsu H, Sugiyama M. Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. J. Biol. Chem. 2006 281:8981-8990.

36. Solomon EI, Sundaram UM, Machonkin TE. Multicopper oxidases and oxygenases. Chem. Rev. 1996 96:2563-2605.

37. Yoon J, Mirica LM, Stack TDP, Solomon EI. Variable-temperature, variable-field magnetic circular dichroism studies of tris-hydroxy- and μ(3)-oxo-bridged trinuclear Cu(II) complexes: Evaluation of proposed structures of the native intermediate of the multicopper oxidases. J. Am. Chem. Soc. 2005 127:13680-13693.

38. Yoshikawa S. Structural chemical studies on the reaction mechanism of cytochrome c oxidase. In: Biophysical and Structural Aspects of Bioenergetics. 2005. Wikstrom M, ed. RSC publishing, Cambridge, UK.

39. Ferguson-Miller S, Babcock GT. Heme/copper terminal oxidases. Chem. Rev. 1996 96:2889-2907.

40. Babcock GT. How oxygen is activated and reduced in respiration. Proc. Natl. Acad. Sci. 1999 96:12971-12973.

41. Budiman K, Kannt A, Lyubenova S, Richter OMH, Ludwig B, Michel H, MacMillan F. Tyrosine 167: the origin of the radical species observed in the reaction of cytochrome c oxidase with hydrogen peroxide in Paracoccus denitrificans. Biochemistry. 2004 43:11709-11716.

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13: Introduction to Biochemistry

Biochemistry is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes. By controlling information flow through biochemical signaling and the flow of chemical energy through metabolism, biochemical processes give rise to the incredible complexity of life.

Over the last decades of the 20th century, biochemistry become so successful at explaining living processes that now almost all areas of the life sciences from botany to medicine to genetics are engaged in biochemical research. Today, the main focus of pure biochemistry is in understanding how biological molecules give rise to the processes that occur within living cells, which in turn relates greatly to the study and understanding of whole organisms.

Biochemistry is closely related to molecular biology, the study of the molecular mechanisms by which genetic information encoded in DNA is able to result in the processes of life. Depending on the exact definition of the terms used, molecular biology can be thought of as a branch of biochemistry, or biochemistry as a tool with which to investigate and study molecular biology.

Much of biochemistry deals with the structures, functions and interactions of biological macromolecules, such as proteins, nucleic acids, carbohydrates and lipids, which provide the structure of cells and perform many of the functions associated with life. The chemistry of the cell also depends on the reactions of smaller molecules and ions. These can be inorganic, for example water and metal ions, or organic, for example the amino acids which are used to synthesize proteins. The mechanisms by which cells harness energy from their environment via chemical reactions are known as metabolism. The findings of biochemistry are applied primarily in medicine, nutrition, and agriculture. In medicine, biochemists investigate the causes and cures of disease. In nutrition, they study how to maintain health and study the effects of nutritional deficiencies. In agriculture, biochemists investigate soil and fertilizers, and try to discover ways to improve crop cultivation, crop storage and pest control. Much of biochemistry deals with the structures and functions of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules&mdashalthough increasingly processes rather than individual molecules are the main focus.

The Chemistry and Biology of Nitroxyl (HNO)

The Chemistry and Biology of Nitroxyl (HNO) provides first-of-its-kind coverage of the intriguing biologically active molecule called nitroxyl, or azanone per IUPAC nomenclature, which has been traditionally elusive due to its intrinsically high reactivity.

This useful resource provides the scientific basis to understand the chemistry, biology, and technical aspects needed to deal with HNO. Building on two decades of nitric oxide and nitroxyl research, the editors and authors have created an indispensable guide for investigators across a wide variety of areas of chemistry (inorganic, organic, organometallic, biochemistry, physical, and analytical) biology (molecular, cellular, physiological, and enzymology) pharmacy and medicine.

