Kinetics of the Superoxide Reductase Catalytic Cycle
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m306488200
ISSN1083-351X
AutoresJoseph P. Emerson, Eric D. Coulter, Robert S. Phillips, Donald M. Kurtz,
Tópico(s)Environmental Toxicology and Ecotoxicology
ResumoThe steady state kinetics of a Desulfovibrio (D.) vulgaris superoxide reductase (SOR) turnover cycle, in which superoxide is catalytically reduced to hydrogen peroxide at a [Fe(His)4(Cys)] active site, are reported. A proximal electron donor, rubredoxin, was used to supply reducing equivalents from NADPH via ferredoxin: NADP+ oxidoreductase, and xanthine/xanthine oxidase was used to provide a calibrated flux of superoxide. SOR turnover in this system was well coupled, i.e. ∼202·¯ reduced:NADPH oxidized over a 10-fold range of superoxide flux. The reduction of the ferric SOR active site by reduced rubredoxin was independently measured to have a second-order rate constant of ∼1 × 106m–1 s–1. Analysis of the kinetics showed that: (i) 1 μm SOR can convert a 10 μm/min superoxide flux to a steady state superoxide concentration of 10–10m, during which SOR turns over about once every 6 s, (ii) the diffusion-controlled reaction of reduced SOR with superoxide is the slowest process during turnover, and (iii) neither ligation nor deligation of the active site carboxylate of SOR limits the turnover rate. An intracellular SOR concentration on the order of 10 μm is estimated to be the minimum required for lowering superoxide to sublethal levels in aerobically growing SOD knockout mutants of Escherichia coli. SORs from Desulfovibrio gigas and Treponema pallidum showed similar turnover rates when substituted for the D. vulgaris SOR, whereas superoxide dismutases showed no SOR activity in our assay. These results provide quantitative support for previous suggestions that, in times of oxidative stress, SORs efficiently divert intracellular reducing equivalents to superoxide. The steady state kinetics of a Desulfovibrio (D.) vulgaris superoxide reductase (SOR) turnover cycle, in which superoxide is catalytically reduced to hydrogen peroxide at a [Fe(His)4(Cys)] active site, are reported. A proximal electron donor, rubredoxin, was used to supply reducing equivalents from NADPH via ferredoxin: NADP+ oxidoreductase, and xanthine/xanthine oxidase was used to provide a calibrated flux of superoxide. SOR turnover in this system was well coupled, i.e. ∼202·¯ reduced:NADPH oxidized over a 10-fold range of superoxide flux. The reduction of the ferric SOR active site by reduced rubredoxin was independently measured to have a second-order rate constant of ∼1 × 106m–1 s–1. Analysis of the kinetics showed that: (i) 1 μm SOR can convert a 10 μm/min superoxide flux to a steady state superoxide concentration of 10–10m, during which SOR turns over about once every 6 s, (ii) the diffusion-controlled reaction of reduced SOR with superoxide is the slowest process during turnover, and (iii) neither ligation nor deligation of the active site carboxylate of SOR limits the turnover rate. An intracellular SOR concentration on the order of 10 μm is estimated to be the minimum required for lowering superoxide to sublethal levels in aerobically growing SOD knockout mutants of Escherichia coli. SORs from Desulfovibrio gigas and Treponema pallidum showed similar turnover rates when substituted for the D. vulgaris SOR, whereas superoxide dismutases showed no SOR activity in our assay. These results provide quantitative support for previous suggestions that, in times of oxidative stress, SORs efficiently divert intracellular reducing equivalents to superoxide. An emerging paradigm for protecting air-sensitive bacteria and Archaea from the toxic reduction products of dioxygen involves reduction of superoxide and hydrogen peroxide, rather than the classical disproportionation route for their removal characteristic of aerobic microoorganisms (1Pianzzola M.J. Soubes M. Touati D. J. Bacteriol. 