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 homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2. Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1. EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species. Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial Ni(III) signals. At the same time the EPR signature (small g = 2.19 signal) of the activated hydrogenase appeared. The detection of a 4Fe-4S cluster signal was noted. Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear. The 4Fe-4S complex intensity was slightly increased. The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases.