Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibriovulgaris: Rubrerythrin and a new, highly active, enzyme

Liu, Ming-Y.; Gall, Jean Le

doi:10.1016/0006-291x(90)91394-8

Cited by 24 publications

(12 citation statements)

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“…P. furiosus rubrerythrin was assayed for pyrophosphatase, superoxide dismutase, ferroxidase, and NADH peroxidase activities, all of which have been attributed to the rubrerythrins of D. vulgaris and/or C. perfringens (4,7,9,(28)(29)(30). However, there was no detectable activity with any of the assays for the P. furiosus protein.…”

Section: Resultsmentioning

confidence: 99%

“…The same assay conditions were used when NADH was the electron donor except that NADH:rubredoxin oxidoreductase, rubredoxin, and rubrerythrin were added in catalytic amounts (Ϸ0.5 M) and NADH was used at a final concentration of 150 M. Its oxidation was measured at 340 nm with an extinction coefficient for NADH of 6.2 cm Ϫ1 mM Ϫ1 (11). Superoxide dismutase activity was measured at 50°C by cytochrome c reduction (33), and pyrophosphatase activity was determined at 80°C by the release of inorganic phosphate from sodium pyrophosphate (30). Inorganic phosphate was estimated as described previously (19).…”

Section: Growth Of Microorganismsmentioning

confidence: 99%

“…Although D. vulgaris rubrerythrin (4, 7-9, 12, 27, 30, 35, 42) and the rubrerythrins from a variety of other microorganisms (2,3,10,16,28,47) have been extensively studied, its cellular function is still not understood. A number of enzyme activities have been reported for rubrerythrin, including pyrophosphatase (30), ferroxidase (4), and NADH peroxidase (7)(8)(9), but which are of physiological significance and the nature of its redox partner proteins have not been established. Even the true metal content of rubrerythrin is still a matter of controversy.…”

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confidence: 99%

“…The oxidized form of the 1Zn/2Fe protein has inorganic pyrophosphatase activity (30), although the reduced protein is inactive (29). On the other hand, the oxidized form of 3Fe-rubrerythrin exhibits ferroxidase activity, the O 2 -dependent oxidation of Fe 2ϩ to Fe 3ϩ (4).…”

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confidence: 99%

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Rubrerythrin from the Hyperthermophilic Archaeon Pyrococcus furiosus Is a Rubredoxin-Dependent, Iron-Containing Peroxidase

et al. 2004

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Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 ؎ 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85°C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 mol of rubredoxin oxidized/min/mg with apparent K m values of 35 and 70 M for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 mol of H 2 O 2 reduced/min/mg of rubrerythrin at 85°C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide: rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

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Section: Resultsmentioning

confidence: 99%

Section: Growth Of Microorganismsmentioning

confidence: 99%

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Rubrerythrin from the Hyperthermophilic Archaeon Pyrococcus furiosus Is a Rubredoxin-Dependent, Iron-Containing Peroxidase

et al. 2004

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“…Rr and Ngr lack activities characteristic of other diiron-oxo proteins and also have no catalase or phosphatase activities (14,33). (The pyrophosphatase activity reported for Rr [24] has not been reproduced [21,33].) Recombinant Rr does have ferroxidase activity (i.e., the ability to catalyze oxidation of Fe 2ϩ to Fe 3ϩ by O 2 ) in vitro, with specific activity comparable to those of apoferritins (5).…”

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A rubrerythrin operon and nigerythrin gene in Desulfovibrio vulgaris (Hildenborough)