This book begins by exploring the unique molecule’s structure and reactivity, including important reactions with small molecules, thiols, porphyrins, and key proteins, before discussing chemical and biological sources of nitroxyl. Advanced chapters discuss methods for both trapping and detecting nitroxyl by spectroscopy, electrochemistry, and fluorescent inorganic cellular probing.

Expanding on the compound’s foundational chemistry, this book then explores its molecular physiology to offer insight into its biological implications, pharmacological effects, and practical issues.

The Chemistry and Biology of Nitroxyl (HNO) provides first-of-its-kind coverage of the intriguing biologically active molecule called nitroxyl, or azanone per IUPAC nomenclature, which has been traditionally elusive due to its intrinsically high reactivity.

This useful resource provides the scientific basis to understand the chemistry, biology, and technical aspects needed to deal with HNO. Building on two decades of nitric oxide and nitroxyl research, the editors and authors have created an indispensable guide for investigators across a wide variety of areas of chemistry (inorganic, organic, organometallic, biochemistry, physical, and analytical) biology (molecular, cellular, physiological, and enzymology) pharmacy and medicine.

This book begins by exploring the unique molecule’s structure and reactivity, including important reactions with small molecules, thiols, porphyrins, and key proteins, before discussing chemical and biological sources of nitroxyl. Advanced chapters discuss methods for both trapping and detecting nitroxyl by spectroscopy, electrochemistry, and fluorescent inorganic cellular probing.

Expanding on the compound’s foundational chemistry, this book then explores its molecular physiology to offer insight into its biological implications, pharmacological effects, and practical issues.

Resonance Raman studies of hemerythrin-ligand complexes

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Conclusion and future challenges

Employing reactive complexes of abundant metals for synthesis, catalysis and energy supply is of great current interest. Selective functionalization of unactivated C–H bonds in organic compounds, for example, is a highly attractive strategy in organic synthesis, and the oxidation of methane and water are considered 'holy grails' in synthetic chemistry 96 . Similarly, energy-efficient production of ammonia is extremely important, as fertilizers generated from ammonia are responsible for sustaining one-third of Earth's population. A range of metalloenyzmes achieve these challenging tasks in biology by activating dioxygen and dinitrogen and using cheap and abundant first-row transition metals, like iron and manganese. Such reactions are carried out under ambient conditions with high efficiency and high stereospecificity. The recent results presented here from the bioinorganic chemistry community lend credence to the participation of high-valent oxo–ion and nitrido–iron complexes in the above-mentioned processes. Oxo and nitridoiron model complexes have now been synthesized using dioxygen or dinitrogen as the oxidant, via mechanisms reminiscent of the O2 and N2 activation process proposed in biology. Many of these complexes show intriguing reactivities, which in turn have provided vital insights into the modelled enzymatic reactions. Among the most significant conclusion of these studies is the observed activation of the model ferryl unit on axial ligand coordination trans to the oxo group. This has been attributed to strong electron donation from the axial thiolate ligand and explains the high reactivity of the natural thiolate-bound P450-I. Another important finding is the recently demonstrated 73,74 increased reactivity of the linear [(O)Fe IV -O-Fe III (OH2)] 2+ model complex, as compared with the ring-like [Fe IV 2(μ-O)2] 2+ core, that provides evidence for a comparable, more ring-opened form of Q with a terminal Fe IV =O unit as the active species in the reactivity of MMO. Additionally, although direct evidence for the involvement of oxoiron(V) intermediates in water oxidation is lacking in the literature, Kundu et al. 97 , has recently demonstrated a O–O bond formation reaction mediated by polynuclear oxoiron(IV) intermediates. Such a metal-mediated O–O bond formation step is considered to be the most critical part of dioxygen evolution in photosystem-II 98 . In N2 activation and transformation chemistry, Lee et al. (ref. 99) have shown that many important intermediates in a variety of oxidation states of a hypothetical N2 to NH3 conversion cycle can be accommodated at a mononuclear iron site. More recently, Rodriguez et al. 100 have demonstrated the potassium-assisted cooperativity of three iron centres in the activation and cleavage of dinitrogen and subsequent generation of ammonia on reaction of the nitride species with dihydrogen.

