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

The results of X-ray absorption spectroscopic studies of Thiocapsa roseopersicina hydrogenase poised in three forms exhibiting EPR signals due to the Ni center (A, B, and C) and two states that are epr silent with respect to the Ni center are reported. These spectra are used to examine the structural changes that occur during the reduction of the enzyme. Analyses of Ni K-edge spectra reveal the presence of weak features 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. The lack of a significant pre-edge peak in the active form of the enzyme 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-coordinate. No observable shift in edge energy occurs upon reduction of the enzyme 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 coordination 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 composed of 3 +/- 1 N(O)-donors at 2.00 +/- 0.06 angstrom and 2 +/- 1 S-donors 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 enzyme. No evidence to support a redox role for Ni in hydrogenase is found in the XAS data.