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 of O2 tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O2 to the active site.
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The authors wish to thank J. Priebe for obtaining preliminary EPR results, J. Hamann and A. Strack for excellent technical assistance and P. Hildebrandt and R. Bittl for generous support. This work was supported by the German Federal ministry of Education and Research (T.G.; BMBF project “H2 Design Cells”), the Deutsche Forschungsgemeinschaft (M. Saggu, N.H., I.Z., B.F., O.L.; Cluster of Excellence “UniCat”), the FP7 of the European Union (J.F.; energy network project SOLAR-H2), the Klaus Tschira Foundation and the Max Planck Society for the Advancement of Science (M. Stein), and the Engineering and Physical Sciences Research Council UK (A.F.W., F.A.A; Grant Supergen 5) and the Biotechnology and Biological Sciences Research Council UK (A.F.W., F.A.A; Grant BB/H003878/1).
The authors declare no competing financial interests.
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Goris, T., Wait, A., Saggu, M. et al. A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase. Nat Chem Biol 7, 310–318 (2011). https://doi.org/10.1038/nchembio.555