AN X-RAY-ABSORPTION SPECTROSCOPIC STUDY OF NICKEL REDOX CHEMISTRY IN HYDROGENASE

The results of X-ray absorption spectroscopic studies of Thiocapsa ros eopersicina hydrogenase poised in three forms exhibiting EPR signals d ue to the Ni center (A, B, and C) and two states that are epr silent w ith respect to the Ni center are reported. These spectra are used to e xamine the structural changes that occur during the reduction of the e nzyme. Analyses of Ni K-edge spectra reveal the presence of weak featu res at ca. 8332 eV in the spectra obtained from forms A and B and the silent intermediate (SI) that are assigned to 1s --> 3d transitions. T he lack of a significant pre-edge peak in the active form of the enzym e and low peak areas in other forms, coupled with the absence of edge features associated with planar four-coordinate Ni complexes, indicate that the Ni site in all states of the enzyme is five- or six-coordina te. No observable shift in edge energy occurs upon reduction of the en zyme to any level. This demonstrates that no significant change in the electron density of the Ni site occurs during reduction. Analyses of the EXAFS spectra obtained from scattering atoms in the first coordina tion sphere of Ni in all rive states of the enzyme that are defined by Ni EPR signals (or lack thereof) are consistent with a Ni site compos ed of 3 +/- 1 N(O)-donors at 2.00 +/- 0.06 angstrom and 2 +/- 1 S-dono rs at 2.23 +/- 0.03 angstrom. These results are discussed in light of various models for the structure and function of the Ni site in the en zyme. No evidence to support a redox role for Ni in hydrogenase is fou nd in the XAS data.