ELECTRON-PARAMAGNETIC-RESONANCE (EPR) STUDIES ON HYDROGENASE-1 (HYD1)PURIFIED FROM A MUTANT STRAIN (AP6) OF ESCHERICHIA-COLI ENHANCED IN HYD1

Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to h omogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2. Plasma emission spectro scopy indicated that 0.93 atom of nickel and 11.4 iron atoms were pres ent in HYD1. EPR studies on the as isolated HYD1 detected a complex 3F e-4S signal and a Ni(III) species. Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial NL(III) signals. At the same time the EPR signature (small g=2.19 signal) of the activa ted hydrogenase appeared. The detection of a 4Fe-4S cluster signal was noted. Reduction of HYD1 with sodium dithionite caused all nickel sig nals to disappear. The 4Fe-4S complex intensity was slightly increased . The EPR responses in the three oxidation-reduction states are consis tent with other known (NiFe)-hydrogenases. (C) 1996 Academic Press, In c.