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

Abstract

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.

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O2-Tolerant H2 Cleavage

“…4A) of the PH in the Ni-A state revealed two main peaks due to Ni-oxygen and Ni-sulfur bonds. Features at higher distances reflected contributions from the Ni-Fe vector (23,60). The best-fit parameters (Table 3-I) showed that the Ni-A spectrum was completely described by close to one short Ni-oxygen distance, four Ni-sulfur bonds from the cysteines at the active site, and a Ni-Fe distance of 2.75 Å, all of which are similar to those found in crystal structures (50,53).…”

“…3). In the oxidized MBH at pH 8.0, a pronounced increase of the primary edge maximum at ~8350 eV suggested additional terminal oxygen ligands at the Ni(II) (23,58,82,85) in the Ni ia -S state and therefore a substantially different site structure compared to Ni-B.…”

Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

“…The resulting unready, inactive enzyme requires a long term reactivation with H 2 to acquire a catalytically active conformation. Two other hydrogenases in R. eutropha, the soluble, hexameric NAD ϩ -reducing hydrogenase and the cytoplasmic H 2 -sensing hydrogenase, share the exceptional ability of being quickly reactivated after exposure to oxygen, and for none of them have Ni u -A signals been detected (25,61).…”

“…Prominent examples are ␤-proteobacteria of the genus Ralstonia, including the well studied chemolithoautotrophic model organism Ralstonia eutropha H16. R. eutropha harbors three distinct [NiFe] hydrogenases that catalyze H 2 oxidation in the presence of ambient oxygen concentrations (11,(25)(26)(27). Their O 2 tolerance is based on at least two molecular mechanisms.…”

“…For the cytoplasmic, hexameric NAD ϩ -reducing hydrogenase, a modified composition of the active site nickel has been described. It is proposed that an extra CN Ϫ ligand bound to the nickel protects the enzyme from O 2 inactivation (25,28,29). O 2 resistance of the H 2 -sensing regulatory [NiFe] hydrogenases from R. eutropha (27) and Rhodobacter capsulatus (30) is likely based on a narrow gas channel that prevents access of O 2 to the active site.…”