CO Disrupts the Reduced H-Cluster of FeFe Hydrogenase. A Combined DFT and Protein Film Voltammetry Study

Baffert, Carole; Bertini, Luca; Lautier, Thomas; Greco, Claudio; Sybirna, Kateryna; Ezanno, Pierre; Étienne, Émilien; Soucaille, Philippe; Bertrand, Patrick; Bottin, Hervé; Meynial-Salles, Isabelle; Gioia, Luca De; Léger, Christophe

doi:10.1021/ja110627b

Cited by 63 publications

(102 citation statements)

References 27 publications

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“…However, a bridging superoxide (O 2 Ϫ ) represents a chemically rather unlikely motif. On the other hand, the elongation of the Fe-Fe distance of 2Fe H , similar to that observed for a bridging carbonyl in CO-inhibited HydA1 (20,60), and the initial increase of N(C,O) are well in agreement with a bridging CO plus a single terminal oxygen species. In a density functional theory study of O 2 binding to the H-cluster, the end-on binding of O 2 to the distal iron ion of 2Fe H energetically was preferred (42).…”

Section: Discussionsupporting

confidence: 80%

“…Furthermore, CO protects against O 2 inhibition probably by occupying the O 2 binding site at 2Fe H (17). However, CO is an even more effective, although mostly reversible, inhibitor of HydA1, which prevents hydrogen coordination at 2Fe H (6,60). Thus, strategies for the protection of the H-cluster against O 2 -induced inhibition may involve diffusible small molecules, which rapidly exchange with H-species but not with O 2 ; alteration of the redox potential of the [4Fe4S] cluster; and blocking of ROS diffusion paths from 2Fe H (i.e.…”

Section: Discussionmentioning

confidence: 99%

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O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

Lambertz

Leidel

Havelius

et al. 2011

Journal of Biological Chemistry

144

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[FeFe]-hydrogenase (H 2 ase) 4 proteins are the most active biological catalysts for the production of molecular hydrogen (H 2 ) from proton reduction, with reported turnover rates of up to 10 4 s Ϫ1 (1, 2). Therefore, these enzymes are of high interest for biotechnology, aiming at the generation of H 2 as a renewable fuel (1, 3-5). However, a severe limitation for such applications is the rapid inactivation of [FeFe]-H 2 ases by dioxygen (O 2 ) (6, 7). Understanding the mechanism of O 2 -induced inactivation may allow the improvement of enzyme features to yield increased O 2 tolerance (e.g. by genetic engineering, which has already been demonstrated for NiFe hydrogenases) (8 -10).[FeFe]-H 2 ases are found in certain bacteria and green algae (11, 12). All of these enzymes contain an active site that consists of an inorganic iron complex, which is denoted as the H-cluster (13-15). The [FeFe]-H 2 ase enzymes from anaerobic bacteria in addition bind several iron-sulfur (FeS) clusters, serving as a relay for electron transfer to and from the active site (13-15).[FeFe]-H 2 ases from green algae represent the minimal unit for biological H 2 production because they contain only the H-cluster, whereas accessory FeS clusters are absent (16). This feature renders these enzymes most suitable for spectroscopic investigations on O 2 -induced inactivation (17), focusing on the active site reactions. The general structure of the H-cluster has been unraveled by crystallography, x-ray absorption spectroscopy (XAS), EPR, and FTIR spectroscopy on H 2 ase protein from various organisms (18). The H-cluster structure in both bacteria and green algae appears to be similar overall (16,19,20). It features a [4Fe4S] cubane cluster, which is bound by four cysteine residues to the protein and is linked by one of them to a binuclear iron unit (2Fe H ) (Fig. 1). The latter carries two cyanide (CN Ϫ ) ligands and three carbon monoxide (CO) ligands (21, 22) and presumably an azadithiolate group (adt; (SCH 2 ) 2 NH) in the metal-bridging position (21,23,24). Both types of diatomic ligands are probably derived from a biosynthetic pathway starting with tyrosine (25-27). The nitrogen atom of the adt has been proposed to be involved in proton transfer at the active site (21, 28). The H-cluster structure is assembled in a complex reaction involving three maturation proteins (29 -34). H 2 formation has been proposed to involve the binding and reduction of a single proton, which transiently creates a hydride ligand, either located in a bridging position between the two iron ions or terminally bound at the distal iron ion of the 2Fe H moiety (Fig. 1). After a second protonation step, H 2 is released from the H-cluster (35)(36)(37) 4 The abbreviations used are: H 2 ase, hydrogenase; adt, azadithiolate; EXAFS, extended x-ray absorption fine structure; NaDT, sodium dithionite; ROS, reactive oxygen species; XANES, x-ray absorption near edge structure; XAS, x-ray absorption spectroscopy; FT, Fourier transform.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

Lambertz

Leidel

Havelius

et al. 2011

Journal of Biological Chemistry

144

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“…(CO binds reversibly to Fe d under oxidizing conditions. 29 ) Replacing F417 (Cp numbering, fig 4B) with a tryptophan unfortunately renders the enzyme inactive (we observed that the F290W mutant of Cr hydrogenase has no activity), but replacing the valine that lines the path from site 7 to the geminate site with a larger phenylalanine (V296F in the Cr enzyme) decreases the rates of CO binding and release about ten-fold (Figure 2E), from

k_{i}^{CO} = 60 \pm 15 s^{- 1} {mM}^{- 1}

and

k_{a}^{CO} = 0.025 \pm 0.006 s^{- 1}

to 6 ± 2 s −1 mM −1 and 0.0014 ± 0.0002 s −1 (n = 3), respectively, at 30°C, pH 7, E = −158 mV. Explicit MD simulations of the corresponding mutant of the Cp enzyme (V423F, see Fig 4B and SI), in either of the two most probable rotameric states of the mutated residue, predict a ~2-fold decrease in k i and k a .…”

Section: Resultsmentioning

confidence: 99%

Mechanism of O2 diffusion and reduction in FeFe hydrogenases

et al. 2016

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FeFe hydrogenases are the most efficient H2 producing enzymes, but inactivation by O2 is an obstacle to using them in biotechnological devices. Here we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We find that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of the highly reactive OH radical and hydroxylated cysteine, consistent with recent crystallographic evidence. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species at prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

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“…A similar process has been found in the context of the inhibitor CO reacting with the H cluster. (42)…”

Section: The Ooh• Radical As Damaging Agentmentioning

confidence: 99%

Regioselectivity of H Cluster Oxidation

2011

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The H2-evolving potential of [FeFe] hydrogenases is severely limited by the oxygen sensitivity of this class of enzymes. Recent experimental studies on hydrogenase from C. reinhardtii point to O2-induced structural changes in the [Fe4S4] subsite of the H cluster. Here, we investigate the mechanistic basis of this observation by means of density functional theory. Unexpectedly, we find that the isolated H cluster shows a pathological catalytic activity for the formation of reactive oxygen species such as O2– and HO2–. After protonation of O2–, an OOH radical may coordinate to the Fe atoms of the cubane, whereas H2O2 specifically reacts with the S atoms of the cubane-coordinating cysteine residues. Both pathways are accompanied by significant structural distortions that compromise cluster integrity and thus catalytic activity. These results explain the experimental observation that O2-induced inhibition is accompanied by distortions of the [Fe4S4] moiety and account for the irreversibility of this process.

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CO Disrupts the Reduced H-Cluster of FeFe Hydrogenase. A Combined DFT and Protein Film Voltammetry Study

Cited by 63 publications

References 27 publications

O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

Mechanism of O2 diffusion and reduction in FeFe hydrogenases

Regioselectivity of H Cluster Oxidation

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