A major obstacle for future biohydrogen production is the oxygen sensitivity of [FeFe]-hydrogenases, the highly active catalysts produced by bacteria and green algae. The reactions of three representative [FeFe]-hydrogenases with 02 have been studied by protein film electrochemistry under conditions of both H-2 oxidation and H2 production, using CO as a complementary probe. The hydrogenases are DdHydAB and CaHydA from the bacteria Desulfovibno desulfuncans and Clostridium acetobutylicum and CrHydA1 from the green alga Chlamydomonas reinhardtii Rates of inactivation depend or) the redox state of the active site 'H-cluster' and on transport through the protein to reach the pocket in which the H-cluster is housed. In all cases CO reacts much faster than 02 In the model proposed, CaHydA shows the most sluggish gas transport and hence little dependence of inactivation rate on H-cluster stata, wheraas DdHydAB shows a large dependence on H-cluster state and the least effective barrier to gas transport All three enzymes show a similar rate of reactivation from CO inhibition, which increases upon illumination the rate-determining step is thus assigned to cleavage of the labile Fe-CO bond, a reaction likely to be intrinsic to the atomic and electronic state of the H-cluster and less sensitive to the surroundrig protein