Cu I and H 2 O 2 Inactivate and Fe II Inhibits [Fe]-Hydrogenase at Very Low Concentrations.

[Fe]-Hydrogenase (Hmd) catalyzes reversible hydride transfer from H ₂ . It harbors an iron-guanylylpyridinol as a cofactor with an Fe ^II that is ligated to one thiolate, two COs, one acyl-C, one pyridinol-N, and solvent. Here, we report that Cu ^I and H ₂ O ₂ inactivate Hmd (half-maximal rates at 1 μM Cu ^I and 20 μM H ₂ O ₂ ) and that Fe ^II inhibits the enzyme with very high affinity (K _i =40 nM). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that Cu ^I exerts its inhibitory effect most probably by binding to the active site iron-thiolate ligand. Using the same methods, it was found that H ₂ O ₂ binds to the active-site iron at the solvent-binding site and oxidizes Fe ^II to Fe ^III . Also it was shown that Fe ^II reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for Fe ^II and is reminiscent of that for the [FeFe]-hydrogenase second iron, which specifically interacts with H ₂ .
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