Resonance Raman studies of iron-only hydrogenases

Abstract

The nature of the iron-sulfur clusters in oxidized and reduced forms of Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritima, and Clostridium pasteurianum has been investigated by resonance Raman spectroscopy. The results indicate the presence of ferredoxin-like [4Fe-4S]2+,+ and [2Fe-2S]2+,+ clusters in both T. maritima hydrogenase and C. pasteurianum hydrogenase I, but only [4Fe-4S]2+,+ clusters in D. vulgaris hydrogenase. This necessitates a reevaluation of the iron-sulfur cluster composition of C. pasteurianum hydrogenase I and indicates that the resonance Raman bands in the oxidized hydrogenase that were previously attributed to the hydrogen activating center [Macor, K. A., Czernuszewicz, R. S., Adams, M. W. W., & Spiro, T. G. (1987) J. Biol. Chem. 262, 9945-9947] arise from an indigenous [2Fe-2S]2+ cluster. No resonance Raman bands that could be uniquely attributed to the oxidized or reduced hydrogen activating center were observed. This suggests that the hydrogen activating center is a novel Fe center that is unrelated to any known type of Fe-S cluster.

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution

“…Bands in the region between 300 and 400 cm –1 are expected to originate mainly from Fe–S modes. For holo-HydA1(adt), comparison with RR spectra of prototypic cubane clusters 11–14,22,23 and the ‘apo-protein’ indicates that these bands are largely due to the [4Fe4S] moiety, while contributions from the [FeFe] sub-site are likely to be small. Bands at higher frequencies (400–700 cm –1 ) can be clearly assigned to the [FeFe] moiety.…”

Characterization of Cluster N5 as a Fast-relaxing [4Fe-4S] Cluster in the Nqo3 Subunit of the Proton-translocating NADH-ubiquinone Oxidoreductase from Paracoccus denitrificans

“…The iron-sulfur clusters of the structurally very similar C. pasteurianum iron-only hydrogenase have been extensively investigated by EPR and magnetic circular dichroism spectroscopy. The dithionite-reduced hydrogenase exhibited a broad featureless EPR resonance in the S ϭ 1/2 region, which arose from magnetically interacting multiple iron-sulfur clusters, one [2Fe-2S] (FS2) and three [4Fe-4S] clusters (FS4A, FS4B, and FS4C) (48,49). In addition, because the midpoint redox potentials of these clusters are very similar (E m,8 ϭ Ϫ400 mV) (48), it was not successful to resolve EPR spectra of the individual iron-sulfur clusters.…”

Concepts in bio-molecular spectroscopy: vibrational case studies on metalloenzymes

“…In particular, metal-ligand coordinates that encode properties and interactions of the metal ion and its ligands can be probed. While the FeS clusters of hydrogenase have been investigated by RR spectroscopy for about three decades, [55][56][57][58] the complex active site remained inaccessible by this technique.…”

“…5). 39,40,[55][56][57] Therefore, Ni-S modes of the active site are normally not accessible, and special requirements have to be met for their detection. The absorbance of FeS clusters strongly decreases with increasing wavelength, which represents an opportunity to detect RR signals that are otherwise masked by spectral contributions from these cofactors.…”