Thorsten Buhrke

Oxygen Tolerance of the H2-sensing [NiFe] Hydrogenase from Ralstonia eutropha H16 Is Based on Limited Access of Oxygen to the Active Site

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.

[NiFe]-Hydrogenases of Ralstonia eutropha H16: Modular Enzymes for Oxygen-Tolerant Biological Hydrogen Oxidation

Recent research on hydrogenases has been notably motivated by a desire to utilize these remarkable hydrogen oxidation catalysts in biotechnological applications. Progress in the development of such applications is substantially hindered by the oxygen sensitivity of the majority of hydrogenases. This problem tends to inspire the study of organisms such as Ralstonia eutropha H16 that produce oxygen-tolerant [NiFe]-hydrogenases. R. eutropha H16 serves as an excellent model system in that it produces three distinct [NiFe]-hydrogenases that each serve unique physiological roles: a membrane-bound hydrogenase (MBH) coupled to the respiratory chain, a cytoplasmic, soluble hydrogenase (SH) able to generate reducing equivalents by reducing NAD+ at the expense of hydrogen, and a regulatory hydrogenase (RH) which acts in a signal transduction cascade to control hydrogenase gene transcription. This review will present recent results regarding the biosynthesis, regulation, structure, activity, and spectroscopy of these enzymes. This information will be discussed in light of the question how do organisms adapt the prototypical [NiFe]-hydrogenase system to function in the presence of oxygen.

Duplication of hyp genes involved in maturation of [NiFe] hydrogenases in Alcaligenes eutrophus H16

Abstract  Alcaligenes eutrophus H16 harbors seven hyp genes (hypA, B, F, C, D, E, and X) as part of the hydrogenase gene cluster on megaplasmid pHG1. Here we demonstrate that three of the hyp genes (hypA, B, and F) are duplicated in A. eutrophus, which explains the lack of a phenotypic change in single-site mutants impaired in one of the two copies. Mutants with lesions in both copies showed clear alterations in hydrogenase activities. Deletions in hypF1 and hypF2 completely abolished activities of the soluble hydrogenase and of the membrane-bound hydrogenase, mutations in hypA1 and hypA2 totally blocked the membrane-bound hydrogenase activity, while residual soluble hydrogenase activity accounted for the extremely slow growth of the strain on H2. Both hydrogenase activities of mutants defective in hypB1 and hypB2 were partially restored by elevating the concentration of nickel chloride in the medium. Reduction of hydrogenase activities in the double mutants correlated with varying degrees of maturation deficiency based upon the amount of unprocessed nickel-free hydrogenase precursor. Despite a high identity between the two copies of hyp gene products, substantial structural differences were identified between the two copies of hypF genes. HypF1, although functionally active, is a truncated version of HypF2, whose structure resembles HypF proteins of other organisms. Interestingly, the N-terminus of HypF2, which is missing in the HypF1 counterpart, contains a putative acylphosphatase domain in addition to a potential metal binding site.

The H2-sensing complex of Ralstonia eutropha: interaction between a regulatory [NiFe] hydrogenase and a histidine protein kinase

Two [NiFe] hydrogenases enable the proteobacterium Ralstonia eutropha H16 to grow on molecular hydrogen as the sole energy source. A third [NiFe] hydrogenase (RH) acts as an H2 sensor in a multiple component signal transduction chain that controls hydrogenase gene transcription. The RH forms a dimeric heterodimer (HoxBC)2 in which HoxC contains the H2‐sensing active site and HoxB the electron‐transferring components including an organic, not yet identified redox cofactor. This oligomer forms a tight complex with the histidine protein kinase HoxJ. Both the sensor and the kinase were analysed by mutagenesis for functional domains that are instrumental in H2 signal transmission. A mutant deleted for a C‐terminal peptide of 55 amino acids in HoxB lost its H2‐sensing ability but still catalysed H2 oxidation. The mutant protein failed to form the dimeric heterodimer and a complex with HoxJ. The organic redox cofactor was no longer detectable in the truncated sensor. H2 sensing was also abolished by deletion of the PAS domain of HoxJ, indicating that this domain is involved in signal transduction. A truncated version of HoxJ consisting of only the input domain of the kinase was still capable of forming a complex with the RH. Mass determination of the purified HoxJ protein revealed that the kinase forms a homotetramer. The unique oligomeric structure of the H2‐sensing complex with respect to its regulatory function is discussed.

