The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain-a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.EXAFS ͉ H-cluster ͉ protein film electrochemistry ͉ biological hydrogen production ͉ green algae

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 O(2) have been studied by protein film electrochemistry under conditions of both H(2) oxidation and H(2) production, using CO as a complementary probe. The hydrogenases are DdHydAB and CaHydA from the bacteria Desulfovibrio desulfuricans and Clostridium acetobutylicum , and CrHydA1 from the green alga Chlamydomonas reinhardtii . Rates of inactivation depend on 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 O(2). In the model proposed, CaHydA shows the most sluggish gas transport and hence little dependence of inactivation rate on H-cluster state, whereas 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 surrounding protein.