# 7.6: Multisite Redox Enzymes (Part 2)

## 3. Iron Hydrogenases

The iron hydrogenases189a generally have higher activities than the NiFe enzymes, with turnover numbers approaching 106 min-1. Iron hydrogenases from four genera of anaerobic bacteria have been isolated: Desulfovibrio,190 Megasphaera,188 Clostridium,191 and Acetobacterium.192 Of these, the enzymes from Desulfovibrio vulgaris, Megasphaera elsdenii, Clostridium pasteurianum (which contains two different hydrogenases), and Acetobacterium woodii192 have been well-characterized (especially the D. vulgaris and C. pasteurianum enzymes). Although Acetobacterium and Clostridium are closely related, the other organisms are only distant cousins.193 Nevertheless, their hydrogenases display significant similarities; all contain two different types of iron-sulfur cluster, called F and H clusters,194 and carbon monoxide is a potent inhibitor (although this has not been reported for the M. elsdenii enzyme). The F clusters are thought to be of the Fe4S4+/2+ thiocubane type, and give S = $$\frac{1}{2}$$ EPR signals when the enzyme is in the reduced form. On the other hand, the H cluster, which is thought to be the hydrogen-activating site, gives an EPR signal only when the enzyme is in the oxidized form. The H-cluster EPR signals of all the enzymes are quite similar (g = 2.09, 2.04, 2.00), and are quite unlike the signals from other oxidized iron-sulfide clusters (such as Fe3S4 clusters and HiPIPs), in that they are observable at relatively high temperatures (> 100 K). Inhibition of the D. vulgaris and both C. pasteurianum enzymes by carbon-monoxide yields a photosensitive species that has a modified H-cluster EPR signal.195,196

The two different hydrogenases of C. pasteurianum, called hydrogenase I and II, have both been quite extensively studied, and can be regarded as prototypical iron-only hydrogenases. Hydrogenase I is active in catalyzing both H2 oxidation and H2 evolution, whereas hydrogenase II preferentially catalyzes H2 oxidation.188 The two enzymes differ in their iron contents: hydrogenase I contains about 20 iron atoms, 16 of which are thought to be involved in four F clusters,194 while the remainder presumably constitute the H cluster, which may contain six Fe atoms.194 Hydrogenase II contains about 14 iron atoms as two F clusters and one H cluster.194 These estimates of iron content result from a recent reappraisal of the metal contents (based on amino-acid analysis) that indicated a rather higher Fe content than previously realized.188,194 It is important to note that much of the spectroscopic work, which will be discussed below, was initially interpreted on the basis of the earlier, erroneous, iron analysis. Of particular interest is the possibility (first suggested197 for the D. vulgaris enzyme) that the H cluster contains six iron atoms.

Carbon-monoxide treatment of the D. vulgaris and both C. pasteurianum enzymes yields a photosensitive species that has a modified H cluster EPR signal.195,196 Interestingly, the C. pasteurianum enzymes also form complexes with O2, in a process that is distinguishable from the deactivation of hydrogenase by O2, which results from a much more prolonged exposure to O2 than that required to form the O2 complex. The O2 complexes have (photosensitive) EPR signals much like those of the CO complex.196 It is important to note that although CO, when in excess, is a potent inhibitor of the enzymes, the hydrogenase I-CO complex is actually quite active.196 With hydrogenase II, the CO complex is dissociated on exposure to H2, restoring the "active" enzyme.196 The EPR spectrum of reduced hydrogenase I is typical of (interacting) Fe4S4+ clusters, and integrates to 3 or 4 spins/protein.194,198 Electrochemical studies199 show that these clusters possess indistinguishable reduction potentials. Recently, MCD and EPR spectroscopies have been used to demonstrate the presence of significant quantities of an S = $$\frac{3}{2}$$ species in reduced hydrogenase I. This signal apparently integrates to about one spin per molecule, and probably originates from an S = $$\frac{3}{2}$$ state of an Fe4S4 cluster.198 No information is yet available on the reduction potential of the S = $$\frac{3}{2}$$ species. However, based on analogy with the nitrogenase iron protein,200 we might expect the S = $$\frac{3}{2}$$ form to have electrochemistry indistinguishable from the S = $$\frac{1}{2}$$ form. EPR signals with high g values (g = 6.1 and 5.0) have also been observed in C. pasteurianum hydrogenase I, and in the D. vulgaris enzyme.198,201 Since there is some uncertainty about the nature and origin of these signals,198 we will not discuss them further. The F clusters of hydrogenase II, on the other hand, give two different EPR signals that integrate to one spin each per protein molecule, and that correspond to sites with different redox potentials.194,198,199 This suggests that hydrogenase II contains two different F clusters, called F and F' (note that the presence of F' was in fact first suggested by Mössbauer spectroscopy202). The EPR spectrum from the F' cluster is unusually broad. The H-cluster EPR signals of active hydrogenase I and II are quite similar and have essentially identical redox potentials.199

