Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe 4 S 4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo(VI), Fe 4 S 4(ox) ] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV), Fe 4 S 4(red) ] form have been determined, revealing a four-domain αβ structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys 140 and His 141 in proton abstraction and the molybdenum, molybdopterin, Lys 44 , and the Fe 4 S 4 cluster in electron transfer.

A general approach for simulation of EPR spectra of mixed-valence dimanganese complexes and proteins is presented, based on the theory of Sage et al. (J. Am. Chem. Soc. 1989, 111, 7239-7247), which overcomes limitations inherent in the theory of strongly coupled ions. This enables explanation of "anomalous" spectral parameters and extraction of accurate g tensors and 55Mn magnetic hyperfine tensors from which the spatial distribution of the unpaired spin density, the electronic configuration, and ligand field parameters have been obtained. This is used to analyze highly accurate simulations of the EPR spectra, obtained by least-squares fits of two mixed valence oxidation states, from a series of dimanganese(II,III) and dimanganese(III,IV) complexes and from the dimanganese catalase enzyme, MnCat(II,III) and MnCat(III,I V), from Thermus thermophilus. The sign of the 55Mn dipolar hyperfine anisotropy ( ) reveals that the valence orbital configuration of the Mn(III) ion in MnCat(III,IV) and all dimanganese(III,IV) complexes possessing sterically unconstrained bis(^-oxo) bridges is dT* 123(dz2)1, with the antibonding dz: electron oriented perpendicular to the plane of the 2(µ-0)2 rhombus. This accounts for the strong Mn-0 bonding and slow ligand exchange kinetics widely observed. The asymmetry of the spin density of Mn(III) increases substantially from / = 0.27 in MnCat(III,I V) to 0.46 in MnCat(II,III), reflecting a change in manganese coordination. Comparison with model complexes suggest this may be due to protonation and opening of the (µ-0)2 bridge upon reduction to yield a single µbridge. The presence of strong Mn-O bonding in an unreactive (µ-0)2 core of MnCat(III,IV) offers a plausible explanation for the 1012 slower kinetics of peroxide dismutation compared to what is observed for the physiologically important oxidation state MnCat(II,II). For the dimanganese(II,III) oxidation state, the theory also provides the first explanation for the anomalously large (~30%) 55Mn(II) hyperfine anisotropy in terms of admixing of the S' = 3/2 excited state into the ground state (S = '/2) via the zero-field splitting interaction of Mn(III). This "transferred" anisotropy obscures the otherwise typical isotropic high-spin 3d5 orbital configuration of Mn(II). An estimate of the ratio of the zero-field splitting to the Heisenberg exchange interaction (D/J) is obtained. The theory also explains the unusual 12-line EPR spectrum for a weakly coupled dimanganese(III,IV) complex (Larson et al. J. Am. Chem. Soc. 1992,114,6263-6265), in contrast to the typical 16-line "multiline" spectra seen in strongly coupled dimanganese(III,IV) complexes. The theory shows this is due to a weak J = -10 cm-1 which results in a D/J ratio approaching unity and not to unusual intrinsic magnetic hyperfine parameters of the Mn ions.

