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

Abstract

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...

Reversible Active Site Sulfoxygenation Can Explain the Oxygen Tolerance of a NAD+-Reducing [NiFe] Hydrogenase and Its Unusual Infrared Spectroscopic Properties

“…Indeed, catalytic water production as a result of reverse electron flow to the active site has recently been demonstrated for oxygen tolerant [NiFe] hydrogenases including the SH. 30,72 The model is also in agreement with the necessity to reactivate fully oxidized SH with low-potential electrons from, e.g., NADH. 45,48,50,52,54,55,63 The FMN-a molecule proposed to be located close to the catalytic center ( Figure 1B) might act as a two-electron transfer unit in analogy to the flavin adenine dinucleotide (FAD) cofactor involved in the sulfur-based detoxification of hydrogen peroxide by NADH peroxidase.…”

“…Thus, biological strategies of oxygen-tolerance may involve (per)oxidase reactions to prevent or detoxify this species. 8,[24][25][26]30,72 In case of the R. eutropha MBH and closely related enzymes, a novel [4Fe3S] cluster has been reported to supply additional electrons to the active site in order to facilitate O 2 detoxification. 8,24−26 The structural basis for O 2 tolerance, however, is presumably different in the SH as no indications for a similar FeS cluster have been observed by Karstens et al 73 Instead, we propose that oxygen is catalytically detoxified by consecutive two-electron reduction steps involving active site sulfenates as central intermediates ( Figure 7).…”

Dissection and engineering of theEscherichia coli formate hydrogenlyase complex

“…It may be difficult to engineer complete O 2 -tolerance into FHL by modification of the HycG subunit alone. This is because HycG already differs considerably from other [NiFe]-hydrogenase small subunits, most notably since it only contains one Fe-S cluster rather than the more usual three [12].…”

“…The molecular basis of hydrogenase O 2 -tolerance has been determined as primarily conferred by a special property of the proximal (and medial) Fe-S clusters within the hydrogenase small subunits [20,21]. A six-cysteine coordination shell allows a stable 4Fe-3S cluster to form, which can change conformation and release two electrons into the [NiFe] active site of the large subunit to reduce O 2 to water [12]. The E. coli FHL Hyd-3 enzyme is a standard hydrogenase and is predicted to bind a single 4Fe-4S cluster coordinated by four cysteines within HycG.…”

“…This special cluster is ligated by six cysteine residues [10], which enable the cluster to release two electrons towards the active site when O 2 attacks, thus reducing O 2 to water [11,12].…”