Abstract
Despite extensive studies on [NiFe]-hydrogenases, the mechanism by which these enzymes produce and activate H2 so efficiently remains unclear. A well-known EPR-active state produced under H2 and known as Ni-C is assigned as a NiIII–FeII species with a hydrido ligand in the bridging position between the two metals. It has long been known that low-temperature photolysis of Ni-C yields distinctive EPR-active states, collectively termed Ni-L, that are attributed to migration of the bridging-H species as a proton; however, Ni-L has mainly been regarded as an artifact with no mechanistic relevance. It is now demonstrated, based on EPR and infrared spectroscopic studies, that the Ni-C to Ni-L interconversion in Hydrogenase-1 (Hyd-1) from Escherichia coli is a pH-dependent process that proceeds readily in the dark—proton migration from Ni-C being favored as the pH is increased. The persistence of Ni-L in Hyd-1 must relate to unassigned differences in proton affinities of metal and adjacent amino acid sites, although the unusually high reduction potentials of the adjacent Fe–S centers in this O2-tolerant hydrogenase might also be a contributory factor, impeding elementary electron transfer off the [NiFe] site after proton departure. The results provide compelling evidence that Ni-L is a true, albeit elusive, catalytic intermediate of [NiFe]-hydrogenases.
Highlights
Hydrogenases are Ni- and Fe-based enzymes that catalyze highly efficient and reversible H2 cycling.[1,2] A major class known as [NiFe]-hydrogenases activate H2 at a buried binuclear site formulated as [(Cys-S)2-Ni-(μ2-Cys-S)2-Fe(CN)2CO] that is served by an electron relay system of Fe−S clusters
Our results reveal that a reaction pathway from Ni-C to Ni-SI via a stable Ni-L state, in which H+ has started its migration from the Ni−Fe bond, is fully appropriate for Hyd-1
The EPR data at high pH show a single Ni-L state, whereas the IR data show two Ni-L states: that more than one Ni-L state exists, albeit depending on enzyme and conditions, is fully in accordance with the findings of others.[7−11,27,36] It has long been tacitly assumed, on the basis that Ni-L is detected only under unusual conditions, that oxidation of Ni-C proceeds in a single proton-coupled electron-transfer (PCET) process. This scenario would either involve synchronous proton−electron transfer or at least require that proton transfer is rate-limiting for no intermediate to be detected
Summary
Hydrogenases are Ni- and Fe-based enzymes that catalyze highly efficient and reversible H2 cycling.[1,2] A major class known as [NiFe]-hydrogenases activate H2 at a buried binuclear site formulated as [(Cys-S)2-Ni-(μ2-Cys-S)2-Fe(CN)2CO] that is served by an electron relay system of Fe−S clusters. Physical techniques such as EPR and IR spectroscopy have played important roles in the characterization of different states of the enzyme, no consensus exists regarding the detailed mechanism. Detailed EPR investigations have indicated that during photolysis, the bridging hydride assigned to Ni-C is released as a proton, which is assumed to migrate to one or more nearby amino acids, including coordinated cysteine, in the Ni-L states.[12,13] Ni-C and Ni-L species, usually assigned as Ni(III) and Ni(I) states, respectively, can be regarded as tautomeric forms of the extended active site at a single oxidation level.[12]
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