The dimanganese (II,II) catalase from Thermus thermophilus, MnCat(II,II), arginase from rat liver, Arg(II,II), and several dimanganese(II,II) compounds, LMn2XY2, which are functional catalase mimics, all possess a pair of coupled Mn(II) ions in their catalytic sites. For each of these, we have measured by EPR spectroscopy the relative energies separating the three lowest electronic states (singlet, triplet, and quintet), described a general method for extracting the individual spectra for these states by multicomponent analysis, and determined the Mn-Mn separation. The triplet-singlet and quintet-singlet energy gaps were modeled well by fitting the temperature dependence of the EPR intensities to a Boltzmann expression for a pair of Mn(II) ions coupled by isotropic Heisenberg spin exchange (-2JS1S2). This dependence indicates diamagnetic ground states with delta E10 (cm-1) = magnitude of 2J = 4 and 11.2 cm-1 for Arg-(II,II)(+borate) and MnCat(II,II)(phosphate), respectively. This large difference in magnitude of 2J reflects either a difference in the bridging ligands or, possibly, a weaker ligand field (larger ionization potential) for the Mn(II) ions in arginase. In n-butanol/CH2Cl2 the triplet-singlet energy gaps for [LMn2(CH3CO2)](C1O4)2 (1), [LMn2(CH3CO2)3] (2), and [LMn2Cl3] (3), where HL = N,N,N',N'-tetrakis(2-methylenebenzimidazole)-1,3-diaminopropan+ ++-2-ol, are 23-24 cm-1. Comparison of the Heisenberg exchange interaction constants for more than 30 dimanganese(II,II) complexes suggests a possible bridging structure of (mu-OH)(mu-carboxylate)1-2 for MnCat(II,II), while the 3-fold weaker coupling in Arg(II,II) suggests mu-aqua in place of mu-hydroxide. EPR spectra of both the triplet and quintet electronic states were extracted and found to exhibit zero-field splittings (ZFS) and resolved 55Mn hyperfine splittings indicating spin-coupled Mn2-(II,II) species. The major ZFS interaction could be attributed to the magnetic dipole-dipole interaction between the Mn(II) ions. A linear correlation is observed between the crystallographically determined Mn-Mn distance and the ZFS of the quintet state (D2) for five dimanganese pairs for which both data sets are available. Using this correlation, the Mn-Mn distance in Arg(II,II) is predicted to be 3.36-3.57 A for the native enzyme (multiple forms) and 3.59 A for MnCat(II,II)(phosphate). Addition of the inhibitor borate to Arg(II,II) simplifies the ZFS, indicative of conversion to a single species with mean Mn-Mn separation of 3.50 A. The second metal ion in dinuclear complexes possessing a shared bridging ligand has been shown to attenuate the strength of the mu-ligand field potential, as monitored by the strength of the single ion ZFS.(ABSTRACT TRUNCATED AT 250 WORDS)

Formate dehydrogenase H, FDH(Se), from Escherichia coli contains a molybdopterin guanine dinucleotide cofactor and a selenocysteine residue in the polypeptide. Oxidation of 13C-labeled formate in 18O-enriched water catalyzed by FDH(Se) produces 13CO2 gas that contains no 18O-label, establishing that the enzyme is not a member of the large class of Mo-pterin-containing oxotransferases which incorporate oxygen from water into product. An unusual Mo center of the active site is coordinated in the reduced Mo(IV) state in a square pyramidal geometry to the four equatorial dithiolene sulfur atoms from a pair of pterin cofactors and a Se atom of the selenocysteine-140 residue [Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C., and Sun, P. D. (1997) Science 275, 1305-1308]. EPR spectroscopy of the Mo(V) state indicates a square pyramidal geometry analogous to that of the Mo(IV) center. The strongest ligand field component is likely the single axial Se atom producing a ground orbital configuration Mo(dxy). The Mo-Se bond was estimated to be covalent to the extent of 17-27% of the unpaired electron spin density residing in the valence 4s and 4p selenium orbitals, based on comparison of the scalar and dipolar hyperfine components to atomic 77Se. Two electron oxidation of formate by the Mo(VI) state converts Mo to the reduced Mo(IV) state with the formate proton, Hf+, transferring to a nearby base Y-. Transfer of one electron to the Fe4S4 center converts Mo(IV) to the EPR detectable Mo(V) state. The Y- is located within magnetic contact to the [Mo-Se] center, as shown by its strong dipolar 1Hf hyperfine couplings. Photolysis of the formate-induced Mo(V) state abolishes the 1Hf hyperfine splitting from YHf, suggesting photoisomerizaton of this group or phototransfer of the proton to a more distant proton acceptor group A-. The minor effect of photolysis on the 77Se-hyperfine interaction with [77Se] selenocysteine suggests that the Y- group is not the Se atom, but instead might be the imidazole ring of the His141 residue which is located in the putative substrate-binding pocket close to the [Mo-Se] center. We propose that the transfer of Hf+ from formate to the active site base Y- is thermodynamically coupled to two-electron oxidation of the formate molecule, thereby facilitating formation of CO2. Under normal physiological conditions, when electron flow is not limited by the terminal acceptor of electrons, the energy released upon oxidation of Mo(IV) centers by the Fe4S4 is used for deprotonation of YHf and transfer of Hf+ against the thermodynamic potential.