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Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase
Zitatschlüssel ISI:000332761500062
Autor Fritsch, Johannes and Siebert, Elisabeth and Priebe, Jacqueline and Zebger, Ingo and Lendzian, Friedhelm and Teutloff, Christian and Friedrich, Baerbel and Lenz, Oliver
Seiten 7982-7993
Jahr 2014
ISSN 0021-9258
DOI 10.1074/jbc.M113.544668
Adresse 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA
Journal JOURNAL OF BIOLOGICAL CHEMISTRY
Jahrgang 289
Nummer 11
Monat MAR 14
Verlag AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Zusammenfassung Background: Biosynthesis of complex metal cofactors in [NiFe] hydrogenase is sensitive toward molecular oxygen. Results: A rubredoxin-like protein is required for hydrogenase maturation under aerobic conditions. Conclusion: The rubredoxin-like protein prevents oxidative damage of metallocenters, including the recently discovered [4Fe3S] center. Significance: Dedicated protection mechanisms enable biosynthesis of sophisticated metal centers in the presence of dioxygen. The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H-2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O-2 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 O-2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Ni-u-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 O-2. 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 Cys(6)[4Fe-3S] cluster.
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