1996; 178: 6736-6742Crossref PubMed Google Scholar, 2Kurtz Jr., D.M. Coulter E.D. Chemtracts. Inorg. Chem. 2001; 14: 407-435Google Scholar, 3Kurtz Jr., D.M. Coulter E.D. J. Biol. Inorg. Chem. 2002; 7: 653-658Crossref PubMed Scopus (34) Google Scholar, 4Liochev S.I. Fridovich I. J. Biol. Chem. 1997; 272: 25573-25575Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 5Jenney Jr., F.E. Verhagen M.F.J.M. Cui X. Adams M.W.W. Science. 1999; 286: 306-309Crossref PubMed Scopus (329) Google Scholar, 6Lombard M. Fontecave M. Touati D. Niviere V. J. Biol. Chem. 2000; 275: 115-121Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 7Lombard M. Touati D. Fontecave M. Niviere V. J. Biol. Chem. 2000; 275: 27021-27026Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 8Lumppio H.L. Shenvi N.V. Summers A.O. Voordouw G. Kurtz Jr., D.M. J. Bacteriol. 2001; 183: 101-108Crossref PubMed Scopus (186) Google Scholar, 9Abreu I.A. Xavier A.V. LeGall J. Cabelli D.E. Teixeira M. J. Biol. Inorg. Chem. 2002; 7: 668-674Crossref PubMed Scopus (29) Google Scholar). The reduction of superoxide via Reaction 1 is catalyzed by a novel class of non-heme iron enzymes called superoxide reductases (SORs). 1The abbreviations used are: SOR, superoxide reductase; SOD, superoxide dismutase; 1Fe-SOR, SOR containing an [Fe(His)4(Cys)] site as the only prosthetic group; 2Fe-SOR, superoxide reductase containing an accessory [Fe(Cys)4] site; 2Fe-SORpink, 2Fe-SOR containing a ferrous [Fe(His)4(Cys)] site and a ferric [Fe(Cys)4] site; E47A 2Fe-SOR, recombinant engineered variant of D. vulgaris 2Fe-SOR in which the glutamate residue providing the ligand to the ferric [Fe(His)4(Cys)] site is substituted by an alanine residue; C13S 2Fe-SOR, recombinant D. vulgaris 2Fe-SOR engineered variant in which the accessory [Fe(Cys)4] site is absent; C13S 2Fe-SORblue, C13S 2Fe-SOR containing a ferric [Fe(His)4(Cys)] site; FNR, spinach ferredoxin:NADP+ oxidoreductase; Rub, rubredoxin; D. vulgaris H, Desulfovibrio vulgaris strain Hildenborough.1The abbreviations used are: SOR, superoxide reductase; SOD, superoxide dismutase; 1Fe-SOR, SOR containing an [Fe(His)4(Cys)] site as the only prosthetic group; 2Fe-SOR, superoxide reductase containing an accessory [Fe(Cys)4] site; 2Fe-SORpink, 2Fe-SOR containing a ferrous [Fe(His)4(Cys)] site and a ferric [Fe(Cys)4] site; E47A 2Fe-SOR, recombinant engineered variant of D. vulgaris 2Fe-SOR in which the glutamate residue providing the ligand to the ferric [Fe(His)4(Cys)] site is substituted by an alanine residue; C13S 2Fe-SOR, recombinant D. vulgaris 2Fe-SOR engineered variant in which the accessory [Fe(Cys)4] site is absent; C13S 2Fe-SORblue, C13S 2Fe-SOR containing a ferric [Fe(His)4(Cys)] site; FNR, spinach ferredoxin:NADP+ oxidoreductase; Rub, rubredoxin; D. vulgaris H, Desulfovibrio vulgaris strain Hildenborough. e-+O2·¯+2H+→SORH2O2Reaction1(Eq. 1) The SORs are characterized by a mononuclear iron active site shown in Scheme 1 with a ligand set consisting of a square plane of histidine nitrogens, an axial cysteine sulfur and, in the ferric form, a glutamate carboxylate (10Coehlo A.V. Matias P. Fülop V. Thomson A. Gonzalez A. Carrondo M.A. J. Biol. Inorg. Chem. 1997; 2: 680-689Crossref Scopus (123) Google Scholar, 11Yeh A.P. Hu Y. Jenney Jr., F.E. Adams M.W. Rees D.C. Biochemistry. 2000; 39: 2499-2508Crossref PubMed Scopus (163) Google Scholar). 