Structural Examination of the Nickel Site in Chromatium vinosum Hydrogenase: Redox State Oscillations and Structural Changes Accompanying Reductive Activation and CO Binding†
2000-06-02T00:00:00Z (GMT) by
An X-ray absorption spectroscopic study of structural changes occurring at the Ni site of Chromatium vinosum hydrogenase during reductive activation, CO binding, and photolysis is presented. Structural details of the Ni sites for the ready silent intermediate state, SIr, 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 SIu; distorted four-coordinate sites in SIr 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 SIr. 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 H2 or H- 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) Eur. J. Biochem. 259, 602−608]; only subtle structural differences between the Ni sites were observed.
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