The nature of the iron-sulfur clusters in oxidized and reduced forms o
f Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritim
a, and Clostridium pasteurianum has been investigated by resonance Ram
an 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 1, 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 indica
tes that the resonance Raman bands in the oxidized hydrogenase that we
re previously attributed to the hydrogen activating center [Macor, K.
A., Czernuszewicz, R. S., Adams, M. W. W., & Spiro, T. G. (1987) J. Bi
ol. Chem. 262,9945-9947] arise from an indigenous [2Fe-2S]2+ cluster.
No resonance Raman bands that could be uniquely attributed to the oxid
ized or reduced hydrogen activating center were observed. This suggest
s that the hydrogen activating center is a novel Fe center that is unr
elated to any known type of Fe-S cluster.