Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus

Friedrich, Bärbel; Heine, Elisabeth; Finck, Andreas; Friedrich, Cornelius G.

doi:10.1128/jb.145.3.1144-1149.1981

Cited by 163 publications

(84 citation statements)

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“…Strains with the initials HF were derived from R. eutropha H16 harbouring the endogenous megaplasmid pHG1. R. eutropha strains were cultivated in mineral salt medium containing 0.2% (w ⁄ v) fructose and 0.2% (v ⁄ v) glycerol (FGN medium) [51]. For protein purification 30 g of cells (wet weight) obtained from 4-L FGN cultures were used.…”

Section: Methodsmentioning

confidence: 99%

Impact of alterations near the [NiFe] active site on the function of the H₂ sensor from Ralstonia eutropha

et al. 2006

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Hydrogenases are redox enzymes that catalyse the reversible oxidation of hydrogen into protons and electrons. Based on their metal content, three classes of hydrogenases are distinguished: [FeFe] In proteobacteria capable of H 2 oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H 2 . The H 2 -sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]-hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni-Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H 2 -oxidizing and D 2 ⁄ H + exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para-⁄ ortho-H 2 conversion activity showed that these centres were still able to bind H 2 . This indicates that H 2 binding at the active site is not sufficient for the regulatory function of H 2 sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy-linked [NiFe]-hydrogenases. The respective RH mutant protein showed only traces of H 2 -oxidizing activity, whereas its D 2 ⁄ H + -exchange activity and H 2 -sensing function were almost unaffected. H 2 -dependent signal transduction in this mutant was less sensitive to oxygen than in the wild-type strain. These results suggest that H 2 turnover is not crucial for H 2 sensing. It may even be detrimental for the function of the H 2 sensor under high O 2 concentrations.Abbreviations MBH, membrane-bound hydrogenase; PAS, Per-Arnt-Sim; RH, regulatory hydrogenase; SH, soluble hydrogenase.

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Section: Methodsmentioning

confidence: 99%

Impact of alterations near the [NiFe] active site on the function of the H₂ sensor from Ralstonia eutropha

et al. 2006

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“…Activities of SH and MBH were determined with cells grown on fructose/glycerol medium to an A,,, of 10-11. Soluble and membrane extracts were prepared as described previously [25]. SH activity was determined in soluble extracts by spectrophotometric measurement of H,-dependent NAD and benzylviologen reduction and NADH-dependent benzylviologen reduction 121.…”

Section: Methodsmentioning

confidence: 99%

“…SH activity of soluble extracts from A. eutrophus strains after growth in various concentrations of NiCI,. Cells were grown on fructose/glycerol medium [25]. H,-dependent NAD reduction was determined.…”

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confidence: 99%

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C‐Terminal Extension of the H₂‐Activating Subunit, HoxH, Directs Maturation of the NAD‐Reducing Hydrogenase in Alcaligenes Eutrophus

