Spectroscopic studies on hydrogenases

The establishment of sustainable economy may be based on molecular hydrogen as an energy-storage molecule and requires the development of cheap catalysts, which are able to work under various conditions. In nature, this most fundamental reaction, i.e. the reversible reduction of protons, is catalysed by a group of enzymes called hydrogenases. Members of this wide family of enzymes exhibit various properties, such as different activity rates, bias towards a specific direction of the catalytic reaction and a potential tolerance towards oxygen. Vibrational spectroscopy can be used as a tool to clarify molecular mechanism related to the catalytic cycle and reactions with other small molecules (e.g. CO and O 2 ). Such insights could provide a blueprint for the synthesis of molecular catalysts for future applications. In the first part of this thesis, resonance Raman (RR) spectroscopy was applied for the first time on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (HydA1), the smallest catalytic unit of this class of hydrogenase, revealing thereby the first catalytic intermediate in the proton reduction reaction. RR spectroscopy was initially used to characterise the in vitro maturation process of this enzyme on the basis of molecular vibrations related to the Fe-S, Fe-CO or Fe-CN bonds located in its active site, which consists of a [FeFe] centre covalently linked to a [4Fe4S] cluster. A comparison of the spectra of redox-tuned samples of artificially maturated holo-HydA1 revealed a photo-induced intramolecular electron transfer occurring between the two metal centres of the active site. Further experiments with a non-native artificial maturated HydA1 could demonstrate that this electron transfer is coupled with a proton transfer to the secondary amine of the aza-thiolate head group also found in the active site of the native enzyme. Following this hypothesis, the stabilisation of newly discovered H red ’ state, characterised by an oxidised [FeFe] centre and a reduced [4Fe4S] cluster, is related to a charge compensation mechanism. Infrared (IR) spectroscopy is a well-established method to study hydrogenases and can discriminate different redox states of these enzymes with respect to the particular band positions of their CO and CN stretching vibrations. Therefore, this technique was used for control measurements both, at cryogenic and at room temperatures to mimic the conditions of the RR experiments and to validate the procedures for redox tuning, respectively. Within these control experiments, a second light-induced reaction was identified. With help of adequate difference spectra, a set of IR bands could be assigned to a new H sred ’ state, which is probably associated to the final step of the catalytic reaction before a H 2 molecule is released from the active site. This study demonstrates the potential of cryogenic vibrational spectroscopy to probe transient intermediates by kinetically trapping thermodynamically instable states. Contrary to most [FeFe]-hydrogenases, the active site of [NiFe]-hydrogenases does not decompose following an exposure to oxygen. However, so called anaerobic “standard” hydrogenases form the unready Ni u -A state, which requires chemical reduction or very long incubation times with hydrogen to achieve full reactivation. On the other hand, oxygen- tolerant enzymes, such as the membrane-bound hydrogenase (MBH) from Ralstonia eutropha (Re), form only the ready Ni r -B state, which is reactivated after a short lag-time following an exposure to hydrogen. These long and short lag-times are the result of “autocatalytic” intermolecular activation mechanisms proposed for [NiFe] hydrogenases. The second part of the thesis comprises three studies on the ReMBH. Unlike the anaerobic “standard” [NiFe]-hydrogenases, which harbour a cubane-like [4Fe4S] cluster in the proximity of the [NiFe] active site, in oxygen tolerant MBH’s, such as ReMBH, two conserved cysteines in the first coordination sphere of this cofactor lead to the formation of an unusual [4Fe3S]-proximal cluster. This unique cluster is capable to perform two redox transitions, which were previously assumed to prevent the formation of unready states. Two variants of ReMBH were generated, in which one of the two cysteine residues (i.e. cysteine 19 or 120) was replaced by a glycine. A combined RR, IR and electron paramagnetic resonance (EPR) spectroscopic characterisation of redox-tuned samples could show that the cluster of both ReMBH variants (i.e. C19G and C120G) could undergo only a single redox transition, corresponding to one of the respective transitions observed in the wildtype. However, no unready states were detected for both variants after exposure to oxygen. Moreover, in pure electrochemical experiments it was shown that both variants exhibit some catalytic activity under aerobic condition. Using surface enhanced infrared absorption (SEIRA) spectroscopy, structural data was obtained to compliment the electrochemical measurements. This spectro- electrochemical approach could show no qualitative difference in terms of redox states composition of the active site, between the two variants and the wildtype after an exposure to oxygen under catalytic conditions. These results suggest that the additional redox transition of the modified proximal cluster of the wildtype plays mainly a regulating role in the kinetic of the oxygen-detoxification mechanism and that the formation of the unready states in ReMBH is prevented by other modifications, presumably near the active site. A study on another variant of ReMBH, namely the D117S variant, with a modified active site environment demonstrates the role of proton transfer as a charge compensation mechanism, which occurs during the activation process of ReMBH’s. Moreover, this study could provide the first spectroscopic evidence for an “autocatalytic” reactivation mechanism in these enzymes. In a third study, a redox titration of the artificially cross-linked heterotrimer of ReMBH as well as IR and EPR spectroscopic characterisation of membrane samples enriched with wildtype and variants of ReMBH could elucidate the role of the native configuration as a super-complex of heterotrimers in de- and reactivation processes. Thereby, it was shown that full reoxidation of the proximal cluster as well as the prevention of the formation of some inactive states of the active site could occur only in the native environment, presumably due to an intact electron relay of ReMBH. These studies contributed to an identification of structural and redox arrangements in ReMBH, which tightly regulate the chemistry occurring at the active site, thereby enabling the oxidation of molecular hydrogen in the presence of oxygen.

Author dc:creator

• Katz, Sagie

Advisor dc:contributor.advisor

• Hildebrandt, Peter

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.14279/depositonce-8312

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/9232