STRUCTURE OF THE NI SITES IN HYDROGENASES BY X-RAY-ABSORPTION SPECTROSCOPY - SPECIES VARIATION AND THE EFFECTS OF REDOX POISE

Structural information obtained from the analysis of nickel K-edge X-r ay absorption spectroscopic data of [NiFe]hydrogenases from Desulfovib rio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC 27774), Escherichia coli (hydrogenase-1), Chromatium vinosum, and Alc aligenes eutrophus H16 (NAD(+)-reducing, soluble hydrogenase), poised in different redox states, is reported. The data allow the active-site structures of enzymes from several species to be compared, and allow the effects of redox poise on the structure of the nickel sites to be examined. In addition, the structure of the nickel site obtained from recent crystallographic studies of the D. gigas enzyme (Volbeda, A.; C haron, M.-H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps , J. C. Nature 1995, 373, 580-587) is compared with the structural fea tures obtained from the analysis of XAS data from the same enzyme. The nickel sites of all but the oxidized (as isolated) sample of A. eutro phus hydrogenase are quite similar. The nickel K-edge energies shift 0 .9-1.5 eV to lower energy upon reduction from oxidized (forms A and B) to fully reduced forms. This value is comparable with no more than a one-electron metal-centered oxidation state change. With the exception of T. roseopersicina hydrogenase, most of the edge energy shift (-0.8 eV) occurs upon reduction of the oxidized enzymes to the EPR-silent i ntermediate redox level (SI). Analysis of the XANES features assigned to 1s-->3d electronic transitions indicates that the shift in energy t hat occurs for reduction of the enzymes to the SI level may be attribu ted at least in part to an increase in the coordination number from fi ve to six. The smallest edge energy shift is observed for the T. roseo persicina enzyme, where the XANES data indicate that the nickel center is always six-coordinate. With the exception of the oxidized sample o f A. eutrophus hydrogenase, the EXAFS data are dominated by scattering from S-donor ligands at similar to 2.2 Angstrom. The enzyme obtained from T. roseopersicina also shows evidence for the presence of O,N-don or ligands. The data from A. eutrophus hydrogenase are unique in that they indicate that a significant structural change occurs upon reducti on of the enzyme. EXAFS data obtained from the oxidized (as isolated) A. eutrophus enzyme indicate that the EXAFS is dominated by scattering from 3-4 N,O-donor atoms at 2.06(2) Angstrom, with contributions from 2-3 S-donor ligands at 2.35(2) Angstrom. This changes upon reduction to a more typical nickel site composed of similar to 4 S-donor ligands at a Ni-S distance of 2.19(2) Angstrom. Evidence for the presence of atoms in the 2.4-2.9 Angstrom distance range is found in most samples, particularly the reduced enzymes (SI, form C, and R). The analysis of these data is complicated by the fact that it is difficult to disting uish between S and Fe scattering atoms at this distance, and by the po tential presence of both S and another metal atom at similar distances . The results of EXAFS analysis are shown to be in general agreement w ith the published crystal structure of the D. gigas enzyme.