Massanz¹,

Fernández²,

Friedrich³

1997

European Journal of Biochemistry

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Formation of enzymatically active [NiFe] hydrogenases is dependent on a number of posttranslational steps, including metal attachment to a precursor of the catalytic subunit, truncation of a small C-terminal peptide from the precursor, and oligomerisation of the subunits. Two amino acid replacements were introduced by site-directed mutagenesis at the C-terminal proteolytic cleavage site of HoxH, the Nicontaining subunit of the cytoplasmic NAD-reducing hydrogenase of Alcciligenes eutrophus H16. Replacement of Ala465, the first residue of the 24-amino-acid cleaved polypeptide, by Pro yielded a form of HoxH that was blocked in C-terminal proteolysis. This HoxH subunit, although capable of binding Ni, was blocked in formation of a stable tetrameric holoenzyme. In the second mutant, the C-terminal extension of HoxH was eliminated by substituting the Ala codon for a translational stop codon. Although this mutant subunit was able to form the oligomeric holoenzyme, it was devoid of Ni. Both mutant proteins contained only traces of H,-activating functions. H2-dependent reduction of NAD and benzylviologen, and D,/H+-exchange activity were almost completely abolished, while the NADH oxidoreductase activity, mediated by the diaphorase moiety of the hydrogenase, was retained. These results allow the following conclusions : the C-terminal extension of HoxH is neccessary to direct specific Ni insertion into the hydrogenase; subunit assembly to the holoenzyme is not dependent on Ni insertion; and a precursor with the C-terminal peptide is not competent for assembly. Spectroscopic data on various [NiFe] hydrogenases indicate that Ni is directly involved in H, activation (reviewed in 1141). The first crystallographic analysis of a periplasmic [NiFe] hydrogenase from DesulJbvihrio gigas uncovered Ni coordination by two pairs of N-terminal and C-terminal cysteine residues in the large subunit, which are fully conserved in the corresponding HoxH subunit of A. eutrophus SH. A second metal, Fe, is located close to the Ni and shares one cysteine of each pair as bridging ligands. In addition, the Fe appears to be coordinated by three diatomic non-protein ligands. This bimetal center is deeply buried inside the hydrogenase dimer [I 51. Moreover, the X-ray data for the D. gigas hydrogenase showed that the small subunit harbours one [3Fe-4S] and two [4Fe-4S] clusters in a linear arrangement. This subunit is acting as an electron-transfer protein. A conesponding function is assigned to HoxY, the sinall subunit of the SH dimer. This polypeptide, however, is substantially smaller than its [NiFe] hydrogenase counterpart, since it only contains the N-terminal [4Fe-4S] cluster domain [6]. KeywordsMutational analyses of hydrogenase-related genes have shown that in various bacteria the structural genes are closely linked to sets of accessory genes that encode auxiliary proteins involved in the biosynthesis of active [NiFe] hydrogenases (reviewed in [I, 16, 171). These proteins appear to participate in Ni insertion, proteolytic C-terminal proces...

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“…Hydrogen evolution from NAD-reducing hydrogenase was measured on Frankia sp. R43 cells by adding NAD and measure NADH formation at 340 nm as earlier described by Friedrich et al [39].…”

Section: Hydrogen Evolutionmentioning

confidence: 99%

A hydrogen-evolving enzyme is present in Frankia sp. R43

Mohapatra

Leul

Mattsson

et al. 2004

FEMS Microbiology Letters

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The ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes Frankia sp. R43 and Frankia sp. KB5. To further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. Under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) Frankia sp. R43 but not Frankia sp. KB5 evolved hydrogen,which was not linked to NAD-reducing activity. Immunological analysis of total protein from Frankia sp. R43 and Frankia sp. KB5 using an antiserum raised against Ralstonia eutropha HoxF, recognized an antigen in Frankia sp. R43 but not in Frankia sp. KB5. Immunogold labeling using antibodies raised against the R. eutropha HoxH recognized sites in both hyphae and vesicles of Frankia sp. R43, but not in Frankia sp. KB5. Based on these physiological and immunological findings, we conclude that Frankia sp. R43 has a hydrogen-evolving hydrogenase.

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Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus

Cited by 163 publications

References 15 publications

Impact of alterations near the [NiFe] active site on the function of the H₂ sensor from Ralstonia eutropha

Impact of alterations near the [NiFe] active site on the function of the H₂ sensor from Ralstonia eutropha

C‐Terminal Extension of the H₂‐Activating Subunit, HoxH, Directs Maturation of the NAD‐Reducing Hydrogenase in Alcaligenes Eutrophus

A hydrogen-evolving enzyme is present in Frankia sp. R43

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Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus

Cited by 163 publications

References 15 publications

Impact of alterations near the [NiFe] active site on the function of the H2 sensor from Ralstonia eutropha

Impact of alterations near the [NiFe] active site on the function of the H2 sensor from Ralstonia eutropha

C‐Terminal Extension of the H2‐Activating Subunit, HoxH, Directs Maturation of the NAD‐Reducing Hydrogenase in Alcaligenes Eutrophus

A hydrogen-evolving enzyme is present in Frankia sp. R43

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Impact of alterations near the [NiFe] active site on the function of the H₂ sensor from Ralstonia eutropha

Impact of alterations near the [NiFe] active site on the function of the H₂ sensor from Ralstonia eutropha

C‐Terminal Extension of the H₂‐Activating Subunit, HoxH, Directs Maturation of the NAD‐Reducing Hydrogenase in Alcaligenes Eutrophus