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Hydrogenases are essential for H₂ cycling in microbial metabolism and serve as valuable blueprints for H₂-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O₂. The O₂-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H₂ uptake in the presence of ambient O₂. Here we show that O₂ tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O₂ as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism …