Spectroscopy

Derivatives of CuZnSOD are known with CuII ion bound either to the native copper site or to the native zinc site. The electronic absorption spectra of these derivatives indicate that the ligand environments of the two sites are very different. Copper(1I) is a d 9 transition-metal ion, and its d-d transitions are usually found in the visible and near-IR regions of the spectrum. 53 Copper(1I) complexes with coordinated nitrogen ligands are generally found to have an absorption band between 500 and 700 nm, with an extinction coefficient below 100 M -Icm -I. Bands in the absorption spectra of complexes with geometries that are distorted away from square planar tend to be red-shifted because of a smaller d-d splitting, and to have higher extinction coefficients because of the loss of centrosymmetry. Thus the optical spectrum of CuZnSOD with an absorption band with a maximum at 680 nm (14,700 cm -I; see Figure 5.18A) and an extinction coefficient of 155 M -Icm -I per Cu is consistent with the crystal structural results that indicate that copper(II) is bound to four imidazole nitrogens and a water molecule in a distorted square-pyramidal geometry. Metalsubstituted derivatives with CuII at the native copper site but with COII, CdII, HgII, or Ni II substituted for ZnII at the native zinc site all have a band at 680 nm, suggesting that the substitution of another metal ion for zinc perturbs the copper site very little, despite the proximity of the two metal sites. The absorption spectra of native CuZnSOD and these CuMSOD derivatives also have a shoulder at 417 nm (24,000 cm -I; see Figure 5.18A), which is at lower energy than normal imidazole-to-Cu II charge-transfer transitions, and has been assigned to 

an imidazolate-to-Cu 1I charge transfer, indicating that the imidazolate bridge between CuII and the metal ion in the native zinc site is present, as observed in the crystal structure of CuZnSOD. Derivatives with the zinc site empty, which therefore cannot have an imidazolate bridge, are lacking this 417 nm shoulder. Small but significant changes in the absorption spectrum are seen when the metal ion is removed from the zinc site, e.g., in copper-only SOD (Figure 5.18B). The visible absorption band shifts to 700 nm (14,300 cm -1), presumably due to a change in ligand field strength upon protonation of the bridging imidazolate. In addition, the shoulder at 417 nm has disappeared, again due to the absence of the imidazolate ligand.
 

The spectroscopic properties due to copper in the native zinc site are best observed in the derivative Ag1CuSOD, which has Ag1 in the copper site and Cu II in the zinc site (see Figure 5.18C), since the d 10 Ag1 ion is spectroscopically silent. In this derivative, the dod transition is markedly red-shifted from the visible region of the spectrum into the near-IR, indicating that the ligand environment of CuII in that site is either tetrahedral or five coordinate. The EPR properties of CuII in this derivative are particularly interesting (as discussed below).
 

The derivative with CUll bound at both sites, CuCuSOD, has a visible-near IR spectrum that is nearly a superposition of the spectra of CuZnSOD and Ag1CuSOD (see Figure 5.19), indicating that the geometry of CUll in each of these sites is little affected by the nature of the metal ion in the other site.

EPR spectroscopy has also proven to be particularly valuable in characterizing the metal environments in CuZnSOD and derivatives. The EPR spectrum of native CuZnSOD is shown in Figure 5.20A. The gil resonance is split by the hyperfine coupling between the unpaired electron on CUll and the I = i nuclear spin of copper. The All value, 130 G, is intermediate between the larger All

typical of square-planar CuII complexes with four nitrogen donor ligands and the lower All observed in blue copper proteins (see Chapter 6). The large linewidthseen in the g -l region indicates that the copper ion is in a rhombic (i.e., distorted) environment. Thus, the EPR spectrum is entirely consistent with the distorted square-pyramidal geometry observed in the x-ray structure.
 

Removal of zinc from the native protein to give copper-only SOD results in a perturbed EPR spectrum, with a narrower g1- resonance and a larger All value (142 G) more nearly typical of CUll in an axial N4 environment (Figure 5.20B). Apparently the removal of zinc relaxes some constraints imposed on the geometry of the active-site ligands, allowing the copper to adopt to a geometry closer to its preferred tetragonal arrangement.
 

The EPR spectrum due to Cu II in the native ZnII site in the Ag ICuSOD derivative indicates that Cu II is in a very different environment than when it is in the native copper site (Figure 5.20C). The spectrum is strongly rhombic, with a low value of All (97 G), supporting the conclusion based on the visible spectrum that copper is bound in a tetrahedral or five-coordinate environment. This type of site is unusual either for copper coordination complexes or for copper proteins in general, but does resemble the CUll EPR signal seen when either laccase or cytochrome c oxidase is partially reduced (see Figure 5.21). Partial

reduction disrupts the magnetic coupling between these CuII centers that makes them EPR-silent in the fully oxidized protein.
 

The EPR spectrum of CuCuSOD is very different from that of any of the other copper-containing derivatives (Figure 5.22) because the unpaired spins on

the two copper centers interact and magnetically couple across the imidazolate bridge, resulting in a triplet EPR spectrum. This spectrum is virtually identical with that of model imidazolate-bridged binuclear copper complexes. 

 

Electronic absorption and EPR studies of derivatives of CuZnSOD containing Cu II have provided useful information concerning the nature of the metal binding sites of those derivatives. IH NMR spectra of those derivatives are generally not useful, however, because the relatively slowly relaxing paramagnetic CuII center causes the nearby proton resonances to be extremely broad. This difficulty has been overcome in two derivatives, CuCoSOD and CuNiSOD, in which the fast-relaxing paramagnetic COIl and Ni II centers at the zinc site interact across the imidazolate bridge and increase the relaxation rate of the CuII center, such that well-resolved paramagnetically shifted IH NMR spectra of the region of the proteins near the two paramagnetic metal centers in the protein can be obtained and the resonances assigned. 1l8,119
 

The use of IH NMR to study CuCoSOD derivatives of CuZnSOD in combination with electronic absorption and EPR spectroscopies has enabled investigators to compare active-site structures of a variety of wild-type and mutant CuZnSOD proteins in order to find out if large changes in active-site structure have resulted from replacement of nearby amino-acid residues.

SOD activity

In the example described above, studies of a metal-substituted derivative helped in the evaluation of mechanistic possibilities for the enzymatic reaction. In addition, studies of such derivatives have provided useful information about the environment of the metal-ion binding sites. For example, metal-ion-substituted derivatives of CuZnSOD have been prepared with Cu II, CuI, ZnII, Ag I, Ni ll , or COli bound to the native copper site, and with Znll , CUll, CuI, COli, HgII, Cdll , Ni ll, or Ag I bound to the native zinc site. 100, 101, 117 The SOD activities of these derivatives are interesting; only those derivatives with copper in the copper site have a high degree of SOD activity, whereas the nature of the metal ion in the zinc site or even its absence has little or no effect.