1Fe-SORs contain this site as the only prosthetic group, whereas 2Fe-SORs contain an additional [Fe(Cys)4] site, the function of which remains enigmatic (12Emerson J.P. Cabelli D.E. Kurtz Jr., D.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3802-3807Crossref PubMed Scopus (42) Google Scholar). Kinetics investigations (13Emerson J.P. Coulter E.D. Cabelli D.E. Phillips R.S. Kurtz Jr., D.M. Biochemistry. 2002; 41: 4348-4357Crossref PubMed Scopus (83) Google Scholar, 14Abreu I.A. Saraiva L.M. Soares C.M. Teixeira M. Cabelli D.E. J. Biol. Chem. 2001; 276: 38995-39001Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) have established that the “resting” ferrous [Fe(His)4(Cys)] site reacts with superoxide in a diffusion-controlled fashion, producing a transient species, formulated as a ferric-(hydro)peroxo (cf. Scheme 1), that at pH ≥ 7 decays in a first-order process to the resting ferric state (14Abreu I.A. Saraiva L.M. Soares C.M. Teixeira M. Cabelli D.E. J. Biol. Chem. 2001; 276: 38995-39001Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Coulter E.D. Emerson J.P. Kurtz Jr., D.M. Cabelli D.E. J. Am. Chem. Soc. 2000; 122: 11555-11556Crossref Scopus (95) Google Scholar, 16Silaghi-Dumitrescu R. Silaghi-Dumitrescu I. Coulter E.D. Kurtz Jr., D.M. Inorg. Chem. 2003; 42: 446-456Crossref PubMed Scopus (67) Google Scholar, 17Lombard M. Houee-Levin C. Touati D. Fontecave M. Niviere V. Biochemistry. 2001; 40: 5032-5040Crossref PubMed Scopus (86) Google Scholar). To complete the catalytic cycle the SOR active site must collect an electron for regeneration of the ferrous state. Although superoxide is thermodynamically capable of reducing the ferric SOR site, no such reactivity is apparent, and, as a consequence, SORs show little or no superoxide dismutase (SOD) activity, even when the ligating glutamate carboxylate is replaced by non-coordinating side chains, such as alanine (5Jenney Jr., F.E. Verhagen M.F.J.M. Cui X. Adams M.W.W. Science. 1999; 286: 306-309Crossref PubMed Scopus (329) Google Scholar, 7Lombard M. Touati D. Fontecave M. Niviere V. J. Biol. Chem. 2000; 275: 27021-27026Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 13Emerson J.P. Coulter E.D. Cabelli D.E. Phillips R.S. Kurtz Jr., D.M. Biochemistry. 2002; 41: 4348-4357Crossref PubMed Scopus (83) Google Scholar, 14Abreu I.A. Saraiva L.M. Soares C.M. Teixeira M. Cabelli D.E. J. Biol. Chem. 2001; 276: 38995-39001Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Neither the kinetics of reduction of the ferric SOR site by other exogenous reducing agents, nor the SOR catalytic turnover cycle have been systematically studied, perhaps because the in vivo electron donor(s) to SORs have generally not been established. Thus, no value for the rate constant, k 3, in Scheme 1 has been reported for any SOR. The small electron transfer protein, rubredoxin, has been proposed to be a proximal electron donor to both 1Fe- and 2Fe-SORs (5Jenney Jr., F.E. Verhagen M.F.J.M. Cui X. Adams M.W.W. Science. 1999; 286: 306-309Crossref PubMed Scopus (329) Google Scholar, 18Brumlik M.J. Voordouw G. J. Bacteriol. 1989; 171: 4996-5004Crossref PubMed Google Scholar), and, consistent with this suggestion, addition of Pyrococcus furiosus 1Fe-SOR resulted in a slight acceleration of rubredoxin oxidation by superoxide (5Jenney Jr., F.E. Verhagen M.F.J.M. Cui X. Adams M.W.W. Science. 1999; 286: 306-309Crossref PubMed Scopus (329) Google Scholar). In at least two bacteria, the genes for 2Fe-SOR and rubredoxin are co-transcribed (18Brumlik M.J. Voordouw G. J. Bacteriol. 1989; 171: 4996-5004Crossref PubMed Google Scholar, 19Das A. Coulter E.D. Kurtz D.M. Ljungdahl L.G. J. Bacteriol. 