The maturation factors HoxR and HoxT contribute to oxygen tolerance of membrane-bound [NiFe] hydrogenase in Ralstonia eutropha H16

Abstract

The membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O(2) and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O(2) levels. MBH-driven growth on H(2) is inhibited or retarded at high O(2) partial pressure (pO(2)) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO(2). Lack of hoxR and hoxT can phenotypically be restored by providing O(2)-limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions.

Fig. 1.

MBH gene cluster and model of the MBH maturation pathway. The genes hoxKGZ encode the small and large hydrogenase subunits (blue) and a membrane-integral b-type cytochrome (gray); the hoxMLOQRTV genes (yellow) code for MBH-specific accessory proteins; the hypA1B1F1CDEX genes (green) are responsible for active site assembly (downstream genes involved in the regulation of hydrogenase gene expression are not shown for simplification). The maturation model represents the current stage of our work. Isc and Suf symbolize the general machinery for assembly and insertion of Fe-S clusters (52) and are likely involved in incorporation of the Fe-S centers into HoxK. For further details and references, see the text.

Fig. 2.

MBH-driven lithoautotrophic growth of mutant strains at different O₂ levels. HF388 (SH⁻) mutant strains with in-frame deletions in the MBH-specific accessory genes hoxO (HF537), hoxQ (HF538), hoxR (HF539), hoxT (HF540), hoxV (HF541), and hoxL (HF536) and the small subunit gene hoxK (HF532) were grown lithoautotrophically on mineral agar plates in the presence of 60% (vol/vol) H₂, 10% (vol/vol) CO₂, and various concentrations of O₂.

Fig. 3.

Hydrogenase activity in the membrane fraction of hoxR and hoxT mutant strains grown under different O₂ partial pressures. Cells (HF361, HF363) were grown heterotrophically in a fermentor continuously aerated with air (21% [vol/vol] O₂), with a gas mixture of 10% (vol/vol) O₂ and 90% (vol/vol) N₂, or under O₂-limited conditions in Erlenmeyer flasks. Hydrogenase activity in the membrane fraction was measured by H₂-dependent methylene blue reduction in an anaerobic assay. Activity of the wild-type strain H16 under the respective conditions was taken as 100% (O₂ limited, 14.3 ± 0.3 U/mg; at 10% O₂, 4.5 ± 0.7 U/mg; at 21% O₂, 3.1 ± 0.4 U/mg; the absolute values of the mutant strains are depicted above the bars). One unit is defined as 1 μmol H₂ min⁻¹. Values are averages of results from three independent replicates, and error bars represent standard deviations.

Fig. 4.

Western blot analysis of cell extracts from mutant strains grown at different pO₂. Wild-type (HF388) and mutant strains defective in hoxR (HF539) and hoxT (HF540) were grown heterotrophically under O₂-limited conditions and under a 10% (vol/vol) or a 20% (vol/vol) oxygen atmosphere. Proteins from crude extracts (an equivalent of 100 μl cell culture at an OD₄₃₆ of 2.5 in each lane) were separated on 10% or 12% SDS-polyacrylamide gels and identified with antibodies raised against HoxK and HoxG.

Fig. 5.

Occurrence of HoxT in cells grown under different O₂ concentrations. Cells harboring the sequence for an N-terminal FLAG tag fused genetically to the hoxT gene on pHG1 (HF841) were grown heterotrophically under oxygen concentrations of 20 to 100% (vol/vol) balanced by N₂. Proteins from crude cell extracts (an equivalent of 100 μl cell culture at an OD₄₃₆ of 2.5 in each lane) were separated on 10% or 12% SDS-polyacrylamide gels, and proteins were identified via Western blot analysis with antibodies raised against HoxK, HoxG, and the FLAG tag.

Fig. 6.

Localization of HoxT in different cell fractions. (A) Crude extract (CE), soluble extract (SE), membrane fraction (MF), and periplasmic fraction (PE) were prepared from the wild-type strain R. eutropha H16 producing FLAG-tagged HoxT from pCH1382. Membrane fraction was washed with 50 mM KPO₄ buffer (pH 7) or alternatively with 100 mM Na₂CO₃ buffer (pH 11). (B) Crude extract, soluble extract, and membrane fraction (washed with 50 mM KPO₄ buffer, pH 7) were prepared from the hydrogenase-free strain R. eutropha HF210 carrying pCH1382. Proteins were separated on a 15% SDS-polyacrylamide gel (25 μg protein per lane) and examined by Western blot analysis with FLAG tag antibody.

Fig. 7.

Copurification experiments with StrepTag II-tagged versions of HoxR and HoxT as baits. HoxR_Strep and HoxT_Strep were enriched from soluble extracts of MBH-overproducing strains (HF757 and HF758) by Strep-Tactin affinity chromatography. In a control experiment, the same purification procedure was done with extracts from R. eutropha HF632 that encodes wild-type HoxR and HoxT. Eluates (2.5 μg of protein) were separated on 10% (for the detection of HoxG) or 15% (for the detection of HoxT, HoxR, HoxK, HoxO, HoxQ, HoxV, HypC, and HypD) SDS-polyacrylamide gels. MBH-related proteins were identified by Western blot analysis with antibodies raised against StrepTag II, HoxK, and HoxG (A) and HoxO, HoxQ, HoxV, HypC, and HypD (B).

Fig. 8.

UV-visible absorption spectroscopy of MBH^WT and MBH^ΔhoxR. MBH was purified from K₃[Fe(CN)₆]-oxidized membranes obtained from the MBH-overproducing strains HF649 (MBH^WT) and HF851 (MBH^ΔhoxR) via affinity chromatography (see Materials and Methods). MBH samples (43 μM, A₂₈₀ = 7.5, pH 5.5) were measured in the as-isolated, oxidized state (ox) and after reduction (15 min) with 100% H₂ (red). (A) Absorption spectra of as-isolated and H₂-reduced MBH proteins. The inset shows difference spectra calculated from oxidized-minus-reduced (ox − red) spectra of MBH^WT and MBH^ΔhoxR. (B) In order to visualize the differences between MBH^WT and MBH^ΔhoxR, double difference spectra were prepared. Wavelengths of relevant absorption maxima or bleachings are marked with a triangle.