Unfortunately, the reactions exhibited by the model complexes are found to be non-catalytic, with activities falling far short of the activity of the biological catalysts. The low reactivity of the model complexes can be explained by the inability of synthetic chemists to exactly reproduce the biological ligand and protein environment. For example, even the ligand set of two histidines and one carboxylate, as observed in the first coordination sphere of non-haem oxygenases, is extremely difficult to synthesize and manipulate. Similarly, it has not yet been possible to generate an oxoiron(IV) porphyrin π-cation radical model complex with an axial thiolate ligand trans to the iron–oxo unit, as observed in Cpd-I of cytochrome-P450. Additionally, no iron-based model complexes mimicking the FeMo cofactor activity of the nitrogenase enzyme are known in the literature, and, as a result, the role of the postulated carbide ligand in dinitrogen activation is far from understood. Thus, new and innovative synthetic strategies are needed to generate superoxidized iron centres in ligand environments that better resemble the active site of the metalloenzymes. This goal may eventually lead to the development of iron-catalysed selective functionalization of organic compounds or ammonia synthesis by using cheap and accessible sources of dioxygen or dinitrogen under ambient conditions.

Biochemistry Online: An Approach Based on Chemical Logic

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Homework Problems

Review: The Cell


Homework Problems - Literature Learning Module:


Homework Problems - Literature Learning Module:


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Chapter 5: BINDING

  1. Introduction to Reversible Binding
  2. Mathematical Analyses of Binding Graphs
  3. Model Binding Systems update on intracellular droplets
  4. Binding and the Control of Gene Transcription update on chromatin structure and phase transitions
  5. New Methods in Drug Development
  6. Immune System Recognition - new section on Inflammasomes

Homework Problems - Literature Learning Module:


Homework Problems - Literature Learning Modules:

Chapter 7: CATALYSIS

Homework Problems - Literature Learning Module:


  1. The Chemistry of Dioxygen
  2. Oxidative Enzymes
  3. ATP and Oxidative Phosphorylation New Jmo/Jsmols for Complex I, III, IV, and ATP synthase New section on Complex III
  4. Photosynthesis: The Light Reaction
  5. Nitrogenase: A Reductive Use of Metal Clusters

Homework Problems - Literature Learning Module:


  1. Active Transport
  2. Neural Signaling
  3. Signaling Proteins 6/14/17 - new section on small G proteins, GAPs and GEFs
  4. mTOR and Nutrient Signaling
  5. Metabolic Control Analysis and Systems Biology
  6. 11/16/17 (just beginning development) Signaling Math : Graphic Analyses of Input and Outputs

Homework Problems - Literature Learning Module:


Chapter 11: ORIGIN OF LIFE


Additional Web Links:

  1. NCBI Biochemistry Texts -search for biochemistry texts
  2. Biochemistry, 5th edition Jeremy M Berg, John L Tymoczko, and Lubert Stryer - searchable but not broweable, from the NCBI.
  3. Martindale's Biochemistry
  4. Biophysical Society: National Lectures | Resources on selected topics
  5. Medical Biochemistry found in Biochemistry textbooks!

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Please send comments to Dr. Henry Jakubowski.