2001; 183: 1560-1567Crossref PubMed Scopus (68) Google Scholar). For one of these bacteria, the sulfate-reducer, Desulfovibrio vulgaris, rubredoxin has been shown to preferentially reduce the [Fe(His)4(Cys)] site of 2Fe-SOR and to function as the proximal electron donor to 2Fe-SOR in an artificial NADPH:superoxide oxidoreductase reaction cycle shown in Scheme 2 and Equation 2, where FNR is the flavoprotein, spinach ferredoxin:NADP+ reductase (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar). In this system a calibrated flux of superoxide was generated from reduction of dissolved dioxygen using xanthine and xanthine oxidase, the well established method used to generate superoxide in SOD activity assays (21McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). x003C;INLINE-FIGx003E;x003C;LINKLOCATOR=“39662react2”x003E;x003C;/INLINE-FIGx003E;(Eq. 2) This report describes the kinetics of Reaction 2 using the catalytic system shown in Scheme 2, and analyzes the kinetics according to Scheme 1. The results demonstrate the kinetic competence of both 1Fe- and 2Fe-SORs to lower the steady state superoxide concentration to sublethal levels. The results also quantitate and clarify several qualitative mechanistic observations, presumptions, and speculations in the SOR literature. Reagents, Proteins, and General Procedures—Reagents and buffers were the highest grade commercially available. All reagents, protein, and media solutions were prepared using water purified with a Millipore ultrapurification system to a resistivity of ∼18 MΩ to minimize trace metal ion contamination. Bovine milk xanthine oxidase was purchased from Biozyme, Inc. as an ammonium sulfate suspension. Horse heart cytochrome c, bovine liver catalase, spinach FNR, Escherichia coli Fe-SOD, and bovine Cu/Zn-SOD were purchased from Sigma. Recombinant rubredoxin (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar), 2Fe-SOR (15Coulter E.D. Emerson J.P. Kurtz Jr., D.M. Cabelli D.E. J. Am. Chem. Soc. 2000; 122: 11555-11556Crossref Scopus (95) Google Scholar), E47A 2Fe-SOR (13Emerson J.P. Coulter E.D. Cabelli D.E. Phillips R.S. Kurtz Jr., D.M. Biochemistry. 2002; 41: 4348-4357Crossref PubMed Scopus (83) Google Scholar), and C13S 2Fe-SOR (12Emerson J.P. Cabelli D.E. Kurtz Jr., D.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3802-3807Crossref PubMed Scopus (42) Google Scholar), all from D. vulgaris, and recombinant Clostridium pasteurianum rubredoxin (22Richie K.A. Teng Q. Elkin C.J. Kurtz Jr., D.M. Protein Sci. 1996; 5: 883-894Crossref PubMed Scopus (44) Google Scholar) was expressed in E. coli and purified as described previously. Recombinant Treponema pallidum (23Jovanovic T. Ascenso C. Hazlett K.R. Sikkink R. Krebs C. Litwiller R. Benson L.M. Moura I. Moura J.J. Radolf J.D. Huynh B.H. Naylor S. Rusnak F. J. Biol. Chem. 2000; 275: 28439-28448Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and Desulfovibrio gigas 1Fe-SORs (24Silva G. LeGall J. Xavier A.V. Teixeira M. Rodrigues-Pousada C. J. Bacteriol. 2001; 183: 4413-4420Crossref PubMed Scopus (39) Google Scholar) were expressed, isolated, and purified by modifications of the published procedures, which are described under Supplemental Materials. Protein Concentrations and Activities—Concentrations of protein stock solutions were calculated using previously determined molar absorptivities: D. vulgaris rubredoxin, ϵ490 = 8,700 m–1 cm–1 (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar); C. pasteurianum rubredoxin, ϵ490 = 8,800 m–1 cm–1 (25Lovenberg W. Walker M.N. Methods Enzymol. 1978; 53: 340-346Crossref PubMed Scopus (21) Google Scholar); D. vulgaris 2Fe-SORpink and E47A 2Fe-SORpink, ϵ502 = 4,300 m–1 cm–1 (13Emerson J.P. Coulter E.D. Cabelli D.E. Phillips R.S. Kurtz Jr., D.M. Biochemistry. 