Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H 2 into protons and electrons or the evolution of H 2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H 2 -sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to

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... tigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application. 1 The abbreviations used are: MBH, membrane-bound hydrogenase; SH, soluble hydrogenase; RH, H 2 -sensing regulatory hydrogenase; ST, Strep-tag.