Involvement of hyp gene products in maturation of the H(2)-sensing [NiFe] hydrogenase of Ralstonia eutropha.

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H(2)-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H(2)-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of (63)Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.

A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit.

The large subunit HoxC of the H2‐sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni–Fe site maturation proteins HypC and HypB. Despite the presence of the Ni–Fe active site in some of the proteins, both forms, which lack the Fe–S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni–Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.

A hydrogen-sensing multiprotein complex controls aerobic hydrogen metabolism in Ralstonia eutropha.

H(2) is an attractive energy source for many microorganisms and is mostly consumed before it enters oxic habitats. Thus aerobic H(2)-oxidizing organisms receive H(2) only occasionally and in limited amounts. Metabolic adaptation requires a robust oxygen-tolerant hydrogenase enzyme system and special regulatory devices that enable the organism to respond rapidly to a changing supply of H(2). The proteobacterium Ralstonia eutropha strain H16 that harbours three [NiFe] hydrogenases perfectly meets these demands. The unusual biochemical and structural properties of the hydrogenases are described, including the strategies that confer O(2) tolerance to the NAD-reducing soluble hydrogenase and the H(2)-sensing regulatory hydrogenase. The regulatory hydrogenase that forms a complex with a histidine protein kinase recognizes H(2) in the environment and transmits the signal to a response regulator, which in turn controls transcription of the hydrogenase genes.

The H(2) sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site.

Abstract. [NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H2-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H2 sensor (RH) of Ralstoniaeutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(ε) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.

hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster

Abstract The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined. The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied. The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase. The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected. We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant. The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor. Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX.

Protein–Protein Complex Formation Affects the Ni–Fe and Fe–S Centers in the H2‐Sensing Regulatory Hydrogenase from Ralstonia eutropha H16

The regulatory Ni-Fe hydrogenase (RH) from the H(2)-oxidizing bacterium Ralstonia eutropha functions as an oxygen-resistant hydrogen sensor, which is composed of the large, active-site-containing HoxC subunit and the small subunit HoxB carrying Fe-S clusters. In vivo, the HoxBC subunits form a dimer designated as RH(wt). The RH(wt) protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RH(wt) (RH(wt)-HoxJ(wt)), located in the cytoplasm. In this study, we used X-ray absorption (XAS), electron paramagnetic resonance (EPR), and Fourier transform infrared (FTIR) spectroscopy to investigate the impact of various complexes between RH and HoxJ on the structural and electronic properties of the Ni-Fe active site and the Fe-S clusters. Aside from the RH(wt) protein and the RH(wt)-HoxJ(wt) complex, we investigated the RH(stop) protein, which consists of only one HoxB and HoxC unit due to the missing C-terminus of HoxB, as well as RH(wt)-HoxJ(Deltakinase), in which the histidine protein kinase lacks the transmitter domain. All constructs reacted with H(2), leading to the formation of the EPR-detectable Ni(III)-C state of the active site and to the reduction of Fe-S clusters detectable by XAS, thus corroborating that H(2) cleavage is independent of the presence of the HoxJ protein. In RH(stop), presumably one Fe-S cluster was lost during the preparation procedure. The coordination of the active site Ni in RH(stop) differed from that in RH(wt) and the RH(wt)-HoxJ complexes, in which additional Ni--O bonds were detected by XAS. The Ni--O bonds caused only very minor changes of the EPR g-values of the Ni-C and Ni-L states and of the IR vibrational frequencies of the diatomic CN(-) and CO ligands at the active-site Fe ion. Both one Fe-S cluster in HoxB and an oxygen-rich Ni coordination seem to be stabilized by RH dimerization involving the C-terminus of HoxB and by complex formation with HoxJ.