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

Abstract

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 ...

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

“…This observation is consistent with the proposition (20,21) that the two oxidation processes associated with the proximal cluster of these enzymes may be assigned to 3FeðIIÞ-1FeðIIIÞ → 2FeðIIÞ-2FeðIIIÞ and 2FeðIIÞ-2FeðIIIÞ → 1FeðIIÞ-3FeðIIIÞ changes. Remarkably, the two redox couples lie within a narrow potential range when compared to that observed for the high-potential iron-sulfur protein (HiPIP), which is about 1 V and involves nonphysiological superreduction of the [4Fe-4S] cluster to the +1 oxidation level (22,23).…”

“…As reported for the O 2 -tolerant Aquifex aeolicus hydrogenase 1 (Aa-Hase 1), the proximal cluster undergoes two one-electron redox processes, and the higher potential redox transition is pH-independent between pHs 6.4 and 7.4 (20). Further adaptations for oxygen tolerance are a much lower K m for H 2 than the K i for O 2 (21), and the more positive potentials of all metal centers relative to O 2 -sensitive [NiFe]-hydrogenases.…”

“…Our corresponding structures agree well with those reported by these authors but also provide further insights into the molecular principles for O 2 tolerance. Using our EcHyd-1 crystal structures, we have (i) identified a glutamic acid carboxylate as the base involved in amide-N deprotonation and (ii) calculated and reproduced previously published Mössbauer and EPR data (20), allowing us to describe the electronic and conformational changes that take place during the superoxidation of the proximal cluster.…”

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).…”

“…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)(2)(3). These enzymes are localized at the cytoplasmic membrane and project into the periplasmic space.…”