The redox behavior of the F and H centers in hydrogenases I and II is nicely consistent with their respective modes of function. As shown in Figure 7.22, the F clusters are presumed to transfer electrons intermolecularly with the external electron carrier and intramolecularly with the H center. In the reversible hydrogenase I, the F and H centers have the same redox potentials (about -400 mV at pH 8), similar to that of the hydrogen electrode (-480 mV at pH 8). Thus, electrons may flow in either direction when a mediator such as methyl viologen (E°' = -440 mV at pH 8) is used as the external electron acceptor (methyl viologen is the 4,4'-bipyridinium ion). On the other hand, for hydrogenase II, the F clusters have E°' (pH 8) = -180 mV and E°' > -300 mV for F and F', respectively. In hydrogenase II, therefore, electrons can only move favorably from H2 to the H cluster, through F' and F, and then to a lower-potential acceptor, such as methylene blue [for which E°' (pH 8) = 11 mV].

Mössbauer spectroscopic studies of both hydrogenase I and II have been reported.202,203 Our discussion focuses primarily on the H cluster. The results are similar for the two enzymes, but better defined for hydrogenase II because of the smaller number of clusters. The H cluster apparently contains only two types of iron, in the ratio of 2:1, with quadrupole splittings reminiscent of Fe3S4 clusters. The oxidized cluster is confirmed to be an S = $$\frac{1}{2}$$ system, also reminiscent of Fe3S4 clusters, and the reduced H cluster is an S = 0 system; this contrasts with reduced Fe3S4 clusters, which have S = 2. In agreement with the Mössbauer studies, ENDOR spectroscopy of 57Fe-enriched protein indicates at least two different types of iron in the H cluster, with metal hyperfine couplings of about 18 and 7 MHz in hydrogenase II. Rather-less-intense ENDOR features were also observed at frequencies corresponding to couplings of about 11 and 15 MHz.204 The H-cluster EPR signals of hydrogenase II and hydrogenase I change on binding carbon monoxide. Although the signals of the uncomplexed enzymes are quite similar, the signals of the CO-bound species are very different (note, however, that C. pasteurianum CO-bound hydrogenase I has EPR similar to that of the CO-bound D. vulgaris enzyme), When produced with 13C-enriched CO, the EPR signal of hydrogenase II shows resolved 13C hyperfine coupling (Aav = 33 MHz) to a single 13C nucleus, indicating that only a single CO is bound, presumably as a metal carbonyl. A slightly smaller coupling of 20 MHz was obtained using ENDOR spectroscopy for the corresponding species of hydrogenase I.205

Recent ESEEM spectroscopy of hydrogenase I indicates the presence of a nearby nitrogen, which may be a nitrogen ligand to the H-cluster. This nitrogen possesses an unusually large nuclear electric quadrupole coupling and a rather novel structure, involving an amide amino-acid side chain connected to an H-cluster sulfide via a bridging proton ligand, has been suggested for it.206 Although the nitrogen in question must come from a chemically novel species, the proposed proton bridge might be expected to be exchangeable with solvent water. The ENDOR-derived result205 that there are no strongly coupled exchangeable protons in the oxidized H cluster may argue against such a structure.

