Abstract
The hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the [NiFe]-hydrogenases in the bacterium. A deletion of the hypX gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble NAD+ -reducing hydrogenase. The enzymatic and infrared spectral properties of the soluble hydrogenase purified from a HypX-negative strain were compared with those from an enzyme purified from a HypX-positive strain. In activity assays under anaerobic conditions both enzyme preparations behaved the same. Under aerobic conditions, however, the mutant enzyme became irreversibly inactivated during H2 oxidation with NAD+ or benzyl viologen as the electron acceptor. Infrared spectra and chemical determination of cyanide showed that one of the four cyanide groups in the wild-type enzyme was missing in the mutant enzyme. The data are consistent with the proposal that the HypX protein is specifically involved in the biosynthetic pathway that delivers the nickel-bound cyanide. The data support the proposal that this cyanide is crucial for the enzyme to function under aerobic conditions.
Highlights
The hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes required for the proper assembly of the active site of the [NiFe]-hydrogenases in the bacterium
Structural data and spectroscopic properties indicate that these hydrogenases are phylogenetically unrelated, they posses some remarkable similarities in the molecular architecture of the active site, namely the presence of cyanide and/or carbon monoxide as ligands to the metals. [FeFe]-hydrogenases are restricted to anaerobic bacteria and lower eukaryotes, whereas [NiFe]-hydrogenases are the dominant hydrogenases in archaea and bacteria, including aerobic organisms
The present study describes that hypX-mutant cells of R. eutropha showed a retarded growth on H2 under standard aerobic conditions, demonstrating that the oxygen tolerance of the SH requires the function of HypX
Summary
Construction of R. eutropha HF480--The mobilizable plasmid pCH630 containing hypX∆ was transferred from E. coli S17-1 to R. eutropha HF359 (hoxG∆) by a spot mating technique [34]. The subsequent protocol was modified as follows: fractions with high NAD+-reducing activity derived from the DEAE column were pooled and concentrated by (NH4)2SO4 precipitation (60% saturation). NADH (5 μM) was added to activate the enzyme, followed by either BV (2.5 mM) or NAD+ (5.0 mM). When anaerobic conditions were required, all solutions were flushed with Ar before use and glucose (50 mM) plus glucose oxidase (9 U/ml) were added to the reaction medium 3 min before the NADH addition. The spin concentration calculated from the EPR signal of the [2Fe-2S]1+ cluster in reduced enzyme (1 bar H2 for 45 min at 30 oC) was used as a measure for the enzyme concentration [42]
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