Superoxide Reactivity of Rubredoxin Oxidoreductase (Desulfoferrodoxin) from Desulfovibrio vulgaris:  A Pulse Radiolysis Study

Coulter, Eric D.; Emerson, Joseph P.; KurtzJr., Donald M.; Cabelli, Diane E.

doi:10.1021/ja005583r

Cited by 102 publications

(169 citation statements)

References 19 publications

Supporting

Mentioning

157

Contrasting

Unclassified

Order By: Relevance

“…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%

“…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%

See 1 more Smart Citation

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

et al. 2004

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

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

et al. 2004

View full text Add to dashboard Cite

show abstract

“…Note that in Compound 0 of peroxidases the protonation of a proximal Oxygen of a Fe III -OOH species is calculated to have a barrier of 9 Kcal/mol (including ZPE correction). 71 However this is still less than the ΔG ± of diffusion of a small ion in solution (11)(12) Kcal/mol) involved in the formation of the Fe II -O 2 − adduct. The following step is cleavage of the weak Fe-(O 2 H 2 ) bond which is endothermic by only E = 8 Kcal/mol and this process is associated with a further 0.03 Å decrease in the Fe-S bond length.…”

Section: Reaction Mechanismmentioning

confidence: 99%

“…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

View full text Add to dashboard Cite

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.

show abstract

“…Additionally, the ability of Mn porphyrins to eliminate peroxynitrite was found to parallel the O 2 •− dismuting activity [6] and is, therefore, governed by closely related SAR. Moreover, as a result of the easy reducibility of the potent SOD mimics, their action in vivo may be coupled to cellular reductants (ascorbic acid, tetrahydrobiopterin, less efficiently with glutathione), converting their function to superoxide or ONOO − reductases [6][7][8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

SOD-like activity of Mn(II) β-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrin equals that of the enzyme itself

DeFreitas-Silva

Rebouças

Spasojević

et al. 2008

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

Mn porphyrins are among the most efficient SOD mimics with potency approaching that of SOD enzymes. The most potent ones, Mn(III) N-alkylpyridylporphyrins bear positive charges in a close proximity to the metal site, affording thermodynamic and kinetic facilitation for the reaction with negatively charged superoxide. The addition of electron-withdrawing bromines onto β-pyrrolic positions dramatically improves thermodynamic facilitation for the O 2 •− dismutation. We have previously characterized the para isomer, Mn II Br 8 TM-4-PyP 4+ [Mn(II) β-octabromo-meso-tetrakis (N-methylpyridinium-4-yl)porphyrin]. Herein we fully characterized its meta analogue, Mn II Br 8 TM-3-PyP 4+ with respect to uv/vis spectroscopy, electron spray mass spectrometry, electrochemistry, O 2 •− dismutation, metal-ligand stability, and the ability to protect SOD-deficient E. coli in comparison with its para analogue. The increased electron-deficiency of the metal center stabilizes Mn in its +2 oxidation state. The metal-centered Mn III /Mn II reduction potential, E ½ = + 468 mV vs NHE, is increased by 416 mV with respect to non-brominated analogue, Mn III TM-3-PyP 5+ and is only 12 mV less positive than for para isomer. Yet, the complex is significantly more stable towards the loss of metal than its para analogue. As expected, based on the structure-activity relationships, a large increase in E ½ results in exceptionally high catalytic rate constant for the O 2 •− dismutation, log k cat ≥ 8.85; 1.5-fold increase with respect to the para isomer. The IC 50 was calculated to be ≤ 3.7 nM. Manipulation of the electron-deficiency of a cationic porphyrin resulted, therefore, in the highest k cat ever reported for a metalloporphyrin, being essentially identical to the k cat of superoxide dismutases (log k cat = 8.84 -9.30). The positive kinetic salt effect points to the unexpected, unique and first time recorded behavior of Mn β-octabrominated porphyrins when compared to other Mn porphyrins studied thus far. When species of opposing charges react, the increase in ionic strength invariably results in the decreased rate constant; with brominated porphyrins the opposite was found to be true. The effect is 3.5 -fold greater with meta than with para isomer, which is discussed with respect to the closer proximity of the quaternary nitrogens of the meta isomer to the metal center than that of the para isomer. The potency of Mn II Br 8 TM-3-PyP 4+ was corroborated by in vivo studies, where 500 nM allows SOD-deficient E. coli to grow ≥ 60% of the growth of wild type; at concentrations ≥5 μM it exhibits toxicity. Our work shows that exceptionally high k cat for the O 2 •− disproportionation can be achieved not only with an N 5 -type *Corresponding authors: Ynara Marina Idemori, Ph. D., Departamento de Química, ICEx, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, MG, 31270-901, Brazil., Tel: +55-31-3499-5762, Fax: +55-31-3499-5700, e-mail: ynara@ufmg.br NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH...

show abstract

Superoxide Reactivity of Rubredoxin Oxidoreductase (Desulfoferrodoxin) from Desulfovibrio vulgaris: A Pulse Radiolysis Study

Cited by 102 publications

References 19 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

SOD-like activity of Mn(II) β-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrin equals that of the enzyme itself

Contact Info

Product

Resources

About