The influence of oxygen on [NiFe]-hydrogenase cofactor biosynthesis and how ligation of carbon monoxide precedes cyanation.
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ABSTRACT:
SUBMITTER: Stripp ST
PROVIDER: S-EPMC4161419 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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Project description:Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.
| S-EPMC3753944 | biostudies-literature
Project description:The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.
| S-EPMC8454700 | biostudies-literature
Project description:'Oxygen-tolerant' [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.
| S-EPMC4771823 | biostudies-literature
Project description:BACKGROUND:Poisoning with carbon monoxide (CO) remains an important cause of accidental and intentional injury worldwide. Several unblinded non-randomized trials have suggested that the use of hyperbaric oxygen (HBO) prevents the development of neurological sequelae. This has led to the widespread use of HBO in the management of patients with carbon monoxide poisoning. OBJECTIVES:To examine randomised trials of the efficacy of hyperbaric oxygen (HBO) compared to normobaric oxygen (NBO) for the prevention of neurologic sequelae in patients with acute carbon monoxide poisoning. SEARCH STRATEGY:We searched the following electronic databases; Cochrane Injuries Group Specialised Register (searched June 2010), Cochrane Central Register of Controlled Trials (The Cochrane Library 2010, Issue 2), MEDLINE (Ovid SP) 1950 to June 2010, EMBASE (Ovid SP) 1980 to June 2010, ISI Web of Science: Science Citation Index Expanded (SCI-EXPANDED) 1970 to June 2010, ISI Web of Science: Conference Proceedings Citation Index-Science (CPCI-S) 1990 to June 2010. SELECTION CRITERIA:All randomised controlled trials of HBO compared to NBO, involving non-pregnant adults who are acutely poisoned with carbon monoxide (regardless of severity). DATA COLLECTION AND ANALYSIS:Two authors independently extracted from each trial information on: the number of randomised patients, types of participants, the dose and duration of the intervention, and the prevalence of neurologic symptoms at follow-up. MAIN RESULTS:Seven randomised controlled trials of varying quality were identified; one was excluded because it did not evaluate clinical outcomes. Of the six remaining trials involving 1361 participants, two found a beneficial effect of HBO for the reduction of neurologic sequelae at one month, while four others did not. One of these is an incomplete publication (an abstract of an interim analysis). Although pooled random effects meta-analysis does not suggest a significant benefit from HBOT (OR for neurological deficits 0.78, 95%CI 0.54 to 1.12), significant methodologic and statistical heterogeneity was apparent among the trials, and this result should be interpreted cautiously. Moreover, design or analysis flaws were evident in all trials. Importantly, the conclusions of one positive trial may have been influenced by failure to adjust for multiple hypothesis testing, while interpretation of the other positive trial is hampered by a high risk of bias introduced during the analysis including an apparent change in the primary outcome. Both were also stopped early 'for benefit', which is likely to have inflated the observed effect. In contrast three negative trials had low power to detect a benefit of HBO due to exclusion of severely poisoned patients in two and very poor follow-up in the other. One trial that was said to be finished around eight years ago has not reported the final analysis in any forum. AUTHORS' CONCLUSIONS:Existing randomised trials do not establish whether the administration of HBO to patients with carbon monoxide poisoning reduces the incidence of adverse neurologic outcomes. Additional research is needed to better define the role, if any, of HBO in the treatment of patients with carbon monoxide poisoning. This research question is ideally suited to a multi-center randomised controlled trial.
| S-EPMC7066484 | biostudies-literature
Project description:BACKGROUND: [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H? as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes (hupSLCDEFGHIJKhypABFCDEX) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen. RESULTS: HupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A ?hupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using StrepTag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex. CONCLUSIONS: The results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen.
| S-EPMC3534401 | biostudies-literature
Project description:This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.
| S-EPMC2713555 | biostudies-literature
Project description:Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen-oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane.
| S-EPMC4073834 | biostudies-literature
Project description:Several accessory proteins are required for the assembly of the metal centers in hydrogenases. In NiFe-hydrogenases, CO and CN- are coordinated to the Fe in the NiFe dinuclear cluster of the active center. Though these diatomic ligands are biosynthesized enzymatically, detail mechanisms of their biosynthesis remain unclear. Here, we report the structural characterization of HypX responsible for CO biosynthesis to assemble the active site of NiFe hydrogenase. CoA is constitutionally bound in HypX. Structural characterization of HypX suggests that the formyl-group transfer will take place from N10-formyl-THF to CoA to form formyl-CoA in the N-terminal domain of HypX, followed by decarbonylation of formyl-CoA to produce CO in the C-terminal domain though the direct experimental results are not available yet. The conformation of CoA accommodated in the continuous cavity connecting the N- and C-terminal domains will interconvert between the extended and the folded conformations for HypX catalysis.
| S-EPMC6802093 | biostudies-literature
Project description:Some carboxydotrophs like Rhodospirillum rubrum are able to grow with CO as their sole source of energy using a Carbone monoxide dehydrogenase (CODH) and an Energy conserving hydrogenase (ECH) to perform anaerobically the so called water-gas shift reaction (WGSR) (CO + H2O-> CO2 + H2). Several studies have focused at the biochemical and biophysical level on this enzymatic system and a few OMICS studies on CO metabolism. Knowing that CO is toxic in particular due to its binding to heme iron atoms, and is even considered as a potential antibacterial agent, we decided to use a proteomic approach in order to analyze R. rubrum adaptation in term of metabolism and management of the toxic effect. In particular, this study allowed highlighting a set of proteins likely implicated in ECH maturation, and important perturbations in term of cofactor biosynthesis, especially metallic cofactors. This shows that even this CO tolerant microorganism cannot avoid completely CO toxic effects associated with its interaction with metallic ions.
2024-09-09 | PXD025489 | Pride
Project description:Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.
| S-EPMC3940037 | biostudies-literature