13.4: The Chemistry and Biochemistry of Dioxygen - Biology

B.A. Washington University, 2008

Ph.D. Northwestern University, 2012

Postdoctoral Researcher, University of California, San Diego 2013-2016

The Machan Group is interested in energy-relevant catalysis, particularly at the interface of molecular electrochemistry and materials. The development of efficient and selective transformations to produce commodity chemical precursors and fuels using CO2, O2, H2, and H2O as reagents remains an ongoing challenge for the storage of electrical energy within chemical bonds. Our approach is inspired by the numerous metalloproteins capable of catalyzing kinetically challenging reactions with significant energy barriers in an efficient manner under ambient conditions. This type of reactivity is achieved through the convergent evolution of active sites with tailored coordination environments and macromolecular structures which can, among other things, transport substrates and products to and from the active site. Our research focuses on developing new inorganic complexes and materials which incorporate co-catalytic moieties, non-covalent secondary sphere interactions, and substrate relays as catalysts.

In order to characterize and optimize these systems, research in the Machan Group uses synthetic inorganic chemistry, electrochemistry, and advanced characterization techniques (spectroelectrochemistry, stopped-flow IR and UV-vis spectroscopies). This enables us to develop an understanding of electronic structure and mechanism in transformations of interest. A brief summary of current projects is listed below.

Electrochemical Reduction of Dioxygen
The reduction of dioxygen (O2) is of vital importance to energy related reactions. In biology, respiration uses O2 reduction as a thermodynamic sink, whereas fuel cells pair the oxygen reduction reaction (ORR) to H2O as a proton-dependent half-reaction to the oxidation of chemical fuels. Catalytic ORR processes mediated by molecular species continue to garner interest as models for more complex heterogeneous systems. We are interested in the understanding selective activation and reduction of O2 to H2O2 or H2O by molecular species through electrochemical, spectrochemical, and spectroelectrochemical studies. Using these studies, we are developing new ligand frameworks for aqeuous and non-aqeuous systems, some of which are models for metalloenzyme active sites.

Molecular Electrocatalysts for the Electrochemical Conversion of Carbon Dioxide

The efficient and cost-effective catalytic reduction of CO2 using renewable energy remains a significant challenge for molecular species. Heterogeneous systems can produce highly reduced products like methane and ethylene from CO2, but these generally suffer from a lack of selectivity. The intrinsic advantage of molecular systems is the relative ease with which they may be characterized and quantified, relative to the distribution of active site morphologies that may be present in a bulk material. Using bioinspired design principles, we are investigating catalytic conversion strategies for producing CO and formic acid from CO2.

Porous Electrocatalyst Materials
Metal-organic frameworks (MOFs) and covalently linked organic frameworks (COFs) continue to attract significant interest in materials chemistry. MOFs and COFs offer many advantages in terms of porosity and stability over more amorphous materials or zeolites. Indeed, the translation of molecular properties to bulk materials in this manner has implications for the development of electrochemically responsive films and membranes. We are focused on developing new methods for synthesizing and processing conducting and semi-conducting 2D MOF and COF materials sensitive to the chemical environment. This is primarily focused on applications in molecular detection, separation, and catalysis. A fundamental understanding of how molecular properties are translated in these systems will enable future studies focusing on other applications in energy storage and optoelectronic devices.

Squire J. Booker (He/Him/His)

Research in the Booker Lab focuses on elucidating the chemical mechanisms by which enzymes containing iron-sulfur clusters catalyze chemical reactions. Most ongoing projects deal with members of the Radical S-adenosylmethionine Superfamily, a diverse group of enzymes that employ radical chemistry to catalyze transformations involved in post-transcriptional and post-translational modifications, cofactor biosynthesis, secondary metabolite biosynthesis, and enzyme activation. Radical SAM enzymes share a [4Fe-4S] cluster cofactor that is used to reductively cleave S-adenosylmethionine and form the 5’-deoxyadenosyl radical, a potent oxidant typically used to abstract a hydrogen atom and initiate radical chemistry on the substrate. These remarkable enzymes utilize this initial shared step to catalyze a wide array of transformations including methylations, sulfur insertions, decarboxylations, and complex rearrangements. Using a combination of biochemical, analytical, structural, and spectroscopic techniques, the Booker Lab works to characterize these complex and intriguing reaction mechanisms to provide insight for applications to human health and disease and gain a greater understanding of how Nature has worked to solve difficult chemical problems.

Selected Publications

Badding, E. D., Grove, T. L., Gadsby, L. K., LaMattina, J. W., Boal, A. K., Booker, S. J. (2017) Rerouting the pathway for the biosynthesis of the side ring system of nosiheptide: the roles of NosI, NosJ, and NosK. J. Am. Chem. Soc., 139, 5896–5905 (PMID: 28343381).

Maiocco, S. J., Arcinas, A. J., Landgraf, B. J., Lee, K. H., Booker S. J., and Elliott, S. J. (2016) Transformation of the FeS clusters of the methylthiotransferases MiaB and RimO, detected by direct electrochemistry. Biochemistry, 55, 5531–5536.

Block, E., Booker S. J., Flores-Penalba, S., George, G. N., Gundala, S., Landgraf, B. J., Liu, J., Lodge, S. N., Pushie, M. J., Rozovsky, S., Vattekkatte, A., Yaghi, R., and Zeng, H. (2016) Trifluoroselenomethionine: A new natural amino acid. Chembiochem, 18, 1738–1751.

McLaughlin, M. I., Lanz, N. D., Goldman, P. J., Lee, K.-H., Booker, S. J., and Drennan, C. L. (2016) Crystallographic snapshots of sulfur insertion by lipoyl synthase. Proc. Natl. Acad. Sci. USA, 113, 9446–94350.

Landgraf, B. J., McCarthy, E. L., and Booker, S. J. (2016) Radical S-adenosylmethionine enzymes in human health and disease. Annu. Rev. Biochem., 85, 485–514 (PMID: 27145839).

Esakova, O. A., Silakov, A., Grove, T. L., Saunders, A. H., McLaughlin, M. I., Yennawar, N. H., and Booker, S. J. (2016) Structure of quinolinate synthase from Pyrococcus horikoshii in the presence of its product, quinolinic acid. J. Am. Chem. Soc,, 138, 7224–7227.

Schwalm, E. L., Grove, T. L., Booker, S. J., and Boal, A. K. (2016) Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA. Science, 352, 309–312.

Landgraf, B. J. and Booker, S. J. (2016) The stereochemical course of the reaction catalyzed by RimO, a radical SAM methylthiotransferase J. Am. Chem. Soc., 138, 2889–2892.

Blaszczyk, A. J., Silakov, A., Zhang, B., Maiocco, S. J., Lanz, N.D., Kelly, W. L., Elliott, S. J., Krebs, C. and Booker, S. J. (2016) Spectroscopic and electrochemical characterization of the iron-sulfur and cobalamin cofactors of TsrM, an unusual radical S-adenosylmethionine methylase. J. Am. Chem. Soc., 138, 3416–3426.

Lanz, N. D., Lee, K.-H., Horstmann, A. K., Pandelia, M.-E., Krebs, C., and Booker, S. J. (2016) Characterization of lipoyl synthase from Mycobacterium tuberculosis. Biochemistry, 55, 1372–1383.

Lanz, N. D., Rectenwald, J., Wang, B., Kakar, E., Laremore, T., Booker, S. J., and Silakov, A. (2015) Characterization of a radical intermediate in lipoyl cofactor biosynthesis. J. Am. Chem. Soc., 137,13216­–13219.

Rajakovich, L. J., Nørgaard, H., Warui, D. M., Chang, W.-C., Li, N., Booker, S. J., Krebs, C., Bollinger, J. M. Jr., Pandelia, M. E. (2015) Rapid reduction of the differic-peroxyhemiacetal intermediate in aldehyde-deformylating oxygenase by a cyanobacterial ferredoxin: Evidence for a free-radical mechanism. J. Am. Chem. Soc., 137, 11695–11709.

