A kinetic and thermodynamic understanding of O 2 tolerance in [NiFe]-hydrogenases

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Iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. In the oxygentolerant Hydrogenase I from Aquifex aeolicus such electron "wires" form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of O 2 to water. Amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. Electron paramagnetic resonance spectra exhibited complex signals that disclose interesting features and spin-coupling patterns; by redox titrations three iron-sulfur clusters were identified in their usual redox states, a ½3Fe4S and two ½4Fe4S , but also a unique high-potential (HP) state was found. On the basis of 57 Fe Mössbauer spectroscopy we attribute this HP form to a superoxidized state of the [4Fe4S] center proximal to the [NiFe] site. The unique environment of this cluster, characterized by a surplus cysteine coordination, is able to tune the redox potentials and make it compliant with the ½4Fe4S 3þ state. It is actually the first example of a biological [4Fe4S] center that physiologically switches between 3þ, 2þ, and 1þ oxidation states within a very small potential range. We suggest that the (1 þ ∕2þ) redox couple serves the classical electron transfer reaction, whereas the superoxidation step is associated with a redox switch against oxidative stress.electrochemistry | EPR | iron-sulfur centers | O2-sensitivity H ydrogenases are metalloproteins occurring in the metabolic pathway of a wide variety of microbial organisms and catalyze the reversible oxidation of dihydrogen: H 2 ⇌ 2H þ þ 2e − (1). The growing interest in alternative sources of energy has focused scientific research on understanding and engineering these enzymes for future applications (2). One of the major limitations of hydrogenases, however, is their sensitivity towards oxygen. Recently, the discovery of hydrogenases that retain catalytic activity in oxygenic environments has potentially opened new applications as "green" vanguard catalysts, in particular as electrocatalysts on electrodes for biofuel cells (3,4).Aquifex aeolicus is a hyperthermophilic Knallgas bacterium with optimum growth temperature of 85°C (5). This microorganism harbors three distinct [NiFe] hydrogenases, among which Hase I is located in the aerobic respiration pathway and attached to the membrane via a diheme cytb (6). Hase I consists of two subunits; the large subunit contains the hetero-bimetallic nickel-iron site and the small subunit the electron transfer cofactors, namely iron-sulfur clusters (6). Based on spectroelectrochemical studies, this enzyme exhibits enhanced thermostability and tolerance for inhibitors (e.g., O 2 and CO) (4, 7).Although the structures of O 2 -sensitive hydrogenases are well characterized (8, 9), such information is still lacking for O 2 -tolerant enzymes. The molecular mechanism and structural determinants for this increased oxygen tolerance remain to ...

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, O 2 -tolerance of enzymes such as Hase I is understood to comprise a concerted effect of redox (thermodynamic) and kinetic factors (7,38). Considering the redox potentials of the "activated" [NiFe] site and the reduced [FeS] clusters (7), the [NiFe] site is likely to be the primary target of O 2 .…”

Section: Discussionmentioning

confidence: 99%

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

Pandelia

Nitschke

Infossi

et al. 2011

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198

“…1, the rate at which O 2 attenuates the H 2 oxidation current (k I ) depends on O 2 concentration but not on potential: under 10% O 2 at 30°C this rate is ∼0.45 s −1 (9). In contrast, the rate of reactivation of Ni-B (k A ) increases exponentially as the potential is lowered: assuming the solution potential sensed by the active site in these experiments is set by the fast H 2 /H + interconversion (and could be as low as −0.4 V), the reactivation rate should exceed 100 s −1 at 20°C and not be rate determining (8,9). Considering the higher temperatures used in the electrochemical experiments, the agreement with the value of 0.28 s −1 now obtained at 20°C for direct four-electron reduction of O 2 by H 2 catalyzed by Hyd-1 is compelling evidence that the two very different experiments are measuring the same process.…”

Section: Assay Of Intermolecular Electron-transfer Kinetics Using Cytmentioning

confidence: 99%

“…In contrast, standard (O 2 sensitive) [NiFe]-hydrogenases react with O 2 to give a mixture of states, including ones variously known as "unready" or Ni-A, in which O 2 is either only partially reduced (possibly trapped as a peroxide) or has oxygenated atoms of the active site (3-7). The unready states are only reactivated very slowly; consequently, their production removes enzyme from the catalyst pool (8,9).…”

mentioning

confidence: 99%

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

Wulff

Day

Sargent

et al. 2014

Self Cite

An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H 2 + O 2 = 2 H 2 O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H 2 O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H 2 and 10% O 2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O 2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H 2 oxidation. The oxidase activity is too slow to be useful in removing O 2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation.hydrogen | mass spectrometry | Fe-S cluster H ydrogenases are enzymes that catalyze the interconversion of H 2 and H + with great efficiency. Containing Fe or Fe and Ni as active metals, they are not only important in biohydrogen production (by fermentative and photosynthetic means) but also provide inspiration for detailed understanding and development of optimal molecular electrocatalysts. The minimal active site motif, common to all hydrogenases, is a low-spin Fe atom coordinated by CO, CN − , and thiolate ligands, a combination expected to be unstable under aerobic conditions. Indeed, most hydrogenases suffer long-term or permanent inactivation when exposed to even traces of O 2 . It is therefore of special interest that certain [NiFe]-hydrogenases have evolved to sustain H 2 oxidation in the continued presence of O 2 , without inactivation: these enzymes are known as O 2 -tolerant [NiFe]-hydrogenases.Most of our current insight into the mechanism of O 2 tolerance stems from studies on respiratory membrane-bound [NiFe]-hydrogenases that couple H 2 oxidation to reduction of quinones (1-3). These enzymes are localized at the cytoplasmic membrane and project into the periplasmic space. A model proposed for the O 2 -tolerance mechanism of these [NiFe]-hydrogenases ( Fig. 1) is based on the following evidence. Oxygen reacts with O 2 -tolerant membrane-bound [NiFe]-hydrogenases to form, exclusively, an inactive state known as Ni-B or "ready," formulated as a Ni(III)-OH species, which is rapidly reactivated by one-electron transfer to rejoin the catalytic cycle of H 2 oxidation. Provided Ni-B is the sole product of O 2 attack, the presence of O 2 merely attenuates the steady-state rate of H 2 oxidation. In contrast, standard (O 2 sensitive) [NiFe]-hydrogenases react with O 2 to give a mixture of states, including ones variously known as "unready" or Ni-A, in which O 2 is either only partially reduced (possibly trapped as a peroxide) or has oxygenated atoms of the active site (3-7). The unready states are only reactivated very slowly; consequently, t...

“…It appears that the formation of either Ni-A or Ni-B states depends on the number of electrons, either two or four, that can rapidly react with O 2 at the active site upon air exposure (10). Therefore, resistance to inactivation by O 2 may depend on the hydrogenase having enough electrons available to avoid formation of unready states (11).…”

mentioning

confidence: 99%

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

Volbeda

Amara

Darnault

et al. 2012

Self Cite

192

269

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