Rather weak MCD198,207 and resonance Raman spectra208 have also been reported for iron hydrogenases. The lack of an intense MCD spectrum198,207 contrasts markedly with results for other biological FeS clusters. The resonance Raman spectra of hydrogenase I resemble, in some respects, spectra from Fe2S2 sites.208 These results further emphasize that the hydrogenase H clusters are a unique class of iron-sulfur clusters. Perhaps most tantalizing of all are the recent EXAFS results on hydrogenase II.209 It is important to remember that for enzymes with multiple sites, the EXAFS represents the sum of all sites present (i.e., the iron of the F, F', and H clusters). Despite this complication, useful information is often forthcoming from these experiments. The EXAFS of oxidized hydrogenase I showed both iron-sulfur and iron-iron interactions; the latter, at about 2.7 Å, is at a distance typical of Fe2S2, Fe3S4, and Fe4S4 clusters, and thus is not unexpected. The reduced enzyme, however, gave an additional, long Fe-Fe interaction at 3.3 Å. This Fe-Fe separation is not found in any of the FeS model compounds reported to date.209 The appearance of the 3.3-Å interaction indicates a change in structure on reduction of the H cluster, again revealing a cluster of unique structure and reactivity. The large structural change of this H cluster on H2 reduction is likely to have significant mechanistic implications.

## 4. Nickel-iron Hydrogenases

The presence of nickel in hydrogenases has only been recognized relatively recently. Purified preparations of the active enzymes were the subject of quite intensive studies for years before the Ni content was discovered by nutritional studies (see Reference 189 for a history). Some workers even tried (in vain) to purify out "impurity" EPR signals that were later found to be from the Ni. In contrast to the Fe hydrogenases discussed in the previous section, the Ni enzymes possess a variety of compositions, molecular weights, activation behavior, and redox potentials.189,210,211 As Table 7.5 shows, some of the Ni hydrogenases contain selenium, likely in the form of selenocysteine, some contain flavin (FMN or FAD), and all contain iron-sulfur centers, but in amounts ranging from 4 to 14 iron atoms per Ni atom.

Among the different Ni hydrogenases there is a common pattern of protein composition, to which many, but not all, seem to conform (especially those enzymes originating from purple eubacteria). There are two protein subunits, of approximate molecular masses 30 and 60 kDa, with the nickel probably residing in the latter subunit. The hydrogenase of the sulfate-reducing bacterium Desulfovibrio gigas is among the best investigated, and we will concentrate primarily on this enzyme. D. gigas hydrogenase contains a single Ni, two Fe4S4 clusters, and one Fe3S4 cluster. Of primary interest is the Ni site, which is thought to be the site of H2 activation.189,210,211

EPR signals attributable to mononuclear Ni [as shown by enrichment with 61Ni (I = $$\frac{3}{2}$$)] have been used in numerous investigations of the role of Ni in hydrogenases.189,210,211 Three major Ni EPR signals are known, which are called Ni-A, Ni-B, and Ni-C. The principal g values of these signals are: 2.32, 2.24, and 2.01 for Ni-A; 2.35, 2.16, and 2.01 for Ni-B; and 2.19, 2.15, and 2.01 for Ni-C. Of these, Ni-C is thought to be associated with the most active form of the enzyme (called active); the other two are thought to originate from less-active enzyme forms.189,210

In the enzyme as prepared (aerobically) the Ni-A EPR is characteristically observed. On hydrogen reduction the Ni-A EPR signal disappears, and the enzyme is converted into a higher-activity form (Ni-B arises from reoxidation of this form). Further progressive reduction of the enzyme gives rise to the Ni-C EPR signal, which also finally disappears. These redox properties show that Ni-C arises from an intermediate enzyme oxidation state. Although the Ni-A and Ni-B EPR signals189,210-212 almost certainly originate from low-spin Ni(III), the formal oxidation state of Ni-C is rather less certain. Both an Ni(I) site189,210 and an Ni(III) hydride213 have been suggested, with the former alternative currently favored because of the apparent absence of the strong proton hyperfine coupling expected for the latter. In the fully reduced enzyme, Ni-C is converted to an EPR-silent species. This has variously been suggested to be Ni(0), Ni(II), or an Ni(II) hydride.214 One possible reaction cycle189,210 is shown in Figure 7.23.

Information on the coordination environment of the nickel has been obtained from both x-ray absorption spectroscopy and EPR spectroscopy. The Ni K-edge EXAFS of several different hydrogenases,215-218 and EPR spectroscopy of 33S enriched Wolinella succinogenes hydrogenase,219 clearly indicate the presence of sulfur coordination to nickel. A recent x-ray absorption spectroscopic investigation of the selenium-containing D. baculatus hydrogenase, using both Ni and Se EXAFS, suggests seienocysteine coordination to Ni.216

ESEEM spectroscopy of the Ni-A and Ni-C EPR signals220,221 indicate the presence of 14N coupling, which probably arises from a histidine ligand to Ni. Interestingly, Ni-C, but not Ni-A, shows coupling to a proton that is exchangeable with solvent water. Although this coupling is too small to suggest a nickel-hydride (consistent with conclusions drawn from EPR), the proton involved could be close enough to the Ni to playa mechanistic role.

