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

Abstract

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...

Rubrerythrin and Rubredoxin Oxidoreductase inDesulfovibrio vulgaris: a Novel Oxidative Stress Protection System

“…The rbr gene in D. vulgaris (Hildenborough) is transcribed with a third gene designated fur (27), which encodes a protein showing highest sequence homology to PerR, a peroxide-responsive regulatory protein in Bacillus subtilis (8,47). The product of the fur-like gene may, therefore, regulate the RboRbr oxidative stress response in D. vulgaris.…”

“…Based on genetic proximity, Rbr could also use rubredoxin-like proteins as redox partners. In D. vulgaris (Hildenborough), Rbr is cotranscribed with Rdl, yet another class of rubredoxin-type protein (23,27).…”

“…D. gigas nlr was cloned from D. gigas chromosomal DNA using a procedure similar to that described by Silva et al (38), and pNLR119 (Table 1) was used as the template. Both D. vulgaris and D. gigas chromosomal DNAs had been isolated as previously described (27). Sequences of the oligonucleotide primers duplicated the N-and complementary C-terminal 14 bases of each gene using sequences from GenBank accession numbers M28848 (rbo), U82323 (rbr), U71215 (ngr), and AF034965 (nlr).…”

“…vulgaris contains a second Rbr-like protein, called nigerythrin (Ngr) (27,32), which also exhibits NADH peroxidase activity (13).…”

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

“…Interestingly, even though the purification of Dv rubredoxin, which presents 57% sequence homology with the one from Tp and the same acidic isoelectric point around 3.9 [20,63], was routinely performed in our laboratory, we had to elaborate a new strategy to obtain good expression levels of Tp rubredoxin, mainly because the gene appeared to be toxic to the cells. Moreover, iron reconstitution was always necessary during the purification of Tp rubredoxin, after the Ni-NTA first chromatography, in order to obtain a protein preparation homogeneous in iron.…”