Anions as Inhibitors

Studies of the interaction of CuZnSOD and its metal-substituted derivatives with anions have been useful in predicting the behavior of the protein in its reactions with its substrate, the superoxide anion, O2 - . 101.102 Cyanide, azide, cyanate, and thiocyanate bind to the copper ion, causing dissociation of a histidyl ligand and the water ligand from the copper. 115 Phosphate also binds to the enzyme at a position close to the Cu II center, but it apparently does not bind directly to it as a ligand. Chemical modification of Arg-141 with phenylglyoxal blocks the interaction of phosphate with the enzyme, suggesting that this positively charged residue is the site of interaction with phosphate.11 6
 

Electrostatic calculations of the charges on the CuZnSOD protein suggest that superoxide and other anions entering into the vicinity of the protein will be drawn toward and into the channel leading down to the copper site by the distribution of positive charges on the surface of the protein, the positively charged lysines at the mouth of the active-site cavity, and the positively charged arginine and copper ion within the active-site region. 112 Some of the anions studied, e.g., CN -, F -, N3 ,and phosphate, have been shown to inhibit the SOD activity of the enzyme. The source of the inhibition is generally assumed to be competition with superoxide for binding to the copper, but it may sometimes result from a shift in the redox potential of copper, which is known to occur sometimes when an anion binds to copper.

Enzymatic Activity and Mechanism

The mechanism of superoxide disproportionation catalyzed by CuZnSOD is generally believed to go by Mechanism I (Reactions 5.96-5.97), i.e., reduction of CuII to CuI by superoxide with the release of dioxygen, followed by reoxidation of CUI to CUll by a second superoxide with the release of HOz- or HzOz. The protonation of peroxide dianion, Oz z-, prior to its release from the enzyme is required, because peroxide dianion is highly basic and thus too unstable to be released in its unprotonated form. The source of the proton that protonates peroxide in the enzymatic mechanism is the subject of some interest.
 

Reduction of the oxidized protein has been shown to be accompanied by the uptake of one proton per subunit. That proton is believed to protonate the bridging imidazolate in association with the breaking of the bridge upon reduction of the copper. Derivatives with CoII substituted for ZnII at the native zinc site have been used to follow the process of reduction of the oxidized CuII form to the reduced Cu1form. The COIl in the zinc site does not change oxidation state, but acts instead as a spectroscopic probe of changes occurring at the native zincbinding site. Upon reduction (Reaction 5.102), the visible absorption band due to CoII shifts in a manner consistent with a change occurring in the ligand environment of CoII. The resulting spectrum of the derivative containing CuI in the copper site and COIl in the zinc site is very similar to the spectrum of the derivative in which the copper site is empty and the zinc site contains COlI. This result suggests strongly that the imidazolate bridge is cleaved and protonated and that the resulting imidazole ligand is retained in the coordination sphere of COIl (Reaction 5.102).

The same proton is thus an attractive possibility for protonation of peroxide as it is formed in the enzymatic mechanism (Reactions 5; 103 and 5. 104).

Attractive as this picture appears, there are several uncertainties about it. For example, the turnover of the enzyme may be too fast for protonation and deprotonation of the bridging histidine to occur. 113 Moreover, the mechanism proposed would require the presence of a metal ion at the zinc site to hold the imidazole in place and to regulate the pKa of the proton being transferred. The observation that removal of zinc gives a derivative with almost full SOD activity is thus surprising and may also cast some doubt on this mechanism. Other criticisms of this mechanism have been recently summarized. 

Studies of CuZnSOD derivatives prepared by site-directed mutagenesis are also providing interesting results concerning the SOD mechanism. For example, it has been shown that mutagenized derivatives of human CuZnSOD with major differences in copper-site geometry relative to the wild-type enzyme may nonetheless remain fully active. 114 Studies of these and similar derivatives should provide considerable insight into the mechanism of reaction of CuZnSOD with superoxide.

Structure

The x-ray crystal structure of the oxidized form of CuZnSOD from bovine erythrocytes shows a protein consisting of two identical subunits held together almost entirely by hydrophobic interactions.loo-I02 Each subunit consists of a flattened cylindrical barrel of {3-pleated sheet from which three external loops of irregular structure extend (Figure 5.15). The metal-binding region of the protein binds Cu II and Zn II in close proximity to each other, bridged by the imidazolate ring of a histidyI side chain. Figure 5.16 represents the metal-binding region. The CuII ion is coordinated to four histidyl imidazoles and a water in a highly distorted square-pyramidal geometry with water at the apical position. The ZnII ion is coordinated to three histidyl imidazoles (including the one shared with

copper) and an aspartyl carboxylate group, forming a distorted tetrahedral geometry around the metal ion.
 

One of the most unusual aspects of the structure of this enzyme is the occurrence of the bridging imidazolate ligand, which holds the copper and zinc ions 6 Aapart. Such a configuration is not unusual for imidazole complexes of metal ions, which sometimes form long polymeric imidazolate-bridged structures.

However, no other imidazolate-bridged bi- or polymetallic metalloprotein has yet been identified.
 

The role of the zinc ion in CuZnSOD appears to be primarily structural. There is no evidence that water, anions, or other potential ligands can bind to the zinc, so it is highly unlikely that superoxide could interact with that site. Moreover, removal of zinc under conditions where the copper ion remains bound to the copper site does not significantly diminish the SOD activity of the enzyme. J10 However, such removal does result in a diminished thermal stability, i.e., the zinc-depleted protein denatures at a lower temperature than the native protein, supporting the hypothesis that the role of the zinc is primarily structural in nature. JJJ
 

The copper site is clearly the site of primary interaction of superoxide with the protein. The x-ray structure shows that the copper ion lies at the bottom of a narrow channel that is large enough to admit only water, small anions, and similarly small ligands (Figure 5.17). In the lining of the channel is the positively charged side chain of an arginine residue,S Aaway from the copper ion

and situated in such a position that it could interact with superoxide and other anions when they bind to copper. Near the mouth of the channel, at the surface of the protein, are two positively charged lysine residues, which are believed to play a role in attracting anions and guiding them into the channel. lIZ Chemical modification of these lysine or arginine residues substantially diminishes the SOD activity, supporting their role in the mechanism of reaction with superoxide. 100-10Z
 

The x-ray structural results described above apply only to the oxidized form of the protein, i.e., the form containing CUll. The reduced form of the enzyme containing CuI is also stable and fully active as an SOD. If, as is likely, the mechanism of CuZnSOD-catalyzed superoxide disproportionation is Mechanism I (Reactions 5.96-5.97), the structure of the reduced form is of critical importance in understanding the enzymatic mechanism. Unfortunately, that structure is not yet available.

Enzymatic Activity

Several transition-metal complexes have been observed to catalyze superoxide disproportionation; in fact, aqueous copper ion, Cu2 +, is an excellent SOD catalyst, comparable in activity to CuZnSOD itselfp7 Free aqueous Cu2 + would not itself be suitable for use as an SOD in vivo, however, because it is too toxic (see Section III) and because it binds too strongly to a large variety of cellular components and thus would not be present as the free ion. (Most forms of complexed cupric ion show much less superoxide dismutase activity than the free ion.) Aside from aqueous copper ion, few other complexes are as effective as the SOD enzymes.
 

Two mechanisms (Reactions 5.96 to 5.99) have been proposed for catalysis of superoxide disproportionation by metal complexes and metalloenzymes.