2002; 41: 4348-4357Crossref PubMed Scopus (83) Google Scholar); C13S 2Fe-SORblue, ϵ645 = 1,900 m–1 cm–1 (12Emerson J.P. Cabelli D.E. Kurtz Jr., D.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3802-3807Crossref PubMed Scopus (42) Google Scholar) (with all 2Fe-SOR extinction coefficients per monomer of the homodimer). FNR concentration, catalase, and SOD activities were assumed to be those provided by Sigma. SOR Activity Assay—The NADPH:superoxide oxidoreductase assay described by Coulter and Kurtz (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar) was modified and used as described below. The buffer for all activity measurements and stock solutions was 50 mm phosphate containing 100 μm EDTA, pH 7.5. All reactions were conducted aerobically at room temperature. In 1 ml of buffer, aliquots of concentrated stock solutions were added to achieve concentrations of 500 μm xanthine, 1 μm FNR, ∼200 units/ml catalase, and 100 μm NADPH, respectively. The high catalase activity was added to remove hydrogen peroxide, which is not only a product of the SOR reaction but also a by-product of the xanthine/xanthine oxidase superoxide generating system (26Fridovich I. J. Biol. Chem. 1970; 245: 4053-4057Abstract Full Text PDF PubMed Google Scholar). After ∼30 s rubredoxin was added to a concentration of 1 μm. Approximately 30 s later either SOR (1 μm in [Fe(His)4(Cys)] sites) or SOD (10, 50, 100 or 250 units/ml) were added. After another ∼30 s, a pre-calibrated amount of xanthine oxidase (typically 5–15 μl of a stock xanthine oxidase solution prepared by diluting the ammonium sulfate suspension 5-fold with buffer) was added to initiate a flux of superoxide. The superoxide flux generated by the added xanthine oxidase was measured separately both before and after the assay using the same concentrations of xanthine and xanthine oxidase and the same buffer as in the assay mixture and measuring the rate of reduction of horse heart cytochrome c (ϵ550 = 21,500 m–1 cm–1), as described previously (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar). The rate of NADPH consumption in the SOR assay was measured as the decrease in absorbance at 340 nm (ϵ340 = 6,220 m–1 cm–1). For correlations with superoxide flux, the small background NADPH consumption rate measured prior to addition of xanthine oxidase was subtracted from that measured after the addition of xanthine oxidase. Variations on this protocol are described under “Results.” Effects of Cyanide and Azide on SOR Activity—The SOR activity assay was conducted as described above, but including small additions of ∼1 m sodium azide or sodium cyanide stock solutions, to achieve concentrations of either 1 or 20 mm azide or cyanide in the assay mixture. The superoxide flux was pre- and post-calibrated as described above but in the presence of the indicated concentrations of azide or cyanide. For some experiments, D. vulgaris 2Fe-SOR or E47A 2Fe-SOR, having concentrations of ∼860 or ∼700 μm in ferrous [Fe(His)4(Cys)] sites, respectively, were pre-incubated with ∼20 mm sodium cyanide for ∼20 h at 4 °C. Aliquots of these cyanide-incubated 2Fe-SOR solutions were added to the assay mixture as candidate SORs in the presence of either 1 or 20 mm cyanide in the assay mixture. Further details are given in the text and Table I.Table ISuperoxide flux and NADPH consumption rates in the absence or presence of azide or cyanide in the SOR assay of either wild-type (WT) or E47A D. vulgaris 2Fe-SORInhibitorSuperoxide fluxaSuperoxide fluxes were independently calibrated with the same concentrations of azide or cyanide as listed in the Inhibitor column.NADPH consumption rateCN- preincubation?WTE47Aμm/minμm/minNone21.5 ± 2.212.4 ± 1.411.4 ± 2.5NoN3- (1 mm)24.7 ± 2.914.3 ± 1.6NDbND, not determined.NoCN- (1 mm)22.1 ± 2.211.9 ± 1.912.2 ± 2.5NoCN- (20 mm)21.3 ± 1.512.5 ± 1.0NDNoCN- (1 mm)26.7 ± 1.912.0 ± 0.411.7 ± 0.6∼20 h/20 mmc2Fe-SOR was preincubated with 20 mm cyanide at 4 °C for 20 h prior to addition to the SOR assay mixture.CN- (20 mm)21.3 ± 1.51.7 ± 0.6ND∼20 h/20 mmc2Fe-SOR was preincubated with 20 mm cyanide at 4 °C for 20 h prior to addition to the SOR assay mixture.a Superoxide fluxes were independently calibrated with the same concentrations of azide or cyanide as listed in the Inhibitor column.b ND, not determined.c 2Fe-SOR was preincubated with 20 mm cyanide at 4 °C for 20 h prior to addition to the SOR assay mixture. Open table in a new tab Stopped-flow Monitoring of Reactions of C13S 2Fe-SORblue with Reduced Rubredoxin—All reactions, column chromatographies, and other manipulations of proteins were conducted in solutions buffered with 50 mm sodium phosphate, pH 7.5, at room temperature, unless otherwise indicated. D. vulgaris C13S 2Fe-SORblue was prepared by aerobically oxidizing ∼1 ml of as-isolated C13S 2Fe-SOR (∼500 μm in [Fe(His)4(Cys)] sites) by the addition of 2 eq of sodium hexachloroiridate (Aldrich). The oxidized C13S 2Fe-SOR was then passed over a 5-ml Hi-Trap desalting column (Amersham Biosciences). The resulting C13S 2Fe-SORblue solution was then diluted anaerobically to ∼400 μm in [Fe(His)4(Cys)] sites. Reduced D. vulgaris rubredoxin solutions were prepared by purging solutions of ∼500 μm oxidized (as-isolated) rubredoxin with argon for 20 min, in a 1-ml quartz cuvette. This sample was then divided into three sealed plastic tubes, and further diluted (∼5 ml total volume) anaerobically to final concentrations of 80, 40, and 20 μm rubredoxin. These solutions were again purged with argon for ∼20 min. These rubredoxin solutions were then reduced by titration with an anaerobic ∼20 mm sodium dithionite stock solution. The titrations were monitored until addition of a drop (∼1 μl) of the sodium dithionite stock solution gave no further decrease in absorbance at 490 nm. The reduced rubredoxin and C13S 2Fe-SORblue solutions were loaded under an argon purge separately into the two 2.5-ml drive syringes of a RSM-1000 stopped-flow spectrophotometer fitted with a rapid scanning monochromator (OLIS, Inc.). Following stopped-flow mixing, absorbance changes between 350 and 770 nm were monitored by rapid scanning at 25 °C. Specificity of the SOR Activity Assay—Previous work from our laboratory (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar) has demonstrated that D. vulgaris 2Fe-SOR can serve as the terminal component of the NADPH:superoxide oxidoreductase described by Reaction 2 and Scheme 2. Fig. 1 contains plots of NADPH consumption rates for this SOR assay using various candidate SORs. As shown in Fig. 1, the dimeric D. vulgaris 2Fe-SOR, the tetrameric D. gigas 1Fe-SOR (a.k.a. neelaredoxin) (27Chen L. Sharma P. Le Gall J. Mariano A.M. Teixeira M. Xavier A. Eur. J. Biochem. 1994; 226: 613-618Crossref PubMed Scopus (78) Google Scholar), and the dimeric T. pallidum 1Fe-SOR (7Lombard M. Touati D. Fontecave M. Niviere V. J. Biol. Chem. 2000; 275: 27021-27026Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 23Jovanovic T. Ascenso C. Hazlett K.R. Sikkink R. Krebs C. Litwiller R. Benson L.M. Moura I. Moura J.J. Radolf J.D. Huynh B.H. Naylor S. Rusnak F. J. Biol. Chem. 2000; 275: 28439-28448Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), all show very similar activities (listed under Supplemental Materials Table IS). The E47A variant of D. vulgaris 2Fe-SOR, in which the ligating glutamate shown in Scheme 1 