X-ray crystallographic and computational studies of the O ₂ -tolerant [NiFe]-hydrogenase 1 from Escherichia coli

Abstract

The crystal structure of the membrane-bound O 2 -tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H 2 -reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O 2 -sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oϵ of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H 2 -reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O 2 to H 2 O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O 2 reduction, located near the active site.[4Fe-3S] cluster | membrane-bound hydrogenase | Mössbauer spectroscopy | QM/MM | structure/function relationships

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

“…In the T18D variant, the H-bond between Glu-25 L and the 18-position residue is lost. Thr-18 S was proposed to be a relay of PT, next to Glu-25 L (11,13,15). Alcoholic residues have frequently been shown, including in [FeFe]-hydrogenases, to act as proton relays or to influence the reactivity of neighboring relay residues (44, 45), but we cannot confirm this function, at least for the Df enzyme.…”

“…We used the nonredox HDE assay to show that changing the residue at this position may moderately affect the rate of gas diffusion in the tunnel (Table 2, for example, the rate constant for H 2 release from the active site in the T18D variant is approximatively 5-fold lower than that in the WT enzyme). Because the H 2 oxidation/production rates and the rates of H/D exchange (k) are affected to the same extent in the T18V, T18Q, and T18D variants, it may have been tempting to assume that S is involved in a step that is involved in the three reactions, namely active site chemistry or proton transfer, as suggested by calculations and crystallographic studies (13)(14)(15)(16). However, a crucial observation is that the T18G variant is almost inactive for H 2 oxidation or production and yet catalyzes HDE at a high rate.…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…[106] This shows that Ni-B reactivates faster in O 2 -tolerant than in O 2 -sensitive Hases. [118,119] Thanks to the resolution of the crystallographic structures of three O 2 -tolerant Hases, [120][121][122] and to genetic engineering both on O 2 -tolerant and O 2 -sensitive Hases, a general scheme is nowadays proposed that may account for O 2 tolerance.…”

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases

“…As illustrated in Fig. 2B, the unusual structure allows it to undergo two consecutive one-electron transfers at similar potentialsthe rapid removal of the second electron (in a proton-coupled reaction) yielding a Fe-N(peptide) bond in a reaction that is (locally at least) electroneutral (1,2,10,(12)(13)(14)(15). The [3Fe-4S] cluster occupying the medial position in respiratory membrane bound [NiFe]-hydrogenases also has a higher reduction potential in O 2 -tolerant hydrogenases (e.g., 190 ± 30 mV at pH 6 in E. coli Hyd-1) (15) than in standard hydrogenases (e.g., −70 mV at pH 7 in Desulfovibrio gigas hydrogenase) (16).…”

“…Fig. 2A shows the structure of an O 2 -tolerant [NiFe]-hydrogenase known as hydrogenase-1 (Hyd-1), which is produced in Escherichia coli (10). Like other respiratory membrane-bound [NiFe]-hydrogenases, it contains a buried [NiFe] catalytic center and FeS clusters that are located in separate α and β subunits.…”