FURTHER CHARACTERIZATION OF THE SPIN COUPLING OBSERVED IN OXIDIZED HYDROGENASE FROM CHROMATIUM-VINOSUM - A MOSSBAUER AND MULTIFREQUENCY EPRSTUDY

Hydrogenase from Chromatium vinosum contains 1 Ni, 11-12 Fe, and ca. 9 sulfides. EPR and Mossbauer studies of the enzyme prepared in four di fferent oxidation states show that the enzyme contains two Fe4S4 and o ne Fe3S4 cluster. In the oxidized (2+) state, the Mossbauer parameters of the two Fe4S4 clusters are typical for this cluster type. Upon red uction, however, these clusters do not exhibit the familiar g = 1.94 s ignal. The unusual nature of the reduced clusters is also borne out by the Mossbauer spectra which exhibit fairly small magnetic hyperfine i nteractions similar to those of centers I and II of the Desulfovibrio gigas enzyme. The Mossbauer spectra of the Fe3S4 cluster in the oxidiz ed (1+) and reduced states are typical for this cluster type. The C. v inosum hydrogenase undergoes a reversible redox reaction at E(m) = + 1 50 mV (vs NHE). Above + 150 mV the EPR spectra exhibit signals (previo usly called signals 2 and 4) that reflect a weak interaction between N i(III) and an Fe-containing moiety. By clamping the Ni in the diamagne tic Ni(II).CO form, we have discovered that signal 2 (X-band resonance s at g = 2.01, 1.974, and 1.963) involves the Fe3S4 cluster and an as yet unidentified paramagnetic moiety. The ''coupled'' system exhibits magnetic hyperfine interactions quite different from those of the unco upled Fe3S4(1+) cluster. We have not yet been able to assign a spin to the coupled state but some of the features of the state are reminis cent of an S = 1 system. The Mossbauer data suggest, but do not prove, that an extra Fe site may be present that shuttles between low-spin F e(III) and low-spin Fe(II) with E(m) = + 150 mV. The Fe(III) may be lo cated between the Ni(III) and the Fe3S4 cluster enabling it to mediate the interaction between the cluster and the Ni site. In this picture, the Fe(III) site is part of the coupled state that gives rise to sign al 2. Other possibilities for signal 2 involve a ligand-based oxidatio n of the Fe3S4(1+) cluster or generation of a nearby radical.