has been changed to alanine, also shows activity indistinguishable from the wild type protein. Confirming the previous results (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar), no NADPH consumption above background occurred upon addition of xanthine oxidase if any of the other protein components were omitted from the assay mixture. Under the standard SOR assay conditions, but substituting either Cu/Zn- or Fe-SOD at 10, 50, 100, or 250 units/ml (corresponding to low micromolar levels of SOD) in place of SOR, NADPH was not consumed above background levels. Classical SODs are, thus, not active in this assay at concentrations comparable with SOR, presumably because the SODs cannot accept electrons from any of the donors present in the assay mixture (i.e. NADPH, FNR, or rubredoxin) at rates sufficient to outcompete the catalytic disproportionation of superoxide by the SOD. Consistent with this interpretation, the NADPH consumption associated with 2Fe-SOR in the assay was found to be inhibited by Cu/Zn- or Fe-SOD (cf. Supplemental Materials Fig. S1). In the case of Cu/Zn-SOD inhibition to background levels could be achieved by ∼1,000 units/ml. Thus, while classical SODs are not detectably active in the SOR assay, they do compete with SOR for superoxide, as expected (6Lombard M. Fontecave M. Touati D. Niviere V. J. Biol. Chem. 2000; 275: 115-121Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Effects of Cyanide and Azide on SOR Activity—Cyanide and azide are known to bind weakly to both the ferric and ferrous [Fe(His)4(Cys)] sites of SORs (28Romao C.V. Liu M.Y. Le Gall J. Gomes C.M. Braga V. Pacheco I. Xavier A.V. Teixeira M. Eur. J. Biochem. 1999; 261: 438-443Crossref PubMed Scopus (56) Google Scholar, 29Silva G. Oliveira S. Gomes C.M. Pacheco I. Liu M.Y. Xavier A.V. Teixeira M. Legall J. Rodrigues-Pousada C. Eur. J. Biochem. 1999; 259: 235-243Crossref PubMed Scopus (60) Google Scholar, 30Clay M.D. Jenney Jr., F.E. Hagedoorn P.L. George G.N. Adams M.W.W. Johnson M.K. J. Am. Chem. Soc. 2002; 124: 788-805Crossref PubMed Scopus (118) Google Scholar), and spectroscopic evidence indicates that these anions occupy the coordination site trans to the axial cysteine ligand. For this reason, cyanide has been presumed to be an inhibitor of SOR (31Shearer J. Fitch S.B. Kaminsky W. Benedict J. Scarrow R.C. Kovacs J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3671-3676Crossref PubMed Scopus (34) Google Scholar), even though no such inhibition data have been published. The data in Table I show that in fact, azide or cyanide at 1 mm caused no substantial inhibition (or activation) of SOR activity using the assay described here. The E47A variant of D. vulgaris 2Fe-SOR also was not inhibited by 1 mm cyanide or azide in the assay mixture. In fact, azide or cyanide added to the assay mixture at up to 20 mm showed neither substantial inhibition nor any significant change in the ratio of NADPH consumption rate to SOR flux (cf. Table I). However, when D. vulgaris 2Fe-SOR (or the E47A variant) was incubated for ∼20 h with 20 mm cyanide, and this cyanide-preincubated SOR was added to the assay mixture, inhibition was observed, but only if 20 mm cyanide was also present in the assay mixture. The preincubation with a large excess of cyanide results in slow air oxidation of the ferrous [Fe(His)4(Cys)] site (which is otherwise air-stable) and formation of the ferric [Fe(His)4(Cys)(CN–)] complex (28Romao C.V. Liu M.Y. Le Gall J. Gomes C.M. Braga V. Pacheco I. Xavier A.V. Teixeira M. Eur. J. Biochem. 