Abstract
[FeFe] hydrogenases are extremely active H2-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called HredH+ and HsredH+. In one model, the HredH+ and HsredH+ states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the HsredH+ and HredH+ states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans, respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the HredH+ and HsredH+ states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.
Highlights
[FeFe] hydrogenases are highly active and efficient H2 conversion catalysts with turnover frequencies up to 10 000 s−1 for H2 production.[1,2] The active site cofactor, the H-cluster, is composed of a unique [2Fe] subcluster ([2Fe]H) linked through a protein cysteine thiolate to a canonical [4Fe-4S] cluster ([4Fe-4S]H).[3,4] The two iron ions are connected by a bridging CO and a 2-azapropane 1,3-dithiolate (ADT) ligand.[5,6] both irons are coordinated by terminal carbon monoxide (CO) and cyanide (CN−) ligands
Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the HsredH+ and HredH+ states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulf uricans, respectively
In order to study the spectral properties of the HredH+ and HsredH+ states in more detail, we chose to apply variabletemperature FTIR spectroscopy
Summary
[FeFe] hydrogenases are highly active and efficient H2 conversion catalysts with turnover frequencies up to 10 000 s−1 for H2 production.[1,2] The active site cofactor, the H-cluster, is composed of a unique [2Fe] subcluster ([2Fe]H) linked through a protein cysteine thiolate to a canonical [4Fe-4S] cluster ([4Fe-4S]H).[3,4] The two iron ions are connected by a bridging CO and a 2-azapropane 1,3-dithiolate (ADT) ligand.[5,6] both irons are coordinated by terminal carbon monoxide (CO) and cyanide (CN−) ligands. The catalytic cycle of [FeFe] hydrogenases (Figure 1A) remains controversial in spite of intensive efforts by many research groups.[13−15] Proton-coupled electron transfer (PCET) between the two subclusters of the H-cluster is believed to be essential for efficient and reversible catalysis.[15,16] It has been demonstrated with pH-dependent FTIR spectroelectrochemistry that, for CrHydA1, two forms of the one-electron reduced state exist where the reducing equivalent is localized on either [4Fe-4S]H or [2Fe]H (Hred and HredH+, respectively).[15] Electron transfer from [4Fe-4S]H to [2Fe]H is coupled to protonation of the bridgehead amine of the ADT ligand This process was shown to occur in DdHydAB, where it is enhanced by redox anticooperativity between [4Fe-4S]H and the F-cluster proximal to it.[17] PCET between the two subclusters is thought to play a role in formation of H2 from a terminal hydride-bound state, Hhyd,[16,18] and activation of the oxygen-stable inactive state Hinact.[19] PCET is disrupted in a sensory [FeFe] hydrogenase HydS from. In 2014, Legeŕ and co-workers demonstrated that CrHydA1 undergoes reversible low potential inactivation[25] and attributed this conversion to the HsredH+ state
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