SELENIUM-CONTAINING FORMATE DEHYDROGENASE-H FROM ESCHERICHIA-COLI - AMOLYBDOPTERIN ENZYME THAT CATALYZES FORMATE OXIDATION WITHOUT OXYGEN-TRANSFER

Formate dehydrogenase H, FDH(Se), from Escherichia coli contains a mol ybdopterin guanine dinucleotide cofactor and a selenocysteine residue in the polypeptide. Oxidation of C-13-labeled formate in O-18-enriched water catalyzed by FDH(Se) produces (CO2)-C-13 gas that contains no O -18-label, establishing that the enzyme is not a member of the large c lass of Mo-pterin-containing oxotransferases which incorporate oxygen from water into product. An unusual Mo center of the active site is co ordinated in the reduced Mo(IV) state in a square pyramidal geometry t o the four equatorial dithiolene sulfur atoms from a pair of pterin co factors 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 axi al Se atom producing a ground orbital configuration Mo(d(xy)). The Mo- Se bond was estimated to be covalent to the extent of 17-27% of the un paired electron spin density residing in the valence 4s and 4p seleniu m orbitals, based on comparison of the scalar and dipolar hyperfine co mponents to atomic Se-77. Two electron oxidation of formate by the Mo( IV) state converts Mo to the reduced Mo(IV) state with the formate pro ton, H-f(+), transferring to a nearby base Y-. Transfer of one electro n to the Fe4S4 center converts Mo(IV) to the EPR detectable Mo(V) stat e. The Y- is located within magnetic contact to the [Mo-Se] center, as shown by its strong dipolar H-1(f) hyperfine couplings. Photolysis of the formate-induced Mo(V) state abolishes the H-1(f) hyperfine splitt ing from YHf, suggesting photoisomerization of this group or phototran sfer of the proton to a more distant proton acceptor group A(-). The m inor effect of photolysis on the Se-77-hyperfine interaction with [Se- 77] selenocysteine suggests that the Y- group is not the Se atom, but instead might be the imidazole ring of the His141 residue which is loc ated in the putative substrate-binding pocket close to the [Mo-Se] cen ter. We propose that the transfer of H-f(+) from formate to the active site base Y- is thermodynamically coupled to two-electron oxidation o f the formate molecule, thereby facilitating formation of CO2. Under n ormal physiological conditions, when electron flow is not limited by t he terminal acceptor of electrons, the energy released upon oxidation of Mo(IV) centers by the Fe4S4 is used for deprotonation of YHf and tr ansfer of H-f(+) against the thermodynamic potential.