1999; 261: 438-443Crossref PubMed Scopus (56) Google Scholar). Consistent with electrochemical data on synthetic model complexes (31Shearer J. Fitch S.B. Kaminsky W. Benedict J. Scarrow R.C. Kovacs J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3671-3676Crossref PubMed Scopus (34) Google Scholar), the binding of cyanide presumably shifts the reduction potential of the ferric [Fe(His)4(Cys)] site sufficiently negative so that the NADPH/FNR/rubredoxin system is unable to reduce it, thereby blocking electron flow at the SORox point of Scheme 2. This pattern of cyanide inhibition provides further confirmation that 2Fe-SOR, and, more specifically, its [Fe(His)4(Cys)] site, is the terminal catalytic component of the NADPH:superoxide oxidoreductase in this assay. Stoichiometry and Component Saturation in the SOR Activity Assay— Fig. 2 shows that, when the superoxide flux in the SOR activity assay of D. vulgaris 2Fe-SOR was varied over a range of 2.5(± 0.4) to 37(± 1.4) μm superoxide/min, a proportional increase occurred in the NADPH consumption rate. According to Reaction 2, the molar stoichiometry of the NADPH consumption rate to superoxide flux in the steady state should be 1:2. The slope of the fitted line in Fig. 2, ∼0.56, is consistent with the expected ratio, and shows that under these SOR turnover conditions, essentially all of the superoxide generated by the xanthine/xanthine oxidase system is consumed via the pathway diagrammed in Scheme 2. These results are consistent with those previously reported over a much smaller superoxide flux range (20Coulter E.D. Kurtz Jr., D.M. Arch. Biochem. Biophys. 2001; 394: 76-86Crossref PubMed Scopus (108) Google Scholar). Fig. 2 also shows that, under these conditions, the superoxide flux is the rate-limiting process for NADPH consumption. The concentration of D. vulgaris 2Fe-SOR in the activity assay was varied from 50 nm to 10 μm in [Fe(His)4(Cys)] sites, while maintaining all other component concentrations and superoxide flux constant at the standard assay levels. A plot of NADPH consumption rate versus 2Fe-SOR concentration under these conditions is shown in Fig. 3. Approximately 0.5 μm 2Fe-SOR is needed to reach maximum consumption of NADPH under the standard conditions. A least-squares fit of the Michaelis-Menten equation to the data gave the solid curve shown in Fig. 3 with V max of 11.6 ± 0.3 μm NADPH consumed per min and apparent Km (app) of ∼100 nm 2Fe-SOR (at a superoxide flux of 21.3 ± 0.7 μm/min). The physical meaning of this Km (app) is ambiguous. However, because from Fig. 2, the rate-limiting step at saturating 2Fe-SOR is production of superoxide, then, at less-than-saturating 2Fe-SOR concentrations (<0.5 μm from Fig. 2), the rate-limiting step must become the reaction of 2Fe-SOR with either superoxide or reduced rubredoxin. An analogous set of experiments was conducted in which the concentration of D. vulgaris rubredoxin was varied between 50 nm and 10 μm while maintaining all other concentrations and superoxide flux constant at the standard levels. A plot of the NADPH consumption rates versus concentration of rubredoxin is shown in Fig. 4. Approximately 0.45 μm rubredoxin was needed for the maximum NADPH consumption rate under these conditions. A fit of the Michaelis-Menten equation to the data gave V max of 11.8 ± 0.2 μm NADPH/min, and Km (app) of ∼100 nm rubredoxin (at a superoxide flux of 19.9 ± 0.8 μm/min). Thus, at <0.45 μm rubredoxin, the rate-limiting step must become either the reduction of rubredoxin by FNR or reduction of 2Fe-SOR by rubredoxin. Varying the concentrations of FNR (0.25–2 μm) or NADPH (50–300 μm) had very little effect on the superoxide-dependent NADPH consumption rates measured in this assay (cf. Supplemental Materials Figs. S2 and S3). The 2Fe-SOR/rubredoxin reaction (corresponding to the
Referência(s)