Structural examination of the nickel site in Chromatium vinosum hydrogenase: Redox state oscillations and structural changes accompanying reductive activation and CO binding
G. Davidson et al., Structural examination of the nickel site in Chromatium vinosum hydrogenase: Redox state oscillations and structural changes accompanying reductive activation and CO binding, BIOCHEM, 39(25), 2000, pp. 7468-7479
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 comp
lex, SI-CO, are presented for the first time in any hydrogenase. Analysis o
f nickel K-edge energy shifts in redox-related samples reveals that reducti
ve activation is accompanied by an oscillation in the electron density of t
he 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 cha
nges 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 revea
ls that the loss of a short Ni-O bond accounts for the change in coordinati
on number from five to four that accompanies formation of SIr. A shortening
of the Ni-Fe distance from 2.85(5) Angstrom in form B to 2.60(5) Angstrom
also occurs at the SI level and is thus associated with the loss of the bri
dging O-donor ligand in the active site. Multiple-scattering analysis of th
e 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-2 or H- was examined by comparin
g 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 act
ive site that accompanies the photoprocess results in a two-electron reduct
ion of the Ni site [Ni(III) --> Ni(I)] [Happe, R. P., Roseboom, W., and Alb
racht, S. P. J. (1999) Eur. J. Biochem. 259, 602-608]; only subtle structur
al differences between the Ni sites were observed.