In Mechanism I, which is favored for the SOD enzymes and most redox-active metal complexes with SOD activity, superoxide reduces the metal ion in the first step, and then the reduced metal ion is reoxidized by another superoxide, presumably via a metal-peroxo complex intermediate. In Mechanism II, which is proposed for nonredox metal complexes but may be operating in other situations as well, the metal ion is never reduced, but instead forms a superoxo complex, which is reduced to a peroxo complex by a second superoxide ion. In both mechanisms, the peroxo ligands are protonated and dissociate to give hydrogen peroxide.
 

Analogues for each of the separate steps of Reactions (5.96) to (5.99) have been observed in reactions of superoxide with transition-metal complexes, thereby establishing the feasibility of both mechanisms. For example, superoxide was shown to reduce CuII(phen)z2+ to give Cul(phen)z + (phen = 1,1O-phenanthroline), 106 a reaction analogous to Reaction (5.96). On the other hand, superoxide reacts with CU II(tet b) 2+ to form a superoxo complex 107 (a reaction analogous to Reaction 5.98), presumably because CUII(tet b) 2+ is not easily reduced to the cuprous state, because the ligand cannot adjust to the tetrahedral geometry that Cu1 prefers.

Reaction of superoxide with a reduced metal-ion complex to give oxidation of the complex and release of hydrogen peroxide (analogous to Reaction 5.97) has been observed in the reaction of FeIIEDTA with superoxide. 108 Reduction of a CoIII superoxo complex by free superoxide to give a peroxo complex (analogous to Reaction 5.99) has also been observed. 

If a metal complex can be reduced by superoxide and if its reduced form can be oxidized by superoxide, both at rates competitive with superoxide disproportionation, the complex can probably act as an SOD by Mechanism I. Mechanism II has been proposed to account for the apparent catalysis of superoxide disproportionation by Lewis acidic nonredox-active metal ions under certain conditionsY However, this mechanism should probably be considered possible for redox metal ions and the SOD enzymes as well. It is difficult to distinguish the two mechanisms for redox-active metal ions and the SOD enzymes unless the reduced form of the catalyst is observed directly as an intermediate in the reaction. So far it has not been possible to observe this intermediate in the SOD enzymes or the metal complexes.

Comparisons of Catalase, Peroxidase, and Cytochrome P-450

The proposal that these three enzymes all go through a similar high-valent oxo intermediate, i.e., 3 or compound I, raises two interesting questions. The first of these is why the same high-valent metal-oxo intermediate gives two very different types of reactions, i.e., oxygen-atom transfer with cytochrome P-450 and electron transfer with catalase and peroxidase. The answer is that, although the high-valent metal-oxo heme cores of these intermediates are in fact very similar, the substrate-binding cavities seem to differ substantially in how much access the substrate has to the iron center. With cytochrome P-450, the substrate is jammed right up against the location where the oxo ligand must reside in the high-valent oxo intermediate. But the same location in the peroxidase enzymes is blocked by the protein structure so that substrates can interact only with the heme edge. Thus oxidation of the substrate by electron transfer is possible for catalase and peroxidase, but the substrate is too far away from the oxo ligand for oxygen-atom transfer.

The second question is about how the the high-valent oxo intermediate forms in both enzymes. For catalase and peroxidase, the evidence indicates that hydrogen peroxide binds to the ferric center and then undergoes heterolysis at the 0-0 bond. Heterolytic cleavage requires a significant separation of positive and negative charge in the transition state. In catalase and peroxidase, analysis of the crystal structure indicates strongly that amino-acid side chains are situated to aid in the cleavage by stabilizing a charge-separated transition state (Figure 5.14). In cytochrome P-450, as mentioned in Section V.C.1, no such groups

are found in the hydrophobic substrate-binding cavity. It is possible that the cysteinyl axial ligand in cytochrome P-450 plays an important role in 0-0 bond cleavage, and that the interactions found in catalase and peroxidase that appear to facilitate such cleavage are therefore not necessary.

CATALASE AND PEROXIDASE Mechanism

The accepted mechanisms for catalase and peroxidase are described in Reactions (5.89) to (5.94).

In the catalase reaction, it has been established by use of HZ I80 Z that the dioxygen formed is derived from hydrogen peroxide, i.e., that 0-0 bond cleavage does not occur in Reaction (5.90), which is therefore a two-electron reduction of compound I by hydrogen peroxide, with the oxo ligand of the former being released as water. For the peroxidase reaction under physiological conditions, it is believed that the oxidation proceeds in one-electron steps (Reactions 5.91 and 5.92), with the final formation of product occurring by disproportionation (Reaction 5.93) or coupling (Reaction 5.94) of the one-electron oxidized intermediate. 94.95

CATALASE AND PEROXIDASE Description of the Enzymes

Catalase and peroxidase are heme enzymes that catalyze reactions of hydrogen peroxide.94,95 In catalase, the enzymatic reaction is the disproportionation of hydrogen peroxide (Reaction 5.82) and the function of the enzyme appears to be prevention of any buildup of that potentially dangerous oxidant (see the discussion of dioxygen toxicity in Section III).

Peroxidase reacts by mechanisms similar to catalase, but the reaction catalyzed is the oxidation of a wide variety of organic and inorganic substrates by hydrogen peroxide (Reaction 5.83).

(The catalase reaction can be seen to be a special case of Reaction 5.83 in which the substrate, AH2, is hydrogen peroxide.) Some examples of peroxidases that have been characterized are horseradish peroxidase, cytochrome c peroxidase, glutathione peroxidase, and myeloperoxidase. 

 

X-ray crystal structures have been determined for beef-liver catalase 80 and for horseradish peroxidase 96 in the resting, high-spin ferric state. In both, there is a single heme b group at the active site. In catalase, the axial ligands are a phenolate from a tyrosyl residue, bound to the heme on the side away from the active-site cavity, and water, bound to heme within the cavity and presumably replaced by hydrogen peroxide in the catalytic reaction. In horseradish peroxidase, the axial ligand is an imidazole from a histidyl residue. Also within the active-site cavity are histidine and aspartate or asparagine side chains that appear to be ideally situated to interact with hydrogen peroxide when it is bound to the iron. These residues are believed to play an important part in the mechanism by facilitating 0-0 bond cleavage (see Section VI.B below).
 

Three other forms of catalase and peroxidase can be generated, which are referred to as compounds I, II, and III. Compound I is generated by reaction of the ferric state of the enzymes with hydrogen peroxide.  Compound I is greenand has spectral characteristics very similar to the FeIV(p' -)(0) + complex prepared at low temperatures by reaction of ferric porphyrins with single-oxygenatom donors (see Section V.c.1.a). Titrations with reducing agents indicate that it is oxidized by two equivalents above the ferric form. It has been proposed (see 5.84) that the anionic nature of the tyrosinate axial ligand in catalase may serve to stabilize the highly oxidized iron center in compound I of that enzyme, 80 and furthermore that the histidyl imidazole ligand in peroxidase may deprotonate, forming imidazolate,52,97 or may be strongly hydrogen bonded,98 thus serving a similar stabilizing function for compound I in that enzyme.

