# 7.8: Multisite Redox Enzymes (Part 4)

## 7. EPR, ENDOR, and ESEEM Studies

The FeMoco or M center has been identified spectroscopically within the FeMo protein;239,248,273 it has a distinctive EPR signal with effective g values of 4.3, 3.7, and 2.01, and originates from an S = $$\frac{3}{2}$$ state of the M center. The signal arises from transitions within the ±$$\frac{1}{2}$$ ground-state Kramers doublet of the S = $$\frac{3}{2}$$ system (D = +5.1 cm-1, E/D = 0.04). The isolated cofactor (FeMoco) gives a similar EPR signal, but with a rather larger rhombicity (E/D = 0.12). Spectra from the C. pasteurianum nitrogenase and cofactor are shown in Figure 7.28, and comparative data are given in Table 7.7. The M-center EPR signal has proved useful in characterizing the nature of the site, especially when more sophisticated magnetic resonance techniques, such as ENDOR or ESEEM, are used.

Extensive ENDOR investigations274,275,275a have been reported using protein samples enriched with the stable magnetic isotopes 2H, 33S, 57Fe, 95Mo, and 97Mo. The 57Fe couplings have been investigated in the most detail. Individual hyperfine tensors of five coupled 57Fe nuclei are discernible, and were evaluated by simulation of the polycrystalline ENDOR spectrum.275 The data from 33S and 95Mo were analyzed in less detail; 33S gave a complex ENDOR spectrum, evidently with quite large hyperfine couplings, although no quantification was attempted because of the complexity of the spectrum.274 On the other hand, 95Mo was shown to possess a small hyperfine coupling, indicating that the molybdenum possesses very little spin density (although the quantitative aspects of the conclusions of the 95Mo ENDOR study have recently been shown to be in error276).

Although no nitrogen splittings were reported in any of the ENDOR studies, evidence for involvement of nitrogen as a cluster component has been forthcoming from ESEEM spectroscopy.277-279 14N modulations are observed in the ESEEM of the M center. The observed 14N is not from the substrate (N2), or from an intermediate or product of nitrogen fixation, because enzyme turnover using 15N as a substrate does not change the ESEEM spectrum. The isolated cofactor (FeMoco) does not show the modulation frequencies observed for the M center in the protein. These experiments suggest that the M-center 14N ESEEM arises from a nitrogen atom that is associated with the M center, and probably from an amino-acid side chain (most likely a histidine) ligated to the cluster.279 Recent evidence from site-directed mutagenesis of the Azotobacter vinelandii protein280 provides strong support for the presence of histidine ligation, and points specifically to His-195 of the $$\alpha$$ subunit as the N ligand.

## 8. Mössbauer Studies

Extensive Mossbauer investigations of nitrogenase271,281-283 and FeMoco283a have been reported. Unlike EPR and EPR-based spectroscopies, which can be used to investigate only the EPR-active S = $$\frac{3}{2}$$ oxidation state, all three available M-center oxidation states are accessible to Mössbauer spectroscopy. The fully reduced site was found to be diamagnetic with S = 0 (but see Reference 284), whereas the oxidized site was found to have S $$\geq$$ 1. The zero-field spectrum of reduced C. pasteurianum nitrogenase is shown in Figure 7.29; the spectrum is comprised of four quadrupole doublets, one of which was concluded to originate from the M site.282 Mössbauer spectra taken in the presence of applied magnetic fields were used to deduce the presence of four types of 57Fe hyperfine coupling; these were called sites A1, A2, and A3, which have negative hyperfine couplings, and B sites, which have positive hyperfine couplings. The A sites were quantitated as a single Fe each; the B sites were estimated to contain three irons. These conclusions were largely confirmed and extended by later ENDOR investigations,274 although the B sites were resolved as two inequivalent, rather than three equivalent, sites. ENDOR is rather more sensitive to the nature of the hyperfine couplings than Mössbauer, although it cannot usually be used to count numbers of exactly equivalent sites. Thus the number of iron atoms in the M center is minimally five, although larger numbers cannot be excluded. Note also that some of the quantitative aspects of the earlier Mossbauer investigations have been criticized.285

## 9. X-ray Absorption Studies

One of the early triumphs of biological x-ray absorption spectroscopy was the deduction that the nitrogenase M center is an Mo-Fe-S cluster.286 (It is also worth noting that nitrogenase was the first enzyme to be studied by x-ray absorption spectroscopy.) Early work on lyophilized protein samples indicated the presence of two major contributions to the Mo K-edge EXAFS, which were attributed to Mo-S ligands, plus a more distant Mo-Fe contribution.286 Subsequently, these conclusions have been confirmed and extended, using samples in solution and with much more sensitive detection systems.

