[NiFe] hydrogenases are important metalloenzymes that carry out the reversible oxidation of dihydrogen. Understanding their mechanism is crucial for the synthesis of biomimetic catalytic systems and future ‘green’ biotechnology. The present work is focused on the study of two distinct types of enzymes; a [NiFe] hydrogenase from the strictly anaerobic sulphate reducing bacterium Desulfovibrio vulgaris Miyazaki F and a [NiFe] hydrogenase from the micro-aerophilic hyperthermophilic bacterium Aquifex aeolicus.
The first part is centred on the study of the [NiFe] enzyme from D. vulgaris, for which the intermediates of the reaction cycle were studied in detail by infrared techniques. Using SEIRA spectroscopy the catalytic activity of this enzyme adsorbed on an electrode was monitored, yielding essentially the same results as FTIR spectroelectrochemistry in solution. The EPR silent inactive state (Ni-SIr)I was shown to be light sensitive and a new state Ni-SL could be detected. Wavelength dependence and time resolved kinetics showed that the light sensitivity corresponds to the dislocation of the hydroxyl ligand present in the active site of (Ni-SIr)I. For the transition from the light-induced Ni-L state to Ni-C a large (H/D) kinetic isotope effect was obtained, showing that the rate limiting step is the binding of a hydron at the active site. Using FTIR spectroelectrochemistry and EPR, the inhibition of the enzyme by carbon monoxide was studied and formation of two CO-adducts demonstrated. Time resolved FTIR studies at low temperatures used the reversible photolysis of the external CO ligand to estimate the activation barrier for its rebinding at the active site.
The second part of this study comprises an in-depth investigation of the Hydrogenase I from Aquifex aeolicus. Chronoamperometric electrochemical measurements provided evidence for the oxygen tolerance of this hydrogenase. FTIR electrochemistry in solution detected only four redox intermediate states with significantly more positive midpoint potentials than those measured for standard hydrogenases. Carbon monoxide was shown to bind to the active site of Hydrogenase I much weaker than to that of D. vulgaris. By an EPR redox titration the types and midpoint potentials of all iron-sulphur centres were determined. Based on these results a model for the oxygen tolerance of Hydrogenase I is proposed The study is concluded by examining the interaction of the active site with the substrate in the catalytically active Ni-C state, in which a weakly bound hydride was found.
|School:||Technische Universitaet Berlin (Germany)|
|Source:||DAI-C 81/4(E), Dissertation Abstracts International|
|Subjects:||Inorganic chemistry, Physical chemistry|
|Keywords:||Aquifex aeolicus, Desulfovibrio vulgaris, FTIR|
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