Structural Examination of the Nickel Site in <i>Chromatium vinosum</i> Hydrogenase: Redox State Oscillations and Structural Changes Accompanying Reductive Activation and CO Binding<sup>†</sup>
2000-06-02T00:00:00Z (GMT) by
An X-ray absorption spectroscopic study of structural changes occurring at the Ni site of <i>Chromatium vinosum </i>hydrogenase during reductive activation, CO binding, and photolysis is presented. Structural details of the Ni sites for the ready silent intermediate state, SI<sub>r</sub>, and the carbon monoxide complex, SI−CO, are presented for the first time in any hydrogenase. Analysis of nickel K-edge energy shifts in redox-related samples reveals that reductive activation is accompanied by an oscillation in the electron density of the Ni site involving formally Ni(III) and Ni(II), where all the EPR-active states (forms A, B, and C) are formally Ni(III), and the EPR-silent states are formally Ni(II). Analysis of XANES shows that the Ni site undergoes changes in the coordination number and geometry that are consistent with five-coordinate Ni sites in forms A, B, and SI<sub>u</sub>; distorted four-coordinate sites in SI<sub>r</sub> and R; and a six-coordinate Ni site in form C. EXAFS analysis reveals that the loss of a short Ni−O bond accounts for the change in coordination number from five to four that accompanies formation of SI<sub>r</sub>. A shortening of the Ni−Fe distance from 2.85(5) Å in form B to 2.60(5) Å also occurs at the SI level and is thus associated with the loss of the bridging O-donor ligand in the active site. Multiple-scattering analysis of the EXAFS data for the SI−CO complex reveals the presence of Ni−CO ligation, where the CO is bound in a linear fashion appropriate for a terminal ligand. The putative role of form C in binding H<sub>2</sub> or H<sup>-</sup> was examined by comparing the XAS data from form C with that of its photoproduct, form L. The data rule out the suggestion that the increase in charge density on the NiFe active site that accompanies the photoprocess results in a two-electron reduction of the Ni site [Ni(III) → Ni(I)] [Happe, R. P., Roseboom, W., and Albracht, S. P. J. (1999) <i>Eur. J. Biochem.</i> <i>259</i>, 602−608]; only subtle structural differences between the Ni sites were observed.