Most EXAFS studies to date have been on the molybdenum K-edge of the protein or of FeMoco, and indicate a very similar Mo environment in both (Table 7.7, Figure 7.30). A consensus of the best available analyses287 indicates that Mo is coordinated by three or four sulfur atoms at 2.4 Å, one to three oxygens or nitrogens at 2.2 Å, with approximately three nearby iron atoms at 2.7 Å. Of these, the EXAFS evidence for the oxygen/nitrogen contribution is weakest. However, comparison of Mo K-edge288 and Mo L-edge XANES289 spectra with model compounds indicates strong similarities with MoFe3S4 thiocubane model compounds possessing MoS3O3 coordination, and provides some support for the presence of O/N ligands.

The iron EXAFS of FeMoco has been independently examined by two groups.290,291 Both groups agree that the iron is coordinated largely to sulfur at about 2.2 Å, with more distant Fe-Fe interactions at about 2.6 Å. They differ, however, concerning the presence of short (1.8 Å) Fe-O interactions. Such interactions were apparently observed in the earlier study,290 but not in the later study.291 One possible explanation for this discrepancy is that the short Fe-O interactions of the earlier study were due to extraneous iron coordinated to solvent, contaminating the FeMoco preparation.291 A final resolution of this discord must, however, await the results of further experiments. Interestingly, a long Fe-Fe interaction at 3.7 Å was also observed in the later study.291

Largely on the basis of the Mo K-edge EXAFS results and model studies discussed below, several proposals for the structure of the M center have been put forward. These are illustrated in Figure 7.31.

The MoFe proteins from Clostridium pasteurianum292 and from Azotobacter vinelandii293 have been crystallized. For the former protein, crystals of space group P21 are obtained, with two molecules per unit cell of dimensions 70 x 151 x 122 Å. There is good evidence for a molecular two-fold axis, which presumably relates equivalent sites in the two $$\alpha \beta$$ dimers that make up the protein molecule.294 Preliminary refinement reveals that the two FeMoco units per protein are about 70 Å apart and the four P clusters are grouped in two pairs.

Single crystal EXAFS studies295 have provided important structural information on the molybdenum site. For different crystal orientations (relative to the polarized x-ray beam), the amplitude of the Mo-Fe EXAFS changes by a factor of 2.5, but the Mo-S EXAFS changes only slightly. Analysis of the anisotropy of the Mo-Fe EXAFS using the available crystallographic information294 is consistent with either a tetrahedral MoFe3 geometry such as that found in thiocubanes (Figure 7.32) or a square-based pyramidal MoFe4 arrangement of metals. This interpretation tends to rule out some of the structural proposals shown in Figure 7.33. The observed orientation-dependence of the iron amplitudes is too small for clusters containing a linear or planar arrangement of iron and molybdenum (e.g., Figure 7.33B,C), and too large for arrangements that involve regular disposition of iron about molybdenum. Moreover, the lack of anisotropy of the sulfur EXAFS (which was apparently not considered in the original interpretation295) argues against an MoS3 (O/N)3 model that has molybdenum coordinated by sulfur atoms that bridge only to Fe atoms disposed to one side of the molybdenum. Significant anisotropy for the Mo-S EXAFS (of opposite polarization, and smaller than that for Mo-Fe) would be expected for such an arrangement of sulfur atoms. However, the cubane model of Figure 7.33, which provides the best model of both geometric and electronic structure, remains viable if one of the nonbridging ligands to molybdenum is a sulfur atom (rather than oxygen or nitrogen) with a bond length similar to that of the bridging sulfides.

## 10. Substrate Reactions

The two-component Mo-nitrogenase enzyme catalyzes the reduction of N2 to 2NH4+ as its physiological reaction. Concomitant with the reduction of N2, H2 evolution occurs, with electrons supplied by the same reductants that reduce N2. The limiting stoichiometry appears to be

$N_{2} + 10 H^{+} + 8 E^{-} \rightarrow 2 NH_{4}^{+} + H_{2} \tag{7.16}$

If N2 is omitted from the assay, all the electrons go to H2 evolution. Indeed, to a first approximation the rate of electron flow through nitrogenase is independent of whether the enzyme is producing only H2, producing both NH4+ and H2, or reducing most of the alternative substrates. As displayed in Table 7.8, many alternative substrates are known for this enzyme.240,243,296 The most important of these from a practical perspective is acetylene, C2H2, which is reduced by the Mo nitrogenase exclusively to ethylene, C2H4. Acetylene can completely eliminate H2 evolution by nitrogenase. Many of the substrates in Table 7.8 have a triple bond. Indeed, the only triple-bonded molecule not reduced by nitrogenase is CO, which nevertheless inhibits all substrate reactions, but not H2 evolution (in the wild type). Triple-bonded molecules such as acetylene (H—C$$\equiv$$C—H) are useful probe molecules for related reactivity as discussed below for simple inorganic systems. All substrate reductions involve the transfer of two electrons or multiples thereof (i.e., 4,6,8 . . .). Multielectron substrate reductions may involve the stepwise execution by the enzyme of two-electron processes. Further, about as many protons as electrons are usually transferred to the substrate. One way of viewing the nitrogenase active site is that it can add the elementary particles (H+ and e-) of H2 to the substrate. This may have mechanistic implications.297

