[Fe]-Hydrogenase (Hmd) catalyzes reversible hydride transfer from H2 . It harbors an iron-guanylylpyridinol as a cofactor with an FeII that is ligated to one thiolate, two COs, one acyl-C, one pyridinol-N, and solvent. Here, we report that CuI and H2 O2 inactivate Hmd (half-maximal rates at 1 μM CuI and 20 μM H2 O2 ) and that FeII inhibits the enzyme with very high affinity (Ki =40 nM). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that CuI exerts its inhibitory effect most probably by binding to the active site iron-thiolate ligand. Using the same methods, it was found that H2 O2 binds to the active-site iron at the solvent-binding site and oxidizes FeII to FeIII . Also it was shown that FeII reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for FeII and is reminiscent of that for the [FeFe]-hydrogenase second iron, which specifically interacts with H2 .
Keywords: EPR spectroscopy; IR spectroscopy; hydrogenases; inhibitors; iron-guanylylpyridinol cofactor.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.