Despite the extensive studies reported to date, there are still many unanswered questions about the mechanism of the NiFe hydrogenases, which remain as exciting topics for future research. Despite our lack of detailed knowledge of enzyme mechanism, it is nevertheless not premature to seek guidance from inorganic chemistry.

## 5. Insights from Inorganic Chemistry

Recent years have brought insights into the way dihydrogen can be bound at a transition-metal site. Unexpectedly, it has been shown that molecular H2 forms simple complexes with many kinds of transition-metal sites.222-224 This finding contrasts with the classical situation, in which H2 interacts with a transition-metal site by oxidative addition to form a dihydride complex.23The H—H bond is largely maintained in the new/nonclassical structures. The dihydrogen and dihydride complexes can exist in simple equilibrium,224 as in Equation (7.9).

$$\tag{7.9}$$

The bonding of dihydrogen to a metal occurs via the $$\sigma^{b}$$ orbital of the H—H bond acting as a donor, with the $$\sigma$$* level of H2 acting as a weak acceptor. If the back donation is too strong, sufficient electron density will build up in the $$sigma$$* level to cause cleavage of the H—H bond, leading to the formation of a dihydride. Dihydrogen complexes therefore require a delicate balance, in which the metal coordination sphere facilitates some back-bonding, but not too much.

The proclivity of a metal center to form H2 complexes can be judged by the stretching frequency of the corresponding N2 complexes: N2 can usually displace H2 from the H2 complex to form an N2 complex without changing the remainder of the coordination sphere. If v(N$$\equiv$$N) is between 2060 and 2160 cm-1, H2 complexes form upon replacement of N2. If v(N$$\equiv$$N) is less than 2060 cm-1, indicative of electron back-donation from the metal center, a dihydride complex should form. For example, v(N$$\equiv$$N) = 1950 cm-1 in MoN2(PCy3)5 and MoH2(PCy3)5 is a dihydride complex, but v(N$$\equiv$$N) = 2090 cm-1 in Mo(Ph2PCH2CH2PPh2)CO(N2) and Mo(Ph2PCH2CH2PPh2)H2(CO)2 is a dihydrogen complex. By comparison, Mo(Et2PCH2CH2PEt2)2(CO)(N2) has v(N$$\equiv$$N) = 2050 cm-1 and forms a dihydride complex, Mo(Et2PCH2CH2-PEt2)2H2(CO). The correlation between v(N$$\equiv$$N) and the type of hydrogen complex formed seems quite useful.

Since Fe-S and Ni-S sites are implied for hydrogenase, the reactivity of transition-metal/sulfide systems with H2 may also be relevant. Interestingly, H2 can react with metal-sulfide systems at S instead of at the metal site. For example,225 (Cp')2Mo2S4 reacts with H2 to form (Cp')2Mo2(SH)4. Here the dihydrogen is cleaved without any evidence for direct interaction with the metal center, and the resulting complex contains bridging SH groups and no direct metal-H bonding.225 In recent work,226 the binuclear rhodium-sulfur complex {RhS[P(C6H5)2CH2CH2]3CH}2 was reported to react with two equivalents of dihydrogen to yield the complex {Rh(H)(SH)[P(C6H5)2CH2CH2]3CH}2, in which two SH groups bridge the two Rh centers, each of which contains a single hydrido ligand. Figure 7.24 illustrates the possibilities for hydrogen activation. Each of these types of reactivity must be considered as possibilities for the hydrogen activation process of hydrogenase.

Recently, a great deal of attention has been given to the chemistry of nickel-sulfur systems, inspired in part by the results showing that many hydrogenases are nickel-sulfur proteins.227-230 A particularly interesting finding is that Ni thiolates can react with O2 to produce sulfinate complexes.231,232 The oxygenated thiolate can be regenerated, thus providing a potential model for the O2 inactivation of Ni hydrogenases.