EPR study of soluble hydrogenase from photosynthetic bacteria Chromatium vinosum

A soluble hydrogenase(SH) was purified from Chromatium vinosum by five step chromatography(DE-23, TSK-DEAE(I), Ultragel AcA-44, TSK-DEAE(I), Superdex TM75) with a specific activity of 8.4 mu mol H-Z/(min.mg prot). The oxidize d SH yield two Ni( I) EPR(electron paramagnetic resonance) signals (g(x,y,z )=2.37, 2.16, 2.016 and g(x,y,z)=2.30, 2.23, 2.016) at 45 K which occurred in the other NiFe-hydrogenases. However, no [3Fe-4S] cluster EPR signal was obtained at 10 K. When the SH was reduced by H-2(over night at 8 degreesC) , the Ni(I) EPR signals disappeared, and an EPR signal from a reduced[4Fe-4 S] cluster appeared (g(x,y,z)=1.88, 1.90, 2.045). The results show that the soluble hydrogenase from C. vinosum is a new NiFe-hydrogenase which cataly zes H-2-production.