Sven T. Stripp

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain—a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.

The [FeFe]‐hydrogenase maturation protein HydF contains a H‐cluster like [4Fe4S]–2Fe site

Formation of the catalytic six‐iron complex (H‐cluster) of [FeFe]‐hydrogenase (HydA) requires its interaction with a specific maturation protein, HydF. Comparison by X‐ray absorption spectroscopy at the Fe K‐edge of HydF from Clostridium acetobutylicum and HydA1 from Chlamydomonas reinhardtii revealed that the overall structure of the iron site in both proteins is highly similar, comprising a [4Fe4S] cluster (Fe–Fe distances of ∼2.7 Å) and a di‐iron unit (Fe–Fe distance of ∼2.5 Å). Thus, a precursor of the whole H‐cluster is assembled on HydF. Formation of the core structures of both the 4Fe and 2Fe units may require only the housekeeping [FeS] cluster assembly machinery of the cell. Presumably, only the 2Fe cluster is transferred from HydF to HydA1, thereby forming the active site.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) to probe monolayers of membrane proteins.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) represents a variation of conventional infrared spectroscopy and exploits the signal enhancement exerted by the plasmon resonance of nano-structured metal thin films. The surface enhancement decays in about 10nm with the distance from the surface and is, thus, perfectly suited to selectively probe monolayers of biomembranes. Peculiar to membrane proteins is their vectorial functionality, the probing of which requires proper orientation within the membrane. To this end, the metal surface used in SEIRAS is chemically modified to generate an oriented membrane protein film. Monolayers of uniformly oriented membrane proteins are formed by tethering His-tagged proteins to a nickel nitrilo-triacetic acid (Ni-NTA) modified gold surface and SEIRAS commands molecular sensitivity to probe each step of surface modification. The solid surface used as plasmonic substrate for SEIRAS, can also be employed as an electrode to investigate systems where electron transfer reactions are relevant, like e.g. cytochrome c oxidase or plant-type photosystems. Furthermore, the interaction of these membrane proteins with water-soluble proteins, like cytochrome c or hydrogenase, is studied on the molecular level by SEIRAS. The impact of the membrane potential on protein functionality is verified by monitoring light-dark difference spectra of a monolayer of sensory rhodopsin (SRII) at different applied potentials. It is demonstrated that the interpretations of all of these experiments critically depend on the orientation of the solid-supported membrane protein. Finally, future directions of SEIRAS including cellular systems are discussed. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Multiple ferredoxin isoforms in Chlamydomonas reinhardtii – Their role under stress conditions and biotechnological implications

The unicellular green alga Chlamydomonas reinhardtii has at least six plant-type ferredoxins (FDX). Besides the long-known photosynthetic ferredoxin PetF the isoforms Fdx2-Fdx6 have been identified. The FDX genes are differentially expressed under various environmental conditions such as the availability of oxygen, copper, iron and ammonium. Recently, the anaerobically induced Fdx5 as well as Fdx2, which is involved in nitrite reduction were characterized in more detail. Moreover, it was shown that PetF, the central and most abundant FDX of C. reinhardtii, is a suitable partner of the hydrogenase HydA1. Using mutant variants of both PetF and HydA1, amino acid residues essential for the interaction of both proteins could be identified. These findings will help to tailor PetF for achieving an optimized photobiotechnological hydrogen production in C. reinhardtii, which might also benefit from new insights into the mechanism of how oxygen attacks the active site metal cluster of HydA1. This review gives an update on recent advances in understanding the function of ferredoxins and the hydrogenase in C. reinhardtii.

Immobilization of the (FeFe)-hydrogenase CrHydA1 on a gold electrode: Design of a catalytic surface for the production of molecular hydrogen

Hydrogenase-modified electrodes are a promising catalytic surface for the electrolysis of water with an overpotential close to zero. The [FeFe]-hydrogenase CrHydA1 from the photosynthetic green alga Chlamydomonas reinhardtii is the smallest [FeFe]-hydrogenase known and exhibits an extraordinary high hydrogen evolution activity. For the first time, we immobilized CrHydA1 on a gold surface which was modified by different carboxy-terminated self-assembled monolayers. The immobilization was in situ monitored by surface-enhanced infrared spectroscopy. In the presence of the electron mediator methyl viologen the electron transfer from the electrode to the hydrogenase was detected by cyclic voltammetry. The hydrogen evolution potential (-290 mV vs NHE, pH 6.8) of this protein modified electrode is close to the value for bare platinum (-270 mV vs NHE). The surface coverage by CrHydA1 was determined to 2.25 ng mm(-2) by surface plasmon resonance, which is consistent with the formation of a protein monolayer. Hydrogen evolution was quantified by gas chromatography and the specific hydrogen evolution activity of surface-bound CrHydA1 was calculated to 1.3 micromol H(2)min(-1)mg(-1) (or 85 mol H(2)min(-1)mol(-1)). In conclusion, a viable hydrogen-evolving surface was developed that may be employed in combination with immobilized photosystems to provide a platform for hydrogen production from water and solar energy with enzymes as catalysts.

HypD is the scaffold protein for Fe-(CN)2CO cofactor assembly in [NiFe]-hydrogenase maturation.

[NiFe]-hydrogenases bind a NiFe-(CN)2CO cofactor in their catalytic large subunit. The iron-sulfur protein HypD and the small accessory protein HypC play a central role in the generation of the CO and CN(-) ligands. Infrared spectroscopy identified signatures on an anaerobically isolated HypCD complex that are reminiscent of those in the hydrogenase active site, suggesting that this complex is the assembly site of the Fe-(CN)2CO moiety of the cofactor prior to its transfer to the hydrogenase large subunit. Here, we report that HypD isolated in the absence of HypC shows infrared bands at 1956 cm(-1), 2072 cm(-1), and 2092 cm(-1) that can be assigned to CO, CN(1), and CN(2), respectively, and which are indistinguishable from those observed for the HypCD complex. HypC could not be isolated with CO or CN(-) ligand contribution. Treatment of HypD with EDTA led to the concomitant loss of Fe and the CO and CN(-) signatures, while oxidation by H2O2 resulted in a positive shift of the CO and CN(-) bands by 35 cm(-1) and 20 cm(-1), respectively, indicative of the ferrous iron as an immediate ligation site for the diatomic ligands. Analysis of HypD amino acid variants identified cysteines 41, 69, and 72 to be essential for maturation of the cofactor. We propose a refined model for the ligation of Fe-(CN)2CO to HypD and the role of HypC in [NiFe]-hydrogenase maturation.

[NiFe]-hydrogenase maturation: Isolation of a HypC–HypD complex carrying diatomic CO and CN− ligands

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)2CO cofactor in the large subunit of [NiFe]‐hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep‐tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN− ligands as well as CO2. Metal and sulphide analyses revealed that along with the [4Fe–4S]2+ cluster in HypD, the complex has two additional oxygen‐labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN)2CO cofactor.

Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

Significance [FeFe]-hydrogenases are H2-forming enzymes with potential in renewable energy applications. Their molecular mechanism of catalysis needs to be understood. A protocol for specific 13CO isotope editing of all carbon monoxide ligands at the six-iron cofactor (H-cluster) was established. Analysis of vibrational modes via quantum chemical calculations implies structural dynamics at the H-cluster in the active-ready state. Site-selective introduction of isotopic reporter groups opens new perspectives to identify intermediates in the catalytic cycle. The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN−) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of 13CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN− at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.

The [NiFe]‐hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron‐ and carbon dioxide‐binding proteins

HybG and HybG bind by comigration in sds page (View interaction)