Reduction of compound I by one electron produces compound II, which has the characteristics of a normal ferryl-porphyrin complex, analogous to 2, i.e., (L)FeIV(p)(O). Reaction of compound II with hydrogen peroxide produces compound III, which can also be prepared by reaction of the ferrous enzyme with dioxygen. It is an oxy form, analogous to oxymyoglobin, and does not appear to have a physiological function. The reactions producing these three forms and their proposed formulations are summarized in Reactions (5.85) to (5.88).

Other metal-containing monooxygenase enzymes

As mentioned above, much less is known about the structural characteristics and mechanisms of the nonheme metal-containing monooxygenase enzymes. From the similarities of the overall stoichiometries of the reactions and the resemblance of some of the enzymes to dioxygen-binding proteins, it is likely that the initial steps are the same as those for cytochrome P-450, i.e., dioxygen binding followed by reduction to form metal-peroxide or hydroperoxide complexes. It is not obvious that the next step is the same, however (i.e., 0-0 bond cleavage to form a high-valent metal-oxo complex prior to attack on substrate). The problem is that such a mechanism would generate metal-oxo complexes that appear to contain metal ions in chemically unreasonable high-oxidation states, e.g., Fe v, CuIII , or CuIV (Reactions 5.79-5.81).

An alternative mechanism is for the peroxide or hydroperoxide ligand to attack the substrate directly; i.e., 0-0 bond cleavage could be concerted with attack on substrate. Another possibility is that the oxygen atom is inserted in a metalligand bond prior to transfer to the substrate. Neither of these alternative mechanisms has been demonstrated experimentally. These various possibilities remain to be considered as more  information about the monooxygenase enzymes becomes available.

Cytochrome P-450

Cytochrome P-450 enzymes are a group of monooxygenase enzymes that oxygenate a wide variety of substrates. 73 Examples of such reactions are:

1. hydroxylation of aliphatic compounds (Reaction 5.59);
2. hydroxylation of aromatic rings (Reaction 5.60);
3. epoxidation of olefins (Reaction 5.61);
4. amine oxidation to amine oxides (Reaction 5.62);
5. sulfide oxidation to sulfoxides (Reaction 5.63); and
6. oxidative dealkylation of heteroatoms (for example, Reaction 5.64).

Some of these reactions have great physiological significance, because they represent key transformations in metabolism, as in lipid metabolism and biosynthesis of corticosteroids, for example. 73 Cytochrome P-450 is also known to catalyze the transformation of certain precarcinogens such as benzpyrene into their carcinogenic forms. 

 

Many of the P-450 enzymes have been difficult to characterize, because they are membrane-bound and consequently relatively insoluble in aqueous solution. However, cytochrome P-450cam ' which is a component of the camphor 5-monooxygenase system isolated from the bacterium Pseudomonas putida, is soluble and has been particularly useful as the subject of numerous spectroscopic and mechanistic studies, as well as several x-ray crystallographic structure determinations. 80 This enzyme consists of a single polypeptide chain, mainly a-helical, with a heme b group (Fe-protoporphyrin IX) sandwiched in between two helices, with no covalent attachments between the porphyrin ring and the protein. One axial ligand complexed to iron is a cysteinyl thiolate. In the resting state, the iron is predominantly low-spin FellI, probably with a water as the other axial ligand. When substrate binds to the resting enzyme, the spin state changes to high-spin, and the non-cysteine axial ligand is displaced. The enzyme can be reduced to an FeU state, which is high-spin, and resembles deoxyhemoglobin or myoglobin in many of its spectroscopic properties. This ferrous form binds dioxygen to make an oxy form or carbon monoxide to make a carbonyl form. The CO derivative has a Soret band (high-energy 7T-7T* transition of the porphyrin ring) at 450 nm, unusually low energy for a carbonyl derivative of a heme protein because of the presence of the axial thiolate ligand. This spectroscopic feature aids in the isolation of the enzyme and is responsible for its name. a. "Active Oxygen" Camphor 5-monooxygenase is a three-component system, consisting of cytochrome P-450cam and two electron-transfer proteins, a  flavoprotein, and an iron-sulfur protein (see Chapters 6 and 7). The role of the electron-transfer proteins is to deliver electrons to theP-450 enzyme, but these may be replaced in vitro by other reducing agents. The reaction sequence is in Figure 5.10.
 

For cytochrome P-450, the question that is possibly of greatest current interest to the bioinorganic chemist is just what mechanism enables activation of dioxygen and its reaction with substrate. It seems clear that dioxygen binds to

the ferrous state of the enzyme-substrate complex, and that the resulting oxy ligand, which presumably is similar to the oxy ligand in oxyhemoglobin and oxymyoglobin, is not sufficiently reactive to attack the bound substrate. The oxy form is then reduced and the active oxidant is generated, but the nature of the active oxidant has not been deduced from studies of the enzyme itself, nor has it been possible to observe and characterize intermediates that occur between the time of the reduction and the release of product. Three species are potential candidates for" active oxygen," the oxygen-containing species that attacks the substrate, in cytochrome P-450. They are:
 

1. (1) a ferric peroxo, la, or hydroperoxo complex, lb, formed from oneelectron reduction of the oxy complex (Reaction 5.65);
2. an iron(1V) oxo complex, 2, formed by homolytic 0-0 bond cleavage of a ferric hydroperoxo complex (Reaction 5.66); and
3. a complex at the oxidation level of an iron(V) oxo complex, 3, formed by heterolytic 0-0 bond cleavage of a ferric hydroperoxo complex (Reaction 5.67).

The hydroxyl radical, HO', although highly reactive and capable of attacking P-450 substrates, is considered to be an unlikely candidate for "active oxygen" because of the indiscriminate character of its reactivity.

An iron(V) oxo complex (or a related species at the same oxidation level), 3, formed via Reaction (5.67), is the favored candidate for "active oxygen" in cytochrome P-450. 81 This conclusion was initially drawn from studies of reactions of the enzyme with alkylhydroperoxides and single-oxygen-atom donors. Single-oxygen-atom donors are reagents such as iodosylbenzene, 01Ph, and periodate, 104 ,capable of donating a neutral oxygen atom to an acceptor, forming a stable product in the process (here, iodobenzene, 1Ph, and iodate, 103 -). It was discovered that ferric cytochrome P-450 could catalyze oxygenation reactions using organic peroxides or single-oxygen-atom donors in place of dioxygen and reducing agents. Usually the same substrates would give the identical oxygenated product. This reaction pathway was referred to as the "peroxide shunt" (see Figure 5.10). The implication of this discovery was that the same form of "active oxygen" was generated in each reaction, and the fact that single- oxygen-atom donors could drive this reaction implied that this species contained only one oxygen atom, i.e., was generated subsequent to 0-0 bond cleavage. The mechanism suggested for this reaction was Reactions (5.68) and (5.69).

