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EPR spectroscopy of oxygen-tolerant [NiFe]-hydrogenases
| Content Provider | Semantic Scholar |
|---|---|
| Author | Saggu, Miguel |
| Copyright Year | 2011 |
| Abstract | Hydrogenases are metalloenzymes and play a pivotal role in the energy metabolism of a variety of microorganisms. They catalyze the reversible cleavage of molecular hydrogen into two protons and two electrons. However, one drawback is their catalytic inactivation upon exposure to oxygen. [FeFe]-hydrogenases are irreversibly inactivated by oxygen, whereas [NiFe]-hydrogenases usually are reversibly inactivated to the so-called ’ready’ and ’unready’ states. Only a few [NiFe]hydrogenases show a remarkable oxygen-tolerance, e.g. the [NiFe]-hydrogenases from the ’Knallgasbacterium’ Ralstonia eutropha H16 investigated in this work. Re H16 harbors three different [NiFe]-hydrogenases, all of them being able to oxidize molecular hydrogen under ambient oxygen concentrations. The goal of this work was to understand the origin of this oxygen-tolerance on a molecular level and to study structure/function-relationships of the involved cofactors using a combination of EPR and FTIR spectroscopy. EPR spectroscopy can be applied to detect all paramagnetic species and to obtain information about their electronic and in its more advanced variant the geometric structure, respectively. FTIR spectroscopy applied to hydrogenases specifically monitors the stretching modes of the inorganic ligands at the Fe to follow redox changes of the [NiFe] center. An important aspect of this thesis was to study biological systems in their native environment, e.g. in whole cells or membrane fragments. The membrane-bound [NiFe]-hydrogenase (MBH) from Re H16 was studied mainly in its native environment, i.e. in membrane fragments that harbor over-produced MBH. All catalytically active redox states of the [NiFe] center were identified. However, the ’unready’ Niu-A, observed in standard [NiFe]-hydrogenases could not be detected, which seems to be a common feature of all oxygen-tolerant [NiFe]-hydrogenases. It was shown, that the MBH contains an additional highpotential paramagnetic center. Concomitant electrochemical experiments revealed that the MBH can be reactivated very fast at high potentials, which might indicate an important role of the additional high-potential center. This center could influence the catalytic cycle by providing electrons to avoid Niu-A formation. The hyperfine structure of the Nir-B state (oxidized enzyme) was investigated in more detail by pulsed EPR methods, namely ENDOR and ESEEM spectroscopy. The results were compared with the standard [NiFe]-hydrogenase from D. vulgaris Miyazaki F. Both hydrogenases showed a similar spin density distribution in their [NiFe] center indicating that the spatial structures of the active sites are identical. Two large hf-couplings arising from the β -protons of one bridging cysteine sulfur were resolved and simulated in orientation-selective ENDOR spectra. With ESEEM spectroscopy a nitrogen located in a histidine residue in the second coordination sphere of the Ni was detected, which is a known property of many [NiFe]-hydrogenases. This histidine is hydrogen-bonded to the same bridging sulfur. Another interesting feature of oxygen-tolerant membrane-bound [NiFe]-hydrogenases is the presence of two additional cysteine residues in the vicinity of the proximal [4Fe4S]-cluster. Muta- |
| File Format | PDF HTM / HTML |
| DOI | 10.14279/depositonce-2736 |
| Alternate Webpage(s) | https://depositonce.tu-berlin.de/bitstream/11303/3033/1/Dokument_54.pdf |
| Alternate Webpage(s) | https://doi.org/10.14279/depositonce-2736 |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
