Structural and oxidation-state changes at its nonstandard Ni-Fe site during activation of the NAD-reducing hydrogenase from Ralstonia eutropha detected by X-ray absorption, EPR, and FTIR spectroscopy.
Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.
Burgdorf
Tanja
T
Humboldt-Universität zu Berlin, Mikrobiologie, Chausseestr. 117, D-10115 Berlin, Germany.
Löscher
Simone
S
Liebisch
Peter
P
Van der Linden
Eddy
E
Galander
Marcus
M
Lendzian
Friedhelm
F
Meyer-Klaucke
Wolfram
W
Albracht
Simon P J
SP
Friedrich
Bärbel
B
Dau
Holger
H
Haumann
Michael
M
D001665:Binding Sites; D020118:Cupriavidus necator / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D013052:Spectrometry, X-Ray Emission; D017550:Spectroscopy, Fourier Transform Infrared
D009243:NAD; D006859:Hydrogen; D010088:Oxidoreductases; C043296:nickel-iron hydrogenase; C021376:hydrogen dehydrogenase; D006864:Hydrogenase