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Impact of Amino Acid Substitutions near the Catalytic Site on the Spectral Properties of an O-2-Tolerant Membrane-Bound [NiFe] Hydrogenase
Citation key ISI:000277666900013
Author Saggu, Miguel and Ludwig, Marcus and Friedrich, Baerbel and Hildebrandt, Peter and Bittl, Robert and Lendzian, Friedhelm and Lenz, Oliver and Zebger, Ingo
Pages 1215-1224
Year 2010
ISSN 1439-4235
DOI 10.1002/cphc.200900988
Address PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY
Journal Chem. Phys. Chem.
Volume 11
Number 6
Month APR 26
Abstract [NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H-2 cycling in the presence of O-2. These enzymes are of special interest for biotechnological applications. To gain further insight into the mechanism(s) responsible for the remarkable O-2 tolerance, we employ FTIR and EPR spectroscopy to study mutant variants of the membrane-bound hydrogenase (MBH) of Re-carrying substitutions of a particular cysteine residue in the vicinity of the [NiFe] active site that is characteristic of O-2-tolerant membrane-bound [NiFe] hydrogenases. We demonstrate that these MBH variants, despite minor changes in the electronic structure and in the interaction behavior with the embedding protein matrix, display all relevant catalytic and noncatalytic states of the wild-type enzyme, as long as they are still located in the cytoplasmic membrane. Notably, in the oxidized Ni-r-B state and the fully reduced forms, the CO stretching frequency increases with increasing polarity of the respective amino acid residue at the specific position of the cysteine residue. We purified the MBH mutant protein with a cysteine-to-alanine exchange to apparent homogeneity as dimeric enzyme after detergent solubilization from the membrane. This purified version displays increased oxygen sensitivity, which is reflected by detection of the oxygen-inhibited Nib-A state, an irreversible inactive redox state, and the light-induced Ni-a-L state even at room temperature.
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