et al. 1997

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Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (Hildenborough). We have sequenced a 3.3-kbp SalI fragment of D. vulgaris chromosomal DNA containing the rubrerythrin gene, rbr, identified additional open reading frames (ORFs) adjacent to rbr, and shown that these ORFs are part of a transcriptional unit containing rbr. One ORF, designated fur, lies just upstream of rbr and encodes a 128-amino-acid-residue protein which shows homology to Fur (ferric uptake regulatory) proteins from other purple bacteria. The other ORF, designated rdl, lies just downstream of rbr and encodes a 74-residue protein with significant sequence homology to rubredoxins but with a different number and spacing of cysteine residues. Overexpression of rdl in Escherichia coli yielded a protein, Rdl, which has spectroscopic properties and iron content consistent with one Fe 3؉ (SCys) 4 site per polypeptide but is clearly distinct from both rubrerythrin and a related protein, nigerythrin. Northern analysis indicated that fur, rbr, and rdl were each present on a transcript of 1.3 kb; i.e., these three genes are cotranscribed. Because D. vulgaris nigerythrin appears to be closely related to rubrerythrin, and its function is also unknown, we cloned and sequenced the gene encoding nigerythrin, ngr. The amino acid sequence of nigerythrin is 33% identical to that of rubrerythrin, and all residues which furnish iron ligands to both the FeS 4 and diiron-oxo sites in rubrerythrin are conserved in nigerythrin. Despite the close resemblance of these two proteins, ngr was found to be no closer than 7 kb to rbr on the D. vulgaris chromosome, and Northern analysis showed that, in contrast to rbr, ngr is not cotranscribed with other genes. Possible redox-linked functions for rubrerythrin and nigerythrin in iron homeostasis are proposed.Rubrerythrin (Rr) is one of a large number of nonheme iron proteins found in anaerobic sulfate-reducing bacteria (16,28). Rr was isolated from Desulfovibrio vulgaris (Hildenborough) as a 44-kDa homodimer which exhibited spectroscopic signatures characteristic of two distinct types of iron sites: one rubredoxin-like FeS 4 site and one nonsulfur, oxo-bridged diiron site. The crystal structure of recombinant D. vulgaris Rr (10) revealed that each subunit is folded into two domains: an Nterminal four-helix bundle surrounding the oxo-bridged diiron site, and a smaller C-terminal rubredoxin-like protein fold surrounding the FeS 4 site. Rr can be structurally classified as a member of a continually expanding class of so-called diironoxo proteins (20,30). This class of proteins contains an oxo-or hydroxo-bridged diiron site connected to the protein by carboxylate and histidine ligands and embedded within a fourhelix bundle protein fold. Other members of this class utilize O 2 for functions ranging from tyrosyl radical generation and hydrocarbon hydroxylation in prokaryotes, fatty acyl desaturation in plants, and reversible O 2 binding in invertebrates (20). The iron storage proteins ferriti...

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Structural genomics of Pyrococcus furiosus: X‐ray crystallography reveals 3D domain swapping in rubrerythrin

et al. 2004

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Introduction. Rubrerythrin was first isolated from the anaerobic bacterium Desulfovibrio vulgaris [Protein DataBank (PDB) entry: 1RYT].1,2 It is a homodimeric protein and each subunit contains two different types of metal sites. One is mononuclear and located in the C-terminal domain. It is rubredoxin-like and consists of 1 metal ion bound to the side-chains of 4 cysteinyl residues. The second site, in the N-terminal domain, is binuclear. It is hemerythrin-like and consists of 2 metal ions bound by the side-chains of 5 glutamyl and 1 histidinyl residue. The nature of the metal ions in the 2 sites of D. vulgaris rubrerythrin is not clear, since both 3Fe 3,4 and 1Zn/2Fe forms 5 of the protein have been isolated. The mononuclear site appears to be occupied only by Fe in both forms.5 A variety of catalytic activities have also been proposed for D. vulgaris rubrerythrin, including pyrophosphatase, 3 ferroxidase, 6 and nicotinamide adenine dinucleotide hydride (NADH) peroxidase. [7][8][9] Its physiological function has also not been established, although a role in oxidative stress has been implicated for the D. vulgaris protein, as well as for those from several other anaerobic and microaerophilic bacteria.

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Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibriovulgaris: Rubrerythrin and a new, highly active, enzyme

Cited by 24 publications

References 14 publications

Rubrerythrin from the Hyperthermophilic Archaeon Pyrococcus furiosus Is a Rubredoxin-Dependent, Iron-Containing Peroxidase

Rubrerythrin from the Hyperthermophilic Archaeon Pyrococcus furiosus Is a Rubredoxin-Dependent, Iron-Containing Peroxidase

A rubrerythrin operon and nigerythrin gene in Desulfovibrio vulgaris (Hildenborough)

Structural genomics of Pyrococcus furiosus: X‐ray crystallography reveals 3D domain swapping in rubrerythrin

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