crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 45, pp. 27204 –27214, November 6, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Globins Scavenge Sulfur Trioxide Anion Radical* Received for publication, July 17, 2015, and in revised form, September 15, 2015 Published, JBC Papers in Press, September 17, 2015, DOI 10.1074/jbc.M115.679621

Paul R. Gardner1, X Daniel P. Gardner2, and X Alexander P. Gardner3 From the Miami Valley Biotech, Dayton, Ohio 45402 Background: Sulfite, an intermediate in sulfur metabolism, can be oxidized to the potentially toxic sulfur trioxide anion radical (STAR). Results: Diverse globins efficiently reduced STAR in vitro, and flavohemoglobin protected yeast from STAR toxicity. Conclusion: Globins can function as STAR scavengers. Significance: The data suggest roles for diverse globins in protecting cells against sulfite stress. Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and > 7,500 ⴛ 106 Mⴚ1 sⴚ1, respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate, and NAD(P)H are also large at ⬃100, 10, 130, and 30 ⴛ 106 Mⴚ1 sⴚ1, respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADPⴙ-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.

Sulfite-derived free radicals may contribute to sulfite toxicity within cells (1). Transition metals, metalloenzymes including cyt c oxidase, O2, 䡠O2⫺, photo-excited chromophores, and UV radiation oxidize (bi)sulfite (pKa ⫽ 7.2) (1–11) forming the mild oxidant sulfur trioxide anion radical (STAR)4 䡠SO3⫺, which can

* The work was supported in part by National Institutes of Health Grants R01 GM65090 (to P. R. G.) and P30 CA016058 and National Science Foundation Grant CHE-1040302 to the Mass Spectrometry and Proteomics Facility at the Ohio State University Campus Chemical Instruments Center. The authors declare that they have no conflicts of interest with the contents of this article. 1 To whom correspondence should be addressed: Miami Valley Biotech, Ste. 2445, 1001 E. Second St., Dayton, OH 45402. E-mail, paul.gardner@ mvbiotech.com. 2 Present address: Chemical Engineering Program, Ohio State University, W. 18th Ave., Columbus, OH 43210. 3 Present address: Biology and Chemistry Program, Illinois College, 1101 W. College Ave., Jacksonville, IL 62650. 4 The abbreviations used are: STAR, sulfur trioxide anion radical; Mb, myoglobin; GSH, glutathione; Cu,ZnSOD, copper and zinc-containing superoxide dismutase; MnSOD, manganese-containing superoxide dismutase; HbA, hemoglobin A; Ngb, neuroglobin; flavoHb, flavohemoglobin; IDH, isocitrate dehydrogenase; cyt c, cytochrome c; cyt b5, cytochrome b5; Sc,

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initiate a O2-consuming free radical chain reaction (5, 10, 12, 13). STAR reacts rapidly with O2 (12, 14) to form peroxysulfate radical, 䡠SO5⫺, a strong oxidant that can be reduced to peroxymonosulfate, HSO5⫺. HSO5⫺ can be further reduced to sulfate radical, 䡠SO4⫺, a stronger oxidant (10). In addition, 