Raman spectral evidence for a .mu.-oxo bridge in the binuclear iron center of ribonucleotide reductase

Sjöberg, Britt‐Marie; Loehr, Thomas M.; Sanders-Loehr, Joann

doi:10.1021/bi00530a017

Cited by 122 publications

(49 citation statements)

References 34 publications

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“…Raman spectra were obtained with a computerized Jarrell-Ash 25-300 Raman spectrometer equipped with a Spectra-Physics 164-05 argon ion laser and a C31034A photomultiplier (RCA Corp.). The sample was sealed in a glass capillary, placed in a cold-finger sample holder maintained at 0°C, and illuminated in a back-scattering geometry as described previously (30).…”

Section: Methodsmentioning

confidence: 99%

Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1)

Actis¹,

Fish²,

Crosa³

et al. 1986

J Bacteriol

148

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Anguibactin, a siderophore produced by cells of Vibrio anguillarum 775 harboring the pJMl plasmid, has now been isolated from the supernatants of iron-deficient cultures. This iron-reactive material was purified by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column. The Many microorganisms in aerobic, iron-limited environments are able to enhance iron uptake by secreting lowmolecular-weight, highly specific iron chelators termed siderophores. These compounds scavenge iron from protein complexes or insoluble hydroxides in the extracellular environment and transport it to the cell cytosol via a membranebound receptor protein in the microorganism (21, 22). Although siderophores exhibit a great diversity of structures, most of the known siderophores are classified as either phenolates or hydroxamates (20,22). The ability to produce and utilize siderophores has been linked to the virulence of certain pathogenic bacteria (5, 10, 34). By using siderophores, these organisms prosper in physiological systems in which most iron is bound by high-affinity proteins such as transferrin and lactoferrin.Vibrio anguillarum is an important fish pathogen responsible for the septicemic disease vibriosis (6,17,27). Virulence in strain 775 of this bacterium has been shown to be conferred by a 65-kilobase plasmid designated pJM1 (13, 14), which specifies an iron-sequestering system (9) that includes the outer membrane protein OM2 (2, 12) and the siderophore anguibactin (11). Virulence was found to be attenuated by plasmid mutations which abolish anguibactin or OM2 production (33) and by elimination of the plasmid from the cells (13). Although the biological function of the pJM1-mediated siderophore is clear, its chemical structure and properties are not. This report describes the isolation of the siderophore anguibactin from the culture medium of ironstarved V. anguillarum 775 and its partial characterization. Anguibactin belongs to the phenolate category of siderophores and is, in fact, a catechol (o-diphenol) rather than a monophenol. Despite its ready classification, its * Corresponding author. molecular composition is unusual, and anguibactin appears to be distinct from other known siderophores (20). MATERIALS AND METHODSCulture of V. anguillarum. V. anguillarum 775 carrying the virulence plasmid pJM1 has been described previously (13,14). Cells were grown for 48 h in M9 minimal medium (9) containing the nonassimilable iron chelator nitrilotriacetic acid at 100 ,uM. To remove traces of contaminating metals, medium salts were passed through a Chelex 100 column prior to use. Bacteria were separated from the medium by centrifugation at 7,000 x g, and supernatants were stored at -200C.Purification of anguibactin. Anguibactin was isolated from V. anguillarum 775 culture supernatants by adsorption onto XAD-7 macroreticular resin (Rohm and Haas). This resin was subjected to Soxhlet extractions with methanol, acetonitrile, and diethylether to remove residual organic impurities prior to being packed in...

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Section: Methodsmentioning

confidence: 99%

Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1)

Actis¹,

Fish²,

Crosa³

et al. 1986

J Bacteriol

148

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“…Thus, we cannot find any evolutionary relationship between these two proteins. The limited number of conserved histidine residues in the B2 proteins support the suggestion that each polypeptide chain of the B2 protein contributes only half of the iron ligands and that the dimeric protein forms the binuclear iron center [9,19].…”

Section: Methodsmentioning

confidence: 66%

“…Protein B2 was recently shown to contain a poxo-bridged binuclear iron center of the same type as is found in met-forms of hemerythrin [19]. In hemerythrin the iron center is liganded by one aspartic, one glutamic and 5 histidine residues [20].…”

Section: Methodsmentioning

confidence: 99%

Identification of the stable free radical tyrosine residue in ribonucleotide reductase

et al. 1985

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The small subunit of ri~nucIeoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center. An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula soiidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important. Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved. We propose that Tyr-122 is responsible for radical stabilization and that His-l 18 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center.Ribonucleotide reductase Secondary structure prediction Sequence comparison Tyrosine radical

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“…The stability ofthe protein B2 radical is closely connected to the presence of the iron center of the enzyme. The iron center of protein B2 has been characterized by Mossbauer spectroscopy (37), light absorption (12,37), magnetic susceptibility (12), and resonance Raman techniques (13,40). It is composed of a pair of high spin (S = 5/2) ferric ions, antiferromagnetically coupled through a ,u-oxo bridge.…”

Section: Stabilitymentioning

confidence: 99%

“…For the first three of these enzymes it has also been shown that the tyrosyl radical coexists with an adjacent binuclear iron center (12)(13)(14). Another remarkable similarity between species is evident in the primary structures of ribonucleotide reductases as deduced from their DNA sequences in E. coli (5), herpes simplex virus (15,16), Epstein Barr virus (17), and the surface clam Spisula solidissima (18) (Fig.…”

Section: Introductionmentioning

confidence: 99%

The Tyrosyl Free Radical in Ribonucleotide Reductase

Gräslund¹,

Sahlin²,

Sjöberg³

1985

Environmental Health Perspectives

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The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins Bi and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of ironradical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase.The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative.In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis.Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical.

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Raman spectral evidence for a .mu.-oxo bridge in the binuclear iron center of ribonucleotide reductase

Cited by 122 publications

References 34 publications

Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1)

Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1)

Identification of the stable free radical tyrosine residue in ribonucleotide reductase

The Tyrosyl Free Radical in Ribonucleotide Reductase

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