b. Metalloporphyrin Model Systems Studies of the reactivities of synthetic metalloporphyrin complexes in oxygen-transfer reactions and characterization of intermediate species observed during the course of such reactions have been invaluable in evaluating potential intermediates and reaction pathways for cytochrome P-450. Logically, it would be most desirable if one could mimic the enzymatic oxygenation reactions of substrates using iron porphyrins, dioxygen, and reducing agents. However, studies of such iron-porphyrin-catalyzed reactions have failed to produce meaningful results that could be related back to the P-450 mechanism. This is perhaps not surprising, since the enzyme system is designed to funnel electrons into the iron-dioxygen-substrate complex, and thus to generate the active oxidant within the confines of the enzyme active site in the immediate proximity of the bound substrate. Without the constraints imposed by the enzyme, however, iron porphyrins generally will either (1) catalyze the oxidation of the reducing agent by dioxygen, leaving the substrate untouched, or (2) initiate free-radical autoxidation reactions (see Section II.C). A different approach was suggested by the observation of the peroxide shunt reaction (Reactions 5.68 and 5.69) using organic peroxides or single-oxygen-atom donors, and the earliest successful studies demonstrated that Fe(TPP)CI (TPP = tetraphenylporphyrin) would catalyze the epoxidation of olefins and the hydroxylation of aliphatic hydrocarbons by iodosylbenzene 81(Reactions 5.70 and 5.71).

Reactions (5.70) and (5.71) were postulated to occur via an iron-bound oxidant such as 3 in Reaction (5.67). This hypothesis was tested by studying the reaction of dioctyl Fe(PPIX)CI with iodosylbenzene, which resulted in 60 percent hydroxylation at positions 4 and 5 on the hydrocarbon tail (see 5.72), positions for which there is no reason to expect increased reactivity except for the fact that those particular locations are predicted from molecular models to come closest to the iron center when the tail wraps around the porphyrin molecule.

The nature of the species produced when single-oxygen-atom donors react with Fe III-porphyrin complexes has been deduced from studies of an unstable, bright-gr,een porphyrin complex produced by reaction of FeIlI(TMP)CI (TMP = 5,1O,15,20-tetramesitylporphyrin) with either iodosylbenzene or peroxycarboxylic
acids in solution at low temperatures. 81 ,83 Titrations of this green porphyrin complex using I - as a reducing agent demonstrated that this species is readily reduced by two electrons to give the ferric complex FeIlI(TMP) + , i.e., that the green complex is two equivalents more oxidized than Fe III. A logical conclusion would be that the green species is Fe V(p2-)(02-) +. However, spectroscopic studies of this species have led to the conclusion that it is, in fact, an Fe lV oxo porphyrin-radical complex, FeIV(p' -)(0 2-) +, and that this formulation is the best description of 3, the product formed from heterolytic cleavage of the hydroperoxy intermediate in Reaction (5.67).81,83 EXAFS studies indicate that the green porphyrin complex contains iron bonded to an atom at an unusually short distance, i.e., 1.6 A, in addition to being bonded to the porphyrin nitrogens at 2 A. This short Fe-O distance is consistent with the formulation of the complex as a "ferryl" complex, i.e., Fe IV= O. In such a complex, the oxo ligand, 0 2-, is bonded to the Fe IV center by a combination of a and 1T bonding, the latter because of overlap of the filled ligand p-orbitals with the partially filled dxz and dyz orbitals of the metal. Confirmation that the oxidation state of iron is indeed Fe IV comes from comparison of the Mossbauer parameters (Ope = 0.06 and flEQ = 1.62) with those of other known Fe IV-porphyrin complexes (see Figure 5.11).83
 

Visible absorption spectra of porphyrin complexes are due largely to 1T-1T* transitions of the porphyrin ligand. The bright green color is unusual for ironporphyrin complexes, which are usually red or purple. (However, this green color has been seen for compound I of catalase and peroxidases; see Section VI below.) The unusually long-wavelength visible absorption bands that account for the green color result from the fact that the porphyrin ring has been oxidized by one electron. Similar visible absorption bands can be seen, for example, in other oxidized porphyrin complexes, such as CoIlI(p' -) +, formed by two-electron oxidation of ColI(p2-)(see 5.73).

Oxidized porphyrin ligands also give characteristic proton NMR spectra, which are seen for the green porphyrin complex as well. 

Magnetic measurements indicate that the green porphyrin complex contains three unpaired electrons. Detailed analysis of the Mossbauer spectra has indicated that the two unpaired electrons on the Fe IV ion are strongly ferromagnetically coupled to the unpaired electron on the porphyrin, accounting for the resulting S = ! state.

Studies of the reactions of this species with P-4S0-type substrates demonstrate that this species is reactive enough to make it an attractive candidate for" active oxygen" in the enzymatic mechanism.

Synthetic analogues for two of the other candidates for "active oxygen" have also been synthesized and their reactivities assessed. For example, FellI and MnIII-porphyrin peroxo complexes analogous to 1a in Reaction (5.65) have been synthesized. The x-ray crystal structure of the Mn complex shows that the peroxo ligand is bound to the metal in a triangular, side-on fashion (see 5.75). The Fe complex is believed to have a similar structure

Studies of this species indicate that 1a in Reaction (5.65) would not have the requisite reactivity to be a candidate for "active oxygen" in the cytochrome P450 mechanism, since it will not even oxidize triphenylphosphine, PPh3 , to triphenylphosphine oxide, OPPh3 , one of the more facile oxygenation reactions known. 87 Attempts to examine the protonated form, 1b in Reaction (5.65), however, indicate that it is highly unstable, and its reactivity has not yet been thoroughly examined. 87 Fe IV-oxo-porphyrin complexes analogous to 2 in Reaction (5.66) have also been prepared in solution and characterized by NMR.60 ,61 Such complexes will react with PPh3 to give OPPh3 , but are relatively unreactive with olefins and totally unreactive with saturated hydrocarbons. Thus 2 is also ruled out as a candidate for "active oxygen" in PA50 mechanisms.
 

These reactivity studies, and the observation of the peroxide shunt described above, indicate that Fe V(p2-)(0) + or FelV(p )(0) + is the most likely candidate for "active oxygen." These two formulations are, of course, isoelectronic, and it is tempting to conclude that the latter is the more likely formulation of the enzymatic intermediate. However, it is important to remember that the model systems lack the axial cysteinylligand present in cytochrome P-450. The effect of the relatively easily oxidized sulfur ligand on the electron distribution within that intermediate is not known, since model systems for high-valent iron-oxo complexes containing axial thiolate ligands have not been synthesized.

The mechanism of reactions of the high-valent oxo complex 3 in Reaction (5.67) with a variety of substrates is an area of active interest. 81 ,88 Such studies are generally carried out by generation of the species in situ from the reaction of a ferric porphyrin with a single-oxygen-atom donor, such as a peracid or iodosylbenzene.89 In hydroxylation reactions of aliphatic hydrocarbons, the initial step appears to be abstraction of a hydrogen atom from the substrate to form a substrate radical and an Fe IV hydroxide complex held together in a cage created by the enzyme active site so that they cannot diffuse away from each other (Reaction 5.76). This step is then followed by recombination of the OH fragment with the substrate radical to make the hydroxylated product (Reaction 5.77). This mechanism is referred to as the "oxygen rebound mechanism.

