Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase |
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Authors: | Johannes Fritsch Elisabeth Siebert Jacqueline Priebe Ingo Zebger Friedhelm Lendzian Christian Teutloff B?rbel Friedrich Oliver Lenz |
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Institution: | From the ‡Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin.;the §Institut für Chemie, Max-Volmer-Laboratorium, Technische Universität Berlin, Strasse des 17 Juni 135, 10623 Berlin, and ;the ¶Institut für Physik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany |
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Abstract: | The membrane-bound NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys64Fe-3S] cluster. |
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Keywords: | Bacterial Metabolism Bioenergetics/Electron Transfer Complex Biophysics Electron Paramagnetic Resonance (EPR) Electron Transfer Energy Metabolism Enzyme Catalysis Membrane Enzymes Metalloproteins Protein Chemistry |
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