Marous, D. R., Lloyd, E. P., Buller, A. R., Mopshos, K. A., Grove, T. L., Blaszczyk, A. J., Booker, S. J., Townsend, C. A. (2015) Consecutive radical S-adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis. Proc. Natl. Acad. Sci. USA, 112, 10354–10358.

Maiocco, S. J., Grove, T. L., Booker, S. J., and Elliott, S. J. (2015) Electrochemical resolution of the [4Fe–4S] centers of the AdoMet radical enzyme BtrN: evidence of proton-coupling and an unusual, low-potential auxiliary cluster. J. Am. Chem. Soc. 137, 8664–8667.

Pandelia, M. E., Lanz, N. D., Booker, S. J., and Krebs, C. (2015) Mössbauer spectroscopy of Fe/S proteins. Biochim. Biophys.Acta, 1853, 1395–1405.

Lanz, N. D., and Booker, S. J. (2015) Auxiliary iron-sulfur cofactors in radical SAM enzymes. Biochim. Biophys. Acta, 1853, 1316–1334.

Bauerle, M.r., Schwalm, E. L. and Booker, S. J. (2015) Mechanistic diversity of radical SAM-dependent methylation. J. Biol. Chem., 290, 3995–4002.

Warui, D. M., Pandelia, M. E., Rajakovich, L. J., Krebs, C., Bollinger, J. M., Jr., and Booker, S. J. (2015) Efficient delivery of long-chain fatty aldehydes from the Nostoc punctiforme acyl–acyl carrier protein reductase to its cognate aldehyde deformylating oxygenase. Biochemistry 54, 1006–10015.

Lanz, N. D., Pandelia, M. E., Kakar, E. S., Lee, K.-H., Krebs, C., and Booker, S. J. (2014) Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase. Biochemistry, 53, 4557–4572.

Silakov, A., Radle, M. I., Grove, T. L., Bauerle, M. R., Green, M. T., Rosenzweig, A. C., Boal, A. K., and Booker, S. J. (2014) Characterization of a cross-linked protein–nucleic acid substrate radical in the reaction catalyzed by RlmN. J. Am. Chem. Soc., 136, 8221–8228.

Goldman, P. J., Grove, T. L., Booker, S. J., and Drennan, C. L. (2013) X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motfis for AdoMet radical chemistry. Proc. Natl. Acad. Sci., USA, 110, 15949–15954.

Landgraf, B. J., Arcinas, A. J., Lee, K.–H., and Booker, S. J. (2013) Identification of an intermediate methyl carrier in the radical SAM methylthiotransferases, RimO and MiaB. J. Am. Chem. Soc., 135, 15404–15416.

Pandelia, M. E., Li, N., Nørgaard, H., Warui, D. M., Rajakovich, L. J. Chang, W.-C., Booker, S. J., Krebs, C., and Bollinger, J. M. (2013) Substrate-triggered addition of dioxygen to the differous cofactor of aldehyde-deformylating oxygenase to form a differic-peroxide intermediate. J. Am. Chem. Soc., 135, 15801–15812.

Christensen, Q. H., Grove, T. L., Booker, S. J., Greenberg, E. P. (2013) A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases. Proc. Natl. Acad. Sci., USA, 110, 13815–13820.

Landgraf, B. J. and Booker, S. J. (2013) The ylide has landed. Nature, 498, 45–47.

Goldman, P. J., Grove, T. L., Sites, L. A., McLaughlin, M. I., Booker, S. J., and Drennan, C. L. (2013) X-ray structure of an S-adenosylmethionine radical activase reveals an anaerobic solution for formylglycine posttranslational modification. Proc. Natl. Acad. Sci., USA, 110, 8519–8524.

Grove, T. L., Ahlum, J. H. Qin, R. M., Lanz, N. D., Radle, M. I., Krebs, C., and Booker, S. J. (2013) Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in mechanism of catalysis. Biochemistry, 52, 2874–2887.

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