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Zusammenfassung:
[Fe]-Hydrogenase (Hmd) catalyzes reversible hydride transfer from H2. It harbors an iron-guanylylpyridinol as cofactor with an FeII that is ligated with one thiolate, two CO, one acyl-C, one pyridinol-N, and a solvent. Here, we report that CuI and H2O2 inactivate Hmd, half maximal rates being observed at 1 µM CuI and 20 µM H2O2 and that FeII inhibits the enzyme with very high affinity (Ki of 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 H2O2 binds to the active site iron at the solvent-binding site and oxidizes FeII to FeIII. Also it was shown that FeII reversibly binds distant to the active site iron, binding being competitive to the organic hydride acceptor; this inhibition is specific for FeII reminiscent to the second iron in [FeFe]-hydrogenases that specifically interacts with H2.
