The need for an environmentally friendly, highly efficient energy source has brought hydrogenases into the center of attention in enzyme research. Hydrogenases catalyze the reversible cleavage of molecular hydrogen into two protons and two electrons and could therefore become a key component of future biological fuel cells. In this thesis, the oxygen-tolerant, membrane bound [Nife] hydrogenase (MBH) from Ralstonia eutropha is investigated by means of Infrared (IR) and Resonance Raman (RR) spectroscopy. This enzyme is of particular technological interest, because it preserves catalytic activity even under ambient oxygen concentrations. With IR spectroscopy, redox transitions of the [NiFe] active site were studied as a function of externally adjusted potentials. Thus, it was possible to make the interconversion of the active site redox states within the catalytic cycle visible. The results demonstrate that the reoxidation process is only reversible if the MBH is still attached to its natural electron acceptor cytochrome b in membrane fragments, as it is in its natural environment. The entire reoxidation process was successfully modeled with a set of coupled mono-exponential functions, confirming that the underlying processes are one-electron transitions. The solubilized MBH is not capable of reacting in a fully reversible fashion with hydrogen and oxygen, since the formation of the irreversible inactive species is also possible during the reoxidation process once the natural electron acceptor is lost. Comparative studies revealed that major fractions of such MBH samples can be reactivated in the presence of redox mediators and sufficiently high overpotentials. Additionally, IR component spectra involving the CO and CN stretching modes were determined for all catalytically active and inactive redox species of the [NiFe] active site. With the present work RR spectroscopy is established as a powerful method complementary to IR and EPR (Electron Paramagnetic Resonance) spectroscopy to investigate the various cofactors of the MBH, i.e. the [NiFe] active site and the FeS clusters. A consistent assignment of the respective vibrational modes in the region from 300 to 700cm-1 was achieved by wavelength- and angle-dependent RR studies, statistical analyses on experimental RR spectra as well as isotopic labeling and quantum chemical calculations by Y. Rippers. The detected vibrational modes of the active site were assigned to Fe-CO/CN modes of the redox states Nia-S and Nia-L. These two active species are formed via light-induced processes in the RR experiment. Due to this photo-induced reactivation of the active site the 'potential window of activity' for H2-oxidation in a fuel cell might be broadened and thus the performance of the biofuel cell increased. The most intense FeS stretching modes of all three FeS clusters were discriminated with angle-dependent RR spectroscopy and RR spectra from samples of MBH variants with genetically engineered FeS clusters. This also yields the first vibrational spectroscopic description of the superoxidized proximal cluster. This proximal [4Fe3S] cluster is unique in oxygen-tolerant membrane bound hydrogenases and is thought to play a key role for protecting the active site from oxidative damage. RR spectroscopy revealed that the native configuration of the superoxidized cluster has a bound hydroxyl group at one Fe ion. This hydroxyl group is rebound in high amounts within the reoxidation process of the MBH. RR spectra of isotopically labeled MBH samples demonstrated that the hydroxyl ligand at the superoxidized proximal cluster is preferentially formed from molecular oxygen, which is reduced in a four-electron three-proton process. Solvent water molecules in the vicinity of the proximal cluster are an alternative source for the ligand, but this binding process is much slower. These mechanistic and structural insights even exceed the information derived from crystallographic data. The advanced understanding of the experimental RR spectra of the MBH from Ralstonia eutropha will be the basis for future investigations of the wildtype enzyme and specific genetically engineered MBH variants with modifications at the [NiFe] active site or the FeS clusters. Further aspects of the catalytic cycle may be elucidated with systematically varied experimental conditions as well.
|School:||Technische Universitaet Berlin (Germany)|
|Source:||DAI-C 81/1(E), Dissertation Abstracts International|
|Subjects:||Molecular chemistry, Biochemistry|
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