It is potentially fruitful to pursue the intimate connection between H2 and the N2 binding site in nitrogenase. It has been shown unequivocally298,299 that one H2 is evolved for each N2 "fixed" even at 50 atm of N2, a pressure of N2 well above full saturation. Moreover, H2 is a potent inhibitor of N2 fixation, and under D2, HD is formed, but only in the presence of N2. These complex relationships between N2 and H2(D2) have elicited a variety of interpretations.255,300-302

Recently, it has been demonstrated that the FeMo protein alone acts as an uptake hydrogenase.303 Dihydrogen in the presence of [FeMo] causes the reduction of oxidizing dyes such as methylene blue or dichlorophenolindophenol in the absence of Fe protein. This is the only known catalytic reaction displayed by the FeMo protein alone. The hydrogen evolution and uptake by [FeMo] suggest that understanding hydrogen interaction with transition-metal/sulfur centers may be crucial to understanding the mechanism of nitrogenase action.

## Table 7.8 - Table 7.8 Nitrogenase substrate reactions.296,374-376

 Two-electron Reductions $$2e^{-} + 2H^{+} \rightarrow H_{2}$$C_{2}H_{2} + 2e^{-} + 2 H^{+} \rightarrow C_{2}H_{4}$$$N_{3}^{-} + 2e^{-} + 3H^{+} \rightarrow NH_{3} + N_{2}$$$N_{2}O + 2e^{-} + 2H^{+} \rightarrow H_{2}O + N_{2}$$Four-electron Reductions$$HCN + 4e^{-} + 4H^{+} \rightarrow CH_{3}NH_{2}RNC + 4e^{-} + 4H^{+} \rightarrow RNHCH_{3}$$Six-electron Reductions$$N_{2} + 6e^{-} + 6H^{+} \rightarrow 2NH_{3}HCN + 6e^{-} + 6H^{+} \rightarrow CH_{4} + NH_{3}HN_{3} + 6e^{-} + 6H^{+} \rightarrow NH_{3}+ N_{2}H_{4}RNC + 6e^{-} + 6H^{+} \rightarrow RNH_{2} + CH_{4}RCN + 6e^{-} + 6H^{+} \rightarrow RCH_{3} + NH_{3}NCNH_{2} + 6e^{-} + 6H^{+} \rightarrow CH_{3}NH_{2} + 2NH_{3}NO_{2}^{-} + 6e^{-} + 6H^{+} \rightarrow NH_{3}$$Multielectron Reductions$$RNC \rightarrow (C_{2}H_{6} , C_{3}H_{6} , C_{3}H_{8}) + RNH_{2}NCNH_{2} + 8e^{-} + 8H^{+} \rightarrow CH_{4} + 2NH_{3}

## 11. The Role of ATP

ATP hydrolysis appears to be mandatory, and occurs during electron transfer from [Fe] to [FeMo]. Dissociation of [Fe] and [FeMo] following electron transfer is probably the rate-limiting step in the overall turnover of the enzyme.255 The fact that reductant and substrate levels do not affect turnover rates is consistent with this finding.

The role of ATP on a molecular level remains one of the great mysteries of the mechanism of nitrogen fixation. As discussed above, the overall thermodynamics of N2 reduction to NH3 by H2 or by its redox surrogate flavodoxin or ferredoxin is favorable. The requirement for ATP hydrolysis must therefore arise from a kinetic necessity. This requirement is fundamentally different from the need for ATP in other biosynthetic or active transport processes, wherein the free energy of hydrolysis of ATP is needed to overcome a thermodynamic limitation.

What is the basis for the kinetic requirement of ATP hydrolysis in nitrogen fixation? To answer this question, we again look at the potential reduction products of the N2 molecule. Of these, only N2H2 (diimide, three potential isomers), N2H4 (hydrazine and its mono and dications), and NH3 (and its protonated form, NH4+) are isolable products. (In the gas phase, other species such as N2H, N2H3, or NH2 also have a "stable" existence.) In the presence of H2, only the formation of ammonia is thermodynamically favored (Figure 7.26). Clearly, the formation of the intermediate species in the free state cannot occur to any reasonable extent. However, this does not mean that nitrogenase must form NH3 directly without the formation of intermediates. It is possible for these reactive intermediates to be significantly stabilized by binding to a metal-sulfur center or centers.

Detailed kinetic studies255,304 have suggested a scheme in which intermediates with bound and probably reduced nitrogen are likely to be present. Rapid quenching experiments in acid solution lead to the detection of hydrazine during nitrogenase turnover.305 Likewise, studies of inhibition of N2 fixation by H2 and the formation of HD under D2 have been interpreted in terms of a bound diimide intermediate.306,307 Although a bound "dinitrogen hydride" is likely to be present, its detailed structure remains unknown.