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Bias from H-2 cleavage to production and coordination changes at the Ni-Fe active site in the NAD(+)-reducing hydrogenase from Ralstonia eutropha
Citation key ISI:000240575900041
Author Loescher, Simone and Burgdorf, Tanja and Zebger, Ingo and Hildebrandt, Peter and Dau, Holger and Friedrich, Baerbel and Haumann, Michael
Pages 11658-11665
Year 2006
ISSN 0006-2960
DOI 10.1021/bi061068f
Address 1155 16TH ST, NW, WASHINGTON, DC 20036 USA
Journal Biochemistry
Volume 45
Number 38
Month SEP 26
Abstract The soluble NAD(+)-reducing Ni-Fe hydrogenase (SH) from Ralstonia eutropha H16 is remarkable because it cleaves hydrogen in the presence of dioxygen at a unique Ni-Fe active site (Burgdorf et al. (2005) J. Am. Chem. Soc. 127, 576). By X-ray absorption (XAS), FTIR, and EPR spectroscopy, we monitored the structure and oxidation state of its metal centers during H-2 turnover. In NADH-activated protein, a change occurred from the (CN)O2NiII(mu-S)(2)Fe-II(CN)(3)(CO) site dominant in the wild-type SH to a standard-like S2NiII(mu-S)(2)Fe-II(CN)(2)(CO) site as the prevailing species in a specific mutant protein, HoxH-H16L. The wild-type SH primarily was active in H2 cleavage. The nonstandard reaction mechanism does not involve stable EPR-detectable trivalent Ni oxidation states, namely, the Ni-A,B,C states as observed in standard hydrogenases. In the HoxH-mutant protein H16L, H-2 oxidation was impaired, but H-2 production occurred via a stable Ni-C state (Ni-III-H–Fe-II), suggesting a reaction sequence similar to that of standard hydrogenases. It is proposed that reductive activation by NADH of both wild-type and H16L proteins causes the release of an oxygen species from Ni and is initiated by electron transfer from a [2Fe-2S] cluster in the HoxU subunit that at first becomes reduced by electrons from NADH. Electrons derived from H-2 cleavage, on the other hand, are transferred to NAD(+) via a different pathway involving a [4Fe-4S] cluster in HoxY, which is reducible only in wild-type SH but not in the H16L variant.
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