Pulse radiolysis studies on superoxide reductase from <i>Treponema pallidum</i>

Nivière, Vincent; Lombard, Murielle; Fontecave, Marc; Houée-Levin⊥, Chantal

doi:10.1016/s0014-5793(01)02468-1

Cited by 52 publications

(86 citation statements)

References 11 publications

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“…These results show that, in presence of a steady-state concentration of the physiological substrate of SOR, neelaredoxin was able to catalyze the reoxidation of dithionite-reduced rubredoxin, in good agreement with similar experiments performed with P. furiosus SOR [7]. When bovine SOD was added to the system, the rate of rubredoxin reoxidation remained unchanged, because SOD is unable to receive electrons from any component of the reaction system at rates able to outcompete with the catalytic reduction of superoxide (10 9 M À1 s À1 ) [6,16,17,18]. However, when SOD was added in place of SOR, reoxidation of rubredoxin was totally inhibited, which confirmed the superoxidemediated character of the observed electron transfer reaction.…”

Section: Discussionsupporting

confidence: 82%

“…In the beginning, classic superoxide dismutase assays, such as the reduction of cytochrome c by superoxide at 550 nm [41], were used to assay SORs, resulting in discrepancies in the literature [4,6,7,9,17,66,67]. It is now currently admitted that superoxide reductases present no, or very low, superoxide dismutase activity that could outcompete the spontaneous disproportionation of superoxide, and that a good assay for superoxide reductases should be specific and allow us to clearly differentiate the dismutation reaction from the reduction of superoxide catalyzed by SORs.…”

Section: Discussionmentioning

confidence: 99%

“…With this method, the activity of superoxide reductase, derived from the NADPH consumption rate, was measured with a detection limit of around 50 nM. Interesting results were also obtained by generating superoxide using pulse radiolysis, but this method is very difficult to use as a routine assay in a laboratory [17]. Here we propose that, with the rate of rubredoxin reoxidation being directly related to the concentration of neelaredoxin, our experiment could be used as an alternative more direct assay, at least for Dv and Tp superoxide reductases, with a detection limit of less than 5 nM.…”

Section: Discussionmentioning

confidence: 99%

“…Only the reduced colorless ferrous form of the active site iron is able to react with superoxide, with a virtually diffusion controlled rate of 10 9 M À1 s À1 , leading to the formation of the blue ferric state of the enzyme, which suggests the existence of an electron donor to regenerate the ferrous active form and complete the catalytic cycle of the enzyme [6,16,17,18]. The in vivo electron donor to SOR has generally not been established but previous work has strongly suggested that a rubredoxin (Rd) could be the proximal electron donor to superoxide reductases during periods of oxidative stress [7,8,19,20,21].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2004

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Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O : 2 into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, e=6.9±0.4 mM À1 cm À1 . Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was À76±5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na 2 IrCl 6 -oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k app =180 min À1 . Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k app =4.5 min À1 . Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2004

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“…10 The reaction mechanism of SOR, involving transfer of an electron and two protons to superoxide to form hydrogen peroxide, has been studied in detail using kinetic and spectroscopic techniques. 12,13,14,15,16,17,18 Using kinetic data, Nivière et al and Kurtz et al have proposed the presence of at least one Fe III intermediate (henceforth referred to as intermediate I, characterized by a CT band at 600 nm) in the reaction. The formation of the intermediate was diffusion controlled and had no pH dependence or deuterium isotope effect and thus was proposed to be an Fe III -μ 2 -O 2 2− species.…”

Section: Introductionmentioning

confidence: 99%

Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

et al. 2007

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Superoxide reductase (SOR) is a non-heme iron enzyme which reduces superoxide to peroxide at a diffusion-controlled rate. Sulfur K-edge x-ray absorption spectroscopy (XAS) is used to investigate the ground state electronic structure of the resting high-spin and CN − bound low-spin Fe III forms of the 1Fe SOR from Pyrococcus furiosus. A computational model with constrained Imidazole rings (necessary for reproducing spin states), H-bonding interaction to the thiolate (necessary for reproducing Fe-S bond covalency of the high-spin and low-spin forms) and H-bonding to axial ligand (necessary to reproduce the ground state of the low-spin form) was developed and then used to investigate the enzymatic reaction mechanism. Reaction of the resting ferrous site with superoxide and protonation leading to a high-spin Fe III -OOH species and its subsequent protonation resulting in H 2 O 2 release is calculated to be the most energetically favorable reaction pathway. Our results suggest that the thiolate acts as a covalent anionic ligand. Replacing the thiolate with a neutral noncovalent ligand makes protonation very endothermic and greatly raises the reduction potential. The covalent nature of the thiolate weakens the Fe III bond to the proximal Oxygen of this hydroperoxo species, which raises its pK a by an additional 5 log units relative to a primarily anionic ligand facilitating its protonation. A comparison with Cytochrome P450 indicates that the stronger equatorial ligand field from the porphyrin results in a low-spin Fe III -OOH species which would not be capable of efficient H 2 O 2 release due to a spin-crossing barrier associated with formation of a high spin 5C Fe III product. Additionally, the presence of the di-anionic porphyrin π ring in Cytochrome P450 allows O-O heterolysis forming a Fe IV -oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally the 5C Fe III site which results from the product release at the end of the O 2 − reduction cycle is calculated to be capable of reacting with a second O 2 − resulting in superoxide dismutase (SOD) activity. However in contrast to FeSOD the 5C Fe III site of SOR which is more positive is calculated to have a high affinity for the binding a 6 th anionic ligand which would inhibit its SOD activity.

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Superoxide Reductase

Nivière¹,

Bonnot²,

Bourgeois³

2004

Handbook of Metalloproteins

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Superoxide reductase (SOR) is a small bacterial metalloprotein, which is present in the cytoplasm of some anaerobic or microaerophilic microorganisms. SOR is involved in the detoxification of the superoxide radical and catalyzes its one‐electron reduction into hydrogen peroxide. Three different classes of SOR have been identified and structurally characterized. They all contain a similar active site, which consists of an unusual nonheme iron center, coordinated in a square‐pyramidal geometry to four nitrogens from four histidine side chains in the equatorial plane and the sulfur of one cysteine residue along the fifth axial position. In the reduced state, the sixth axial coordinating site of the iron is vacant, suggesting the most obvious site for initial binding of the superoxide substrate. The reaction mechanism of SOR has been studied by pulse radiolysis. SOR reacts specifically at a nearly diffusion‐controlled rate with O 2 •− , generating H 2 O 2 and the oxidized Fe 3+ active site. Reaction intermediates have been observed and proposed to be Fe 3+ ‐(hydro)peroxo species, resulting from the fast electron transfer from the ferrous iron to the superoxide anion and from protonation step. Formation of Fe 3+ ‐(hydro)peroxo intermediate species in the active site of SOR has been supported by resonance Raman spectroscopy and X‐ray structures of iron(III) peroxide species in the SOR from Desulfoarculus baarsii .

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Pulse radiolysis studies on superoxide reductase from Treponema pallidum

Cited by 52 publications

References 11 publications

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

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

Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

Superoxide Reductase

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