The radical character of the intermediates formed in this reaction is supported by the observation that such reactions carried out using synthetic porphyrins and single-oxygen-atom donors in the presence of BrCCh give substantial amounts of alkyl bromides as products, a result that is consistent with radical intermediates and inconsistent with either carbanion or carbonium-ion intermediates.83 In the enzymatic reactions themselves, there is also strong evidence to support a stepwise mechanism involving free-radical intermediates. For example, cytochrome P-450cam gives hydroxylation of d-camphor only in the 5-exo position,but deuterium-labeling studies show that either the 5-exo or the 5-endo hydrogen is lost (Reaction 5.78).

Such results are obviously inconsistent with a concerted mechanism in which the oxygen atom would be inserted into the 5-exo C-H bond in one step; so there would be no chance for the hydrogens in the two positions to exchange. (Remember that alcohol protons exchange rapidly with water and therefore are not expected to remain deuterated when the reaction is carried out in H20.)
 

The crystal structure of reduced cytochrome P-450cam with CO bound to the iron and the substrate camphor bound 90 adjacent to it has been examined and compared with the crystal structure of the oxidized enzyme with camphor bound. The former is expected to be similar in structure to the less-stable oxy complex. The comparison shows that the substrate camphor is closer to the iron center in the oxidized enzyme. It is therefore possible that a similar movement of the substrate occurs during the catalytic reaction after either a 5-exo or a 5-endo hydrogen is abstracted, and that the new position of the camphor molecule then restricts the hydroxylation step to the 5-exo position. It is interesting to note that the 5-exo position on the camphor that is hydroxylated is held in very close proximity to the FeIII center, and therefore to the presumed location of the oxo ligand in the high-valent oxo intermediate in the structure of the ferric enzyme plus camphor derivative (Figure 5.12). Crystal structures of the ferric form of cytochrome P-450cam with norcamphor and adamantanone bound in place of camphor have also been determined. 90 These alternative substrates are smaller than camphor, and appear to fit more loosely than camphor. It is therefore reasonable to assume that they "rattle around" to a certain extent in the substrate

binding site, which probably accounts for the less-specific pattern of hydroxylation observed for these alternative substrates.
 

Mechanisms for olefin epoxidations catalyzed either by the enzyme or by model porphyrin complexes are not as well understood as those for hydroxylation of aliphatic hydrocarbons. Some of the possibilities that have been proposed 88.91 are represented schematically in Figure 5.13. c. 0-0 Bond Cleavage The evidence is persuasive that the "active oxygen" species that attacks substrate in cytochrome P-450 is a high-valent ironoxo complex. However, the mechanism of formation of that species in the catalytic reaction with dioxygen is less well-understood. Heterolytic 0-0 bond cleavage of a ferric porphyrin hydroperoxide complex, Ib (Reaction 5.67), is the logical and anticipated route, but it has not yet been unequivocally demonstrated in a model complex.92,93 The catalase and peroxidase enzymes catalyze heterolytic 0-0 bond cleavage in reactions of hydrogen peroxide, but in them the active sites contain amino-acid side chains situated to facilitate the devel

oping charge separation that occurs in heterolytic cleavage (see Section VI). The crystal structure of cytochrome P-450cam shows no such groups in the activesite cavity, nor does it give any clue to the source of a proton to protonate the peroxide ligand when it is produced. 8o Also, we have little experimental evidence concerning possible roles that the cysteinyl sulfur axial ligand might play in facilitating 0-0 bond cleavage. These issues remain areas of active interest for researchers interested in cytochrome P-450 mechanisms.

Monooxygenases

Metal-containing monooxygenase enzymes are known that contain heme iron, nonheme iron, or copper at their active sites. 2 For most of these enzymes, there is only limited information about the nature of the active site and the mode of interaction with dioxygen or substrates. But there are three monooxygenase enzymes that strongly resemble well-characterized reversible dioxygen-carrying proteins (see preceding chapter), suggesting that dioxygen binding to the metalloenzyme in its reduced state is an essential first step in the enzymatic mechanisms, presumably followed by other steps that result in oxygenation of substrates. The enzymes are:
 

1. cytochrome P-450,73 a heme-containing protein whose active site resembles the dioxygen-binding sites of myoglobin or hemoglobin in many respects, except that the axial ligand to iron is a thiolate side chain from cysteine rather than an imidazole side chain from histidine;
2. tyrosinase,74 which contains two copper ions in close proximity in its active site and which has deoxy, oxy, and met states that closely resemble comparable states of hemocyanin in their spectroscopic properties; 
3. methane monooxygenase,75,76 which contains two nonheme iron ions in close proximity and which resembles hemerythrin in many of its spectroscopic properties.

In addition to these three, there are also monooxygenase enzymes containing single nonheme iron 77 or copper ions,78 or nonheme iron plus an organic cofactor such as a reduced pterin at their active sites. 79 Just as with the dioxygenase enzymes, we do not know how similar the mechanisms of the different metal-containing monooxygenase enzymes are to one another. The enzyme for which we have the most information is cytochrome P-450, and we will therefore focus our discussion on that system. Speculations about the mechanisms for the other systems are discussed at the end of this section.

Intradiol catechol dioxygenases

The role of these nonheme iron-containing enzymes is to catalyze the degradation of catechol derivatives to give muconic acids (Reaction 5.55, for example). The enzymes are induced when the only carbon sources available to the bacteria are aromatic molecules. The two best-characterized members of this class are catechol 1,2-dioxygenase (CTD) and protocatechuate 3,4-dioxygenase (PCD),

a. Characterization of the Active Sites Even before the x-ray crystal structure of PCD was obtained, a picture of the active site had been constructed by detailed spectroscopic work using a variety of methods. The success of the spectroscopic analyses of these enzymes is a particularly good example of the importance and usefulness of such methods in the characterization of metalloproteins. The two enzymes referred to in Reaction (5.55) have different molecular weights and subunit compositions,66 but apparently contain very similar activesite structures and function by very similar mechanisms. In both, the resting state of the enzyme contains one Fe III ion bound at the active site. EPR spectra show a resonance at g = 4.3, characteristic of high-spin Fe III in a so-called rhombic (low symmetry) environment,66 and the Mossbauer parameters are also characteristic of high-spin ferric. 66-68 Reactions with substrate analogues (see below) cause spectral shifts of the iron chromophore, suggesting strongly that the substrate binds directly to the iron center in the course of the enzymatic reaction.
 

