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 Cys6[4Fe-3S] cluster.
Highlights
Biosynthesis of complex metal cofactors in [NiFe] hydrogenase is sensitive toward molecular oxygen
Catalytic Features of membrane-bound [NiFe] hydrogenase (MBH) Protein Purified from hoxR Mutant Cells—Heterodimeric MBH protein was solubilized and purified from ferricyanide-oxidized membranes prepared from wild-type (MBHWT) and ⌬hoxR mutant (MBH⌬hoxR) cells, which were cultivated either under well aerated or O2-limited conditions
No significant increase of MBH activity was visible for both the MBHWT and MBH⌬hoxR proteins from O2-deprived cells (Fig. 2A), and only a moderate increase of 25% was observed for the MBHWT protein isolated from the well aerated cells
Summary
Biosynthesis of complex metal cofactors in [NiFe] hydrogenase is sensitive toward molecular oxygen. In contrast to the MBH from R. eutropha, structurally related O2-sensitive “standard” [NiFe] hydrogenases, which have been initially characterized in sulfate-reducing bacteria, are rapidly inactivated in the presence of O2 giving rise to a mixture of inactive oxidized states of the catalytic site [11, 12]. Two of these states, denoted Niu-A and Nir-B, can be detected by electron paramagnetic resonance (EPR) spectroscopy.
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