It is straightforward to rule out the presence of heme in these enzymes, because the heme chromophore has characteristic electronic-absorption bands in the visible and ultraviolet regions with high extinction coefficients, which are not observed for these proteins. Likewise, the spectral features characteristic of other known cofactors or iron-sulfur centers are not observed. Instead, the dominant feature in the visible absorption spectrum is a band with a maximum near 460 nm and a molar extinction coefficient of 3000 to 4000 M - Jcm - 1 per iron (see Figure 5.5). This type of electronic absorption spectrum is characteristic of a class of proteins, sometimes referred to as iron-tyrosinate proteins, that contain tyrosine ligands bound to iron(III) in their active sites, and which consequently show the characteristic visible absorption spectrum due to phenolate-toiron( III) charge-transfer transitions. This assignment can be definitively proven by examination of the resonance Raman spectrum, which shows enhancement of the characteristic tyrosine vibrational modes (typically ~ 1170, 1270, 1500, and 1600 cm 1) when the sample is irradiated in the charge-transfer band described above. Ferric complexes of phenolate ligands may be seen to give almost identical resonance Raman spectra (see Figure 5.6). These bands have been assigned as a C-H bending vibration and a C-O and two C-C stretching vibrations of the phenolate ligand. 69 In addition, NMR studies of the relaxation rates of the proton spins of water indicate that water interacts with the paramagnetic FeIII center in the enzyme. This conclusion is supported by the broadening of the FeIII EPR signal in the presence of Hz 170, due to interaction

with the I = i nuclear spin of 170. Thus numerous spectroscopic studies of the catechol dioxygenases led to the prediction that the high-spin ferric ion was bound to tyrosine ligands and water. In addition, EXAFS data, as well as the resemblance of the spectral properties to another, better characterized iron-tyrosinate protein, i.e., transferrin (see Chapter 1), suggested that histidines would also be found as ligands to iron in these proteins. 66 ,67 Preliminary x-ray crystallographic results on protocatechuate 3,4-dioxygenase completely support the earlier predictions based on spectroscopic studies. 70 The Fe IlI center is bound to two histidine and two tyrosine ligands and a water, the five ligands being arranged in a trigonal bipyramidal arrangement, with a tyrosine and a histidine located in axial positions, and with the equatorial water or hydroxide ligand facing toward a cavity assumed to be the substrate-binding cavity. The cavity also contains the positively charged guanidinium group of an arginine side chain, in the correct position to interact with the negatively charged carboxylate group on the protocatechuate substrate (see Figure 5.7).

b. Mechanistic Studies As mentioned above, substrates and inhibitors that are substrate analogues bind to these enzymes and cause distinct changes in the spectral properties, suggesting strongly that they interact directly with the Fe III center. Nevertheless, the spectra remain characteristic of the Fe IIl oxidation state, indicating that the ferric center has not been reduced. Catecholates are excellent ligands for Fe III (see, for example, the catecholate siderophores, Chapter 1) and it might therefore be assumed that the catechol substrate would bind to iron using both oxygen atoms (see 5.56).

However, the observation that phenolic inhibitors p-X-C6H4-OH bind strongly to the enzymes suggested the possibility that the substrate binds to the iron center through only one oxygen atom (see 5.57).

Paramagnetic NMR studies have been invaluable in distinguishing these two possibilities. 67 The methyl group of the 4-methylcatecholate ligand is shifted by the paramagnetic ferric ion to quite different positions in the lH NMR spectra, depending on whether the catecholate is monodentate or bidentate. Comparison of the positions of the methyl resonances in the lH NMR spectra of the model complexes with those of the substrate 4-methylcatechol bound to the enzymes CTD and PCD indicates quite clearly that the substrate is bound to CTD in a monodentate fashion and to PCD in a bidentate fashion (see Figure 5.8). These 

results contradict an early hypothesis that the mode of substrate binding, i.e., monodentate versus bidentate, might be a crucial factor in activating the substrate for reaction with dioxygen. 67
 

Spectroscopic observations of the enzymes during reactions with substrates and substrate analogues have enabled investigators to observe several intermediates along the catalytic pathway. Such studies have led to the conclusion that the iron center remains high-spin Fe III throughout the entire course of the reaction. This conclusion immediately presents a problem in understanding the nature of the interaction of dioxygen with the enzyme, since dioxygen does not in general interact with highly oxidized metal ions such as Fe III. The solution seems to be that this reaction represents an example of substrate rather than dioxygen activation.
 

Studies of the oxidation of ferric catecholate coordination complexes have been useful in exploring mechanistic possibilities for these enzymes.71 A series of ferric complexes of 3,5-di-t-butyl-catechol with different ligands L have been found to react with Oz to give oxidation of the catechol ligand (Reaction 5.58).

The relative reactivities of these complexes appear to vary with the donating properties of the ligand L. Ligands that are the poorest donors of electron density tend to increase the reactivities of the complexes with Oz. These results suggest that the reactivity of these complexes with Oz is increased by an increase in the contribution that the minor resonance form B makes to the ground state of the complex (see Figure 5.9) and that complexes of ligands that are poor donors tend to favor electron donation from catechol to Fe III, thus increasing the relative amount of minor form B. It should be noted that the spectroscopic characteristics of these complexes are nevertheless dominated by the major resonance form A, regardless of the nature of L.
 

All these studies of the enzymes and their model complexes have led to the mechanism summarized in Figure 5.9. 66 In this proposed mechanism, the catechol substrate coordinates to the ferric center in either a monodentate or a bidentate fashion, presumably displacing the water or hydroxide ligand. The resulting catechol complex then reacts with dioxygen to give a peroxy derivative of the substrate, which remains coordinated to Fe Ill. The subsequent rearrangement of this peroxy species to give an anhydride intermediate is analogous to well-characterized reactions that occur when catechols are reacted with alkaline hydrogen peroxide. 72 The observation that both atoms of oxygen derived from Oz are incorporated into the product requires that the ferric oxide or hydroxide complex formed in the step that produces the anhydride does not exchange with external water prior to reacting with the anhydride to open it up to the product diacid.

It is interesting to consider how the intradiol dioxygenase enzymes overcome the kinetic barriers to oxidations by dioxygen, and why this particular mechanism is unlikely to be applicable to the monooxygenase enzymes. The first point is that the ferric catechol intermediate is paramagnetic, with resonance forms that put unpaired electron density onto the carbon that reacts with dioxygen. The spin restriction is therefore not a problem. In addition, the catechol ligand is a very good reducing agent, much more so than the typical substrates of the monooxygenase enzymes (see next section). It is possible, therefore, that the reaction of dioxygen with the ferric catechol complex results in a concerted two-electron transfer to give a peroxy intermediate, thus bypassing the relatively unfavorable one-electron reduction of O2 .

Dioxygenases

Dioxygenase enzymes are known that contain heme iron, nonheme iron, copper, or manganese. 66 ,67 The substrates whose oxygenations are catalyzed by these enzymes are very diverse, as are the metal-binding sites; so probably several, possibly unrelated, mechanisms operate in these different systems, For many of these enzymes, there is not yet much detailed mechanistic information. However, some of the intradiol catechol dioxygenases isolated from bacterial sources have been studied in great detail, and both structural and mechanistic information is available. 66,67 These are the systems that will be described here.

Mechanistic studies of the enzyme

A single turnover in the reaction of cytochrome c oxidase involves (1) reduction of the four metal centers by four equivalents of reduced cytochrome c, (2) binding of dioxygen to the partially or fully reduced enzyme, (3) transfer of four electrons to dioxygen, coupled with (4) protonation by four equivalents of protons to produce two equivalents of water, all without the leakage of any substantial amount of potentially harmful partially reduced dioxygen byproducts such as superoxide or hydrogen peroxide. 44 - 46 At low temperatures, the reaction can be slowed down, so that the individual steps in the dioxygen reduction can be observed. Such experiments are carried out using the fully reduced enzyme to which CO has been bound. Binding of CO to the Fell heme center in reduced cytochrome c oxidase inhibits the enzyme and makes it unreactive to dioxygen. The CO-inhibited derivative can then be mixed with dioxygen and the mixture cooled. Photolysis of metal-CO complexes almost always leads to dissociation of CO, and CO-inhibited cytochrome c oxidase is no exception. Photolytic dissociation of CO frees the Fell heme, thereby initiating the reaction with dioxygen, which can then be followed spectroscopically. 44-46 Dioxygen reacts very rapidly with the fully reduced enzyme to produce a species that appears to be the dioxygen adduct of cytochrome a3 (Reaction 5.48). Such a species is presumed to be similar to other mononuclear oxyheme derivatives. The dioxygen ligand in this species is then rapidly reduced to peroxide by the nearby CUB, forming what is believed to be a binuclear /-L-peroxo species (Reaction 5.49). These steps represent a two-electron reduction of dioxygen to the peroxide level, and are entirely analogous to the model reactions discussed above (Reactions 5.36 to 5.46), except that the binuclear intermediates contain one copper and one heme iron. The /-L-peroxo FellI - (02 2 -) - CUll species is then reduced by a third electron, resulting in cleavage of the 0-0 bond (Reaction 5.50). One of the oxygen atoms remains with iron in the form of a ferryl complex, i. e., an Fe IV oxo, and the other is protonated and bound to copper in the form of a CuII aquo complex. 65 Reduction by another electron leads to hydroxo complexes of both the Fe III heme and the CuII centers (Reaction 5.51).65 Protonation then causes dissociation of two water molecules from the oxidized cytochrome a3-CuB center (Reaction 5.52).

Several important questions remain to be resolved in cytochrome c oxidase research. One is the nature of the ligand bridge that links cytochrome a3 and CUB in the oxidized enzyme. Several hypotheses have been advanced (imidazolate, thiolate sulfur, and various oxygen ligands), but then discarded or disputed, and there is consequently no general agreement concerning its identity. However, EXAFS measurements of metal-metal separation and the strength of the magnetic coupling between the two metal centers provide evidence that a single atom bridges the two metals. 45 ,46 Another issue, which is of great importance, is to find out how the energy released in the reduction of dioxygen is coupled to the synthesis of ATP. It is known that this occurs by coupling the electron-transfer steps to a proton-pumping process, but the molecular mechanism is unknown.46 Future research should provide some interesting insights into the mechanism of this still mysterious process.

Molecular Mechanisms of Dioxygen Toxicity

What has been left out of the preceding discussion is the identification of the species responsible for oxidative damage, i.e., the agents that directly attack the various vulnerable targets in the cell. They were left out because the details of "- the chemistry responsible for dioxygen toxicity are largely unknown. In 1954, Rebeca Gerschman formulated the "free-radical theory of oxygen toxicity" after noting that tissues subjected to ionizing radiation resemble those exposed to elevated levels of dioxygen. 35 Fourteen years later, Irwin Fridovich proposed that the free radical responsible for dioxygen toxicity was superoxide, O2 , based on his identification of the first of the superoxide dismutase enzymes. 

Today it is still not known if superoxide is the principal agent of dioxygen toxicity, and, if so, what the chemistry responsible for that toxicity is. 6 There is no question that superoxide is formed during the normal course of aerobic metabolism,121 although it is difficult to obtain estimates of the amount under varying conditions, because, even in the absence of a catalyst, superoxide disproportionates quite rapidly to dioxygen and hydrogen peroxide (Reaction 5.4) and therefore never accumulates to any great extent in the cell under normal conditions of pH. 37

One major problem in this area is that a satisfactory chemical explanation for the purported toxicity of superoxide has never been found, despite much indirect evidence from in vitro experiments that the presence of superoxide can lead to undesirable oxidation of various cell components and that such oxidation can be inhibited by superoxide dismutase. 38 The mechanism most commonly proposed is production of hydroxyl radicals via Reactions (5.28) to (5.30) with Red - = O2-, which is referred to as the "Metal-Catalyzed Haber-Weiss Reaction". The role of superoxide in this mechanism is to reduce oxidized metal ions, such as Cu2 + or Fe 3+ , present in the cell in trace amounts, to a lower oxidation stateY Hydroxyl radical is an extremely powerful and indiscriminate oxidant. It can abstract hydrogen atoms from organic substrates, and oxidize most reducing agents very rapidly. It is also a very effective initiator of freeradical autoxidation reactions (see Section II.C above). Therefore, reactions that produce hydroxyl radical in a living cell will probably be very deleterious. 6

The problem with this explanation for superoxide toxicity is that the only role played by superoxide here is that of a reducing agent of trace metal ions. The interior of a cell is a highly reducing environment, however, and other reducing agents naturally present in the cell such as, for example, ascorbate anion can also act as Red - in Reaction (5.28), and the resulting oxidation reactions due to hydroxyl radical are therefore no longer inhibitable by SOD.39 Other possible explanations for superoxide toxicity exist, of course, but none has ever been demonstrated experimentally. Superoxide might bind to a specific enzyme and inhibit it, much as cytochrome oxidase is inhibited by cyanide or hemoglobin by carbon monoxide. Certain enzymes may be extraordinarily sensitive to direct oxidation by superoxide, as has been suggested for the enzyme aconitase, an iron-sulfur enzyme that contains an exposed iron atom. 122 Another possibility is that the protonated and therefore neutral form of superoxide, H02 , dissolves in membranes and acts as an initiator of lipid peroxidation. It has also been suggested that superoxide may react with nitric oxide, NO, in the cell producing peroxynitrite, a very potent oxidant. 123 One particularly appealing mechanism for superoxide toxicity that has gained favor in recent years is the "Site-Specific Haber-Weiss Mechanism." 40,41 The idea here is that traces of redox-active metal ions such as copper and iron are bound to macromolecules under normal conditions in the cell. Most reducing agents in the cell are too bulky to come into close proximity to these sequestered metal ions. Superoxide, however, in addition to being an excellent reducing agent, is very small, and could penetrate to these metal ions and reduce them. The reduced metal ions could then react with hydrogen peroxide, generating hydroxyl radical, which would immediately attack at a site near the location of the bound metal ion. This mechanism is very similar to that of the metal complexes that cause DNA cleavage; by reacting with hydrogen peroxide while bound to DNA, they generate powerful oxidants that react with DNA with high efficiency because of their proximity to it (see Chapter 8).

Although we are unsure what specific chemical reactions superoxide might undergo inside of the cell, there nevertheless does exist strong evidence that the superoxide dismutases play an important role in protection against dioxygeninduced damage. Mutant strains of bacteria and yeast that lack superoxide dismutases are killed by elevated concentrations of dioxygen that have no effect on the wild-type cells. This extreme sensitivity to dioxygen is alleviated when the gene coding for a superoxide dismutase is reinserted into the cell, even if the new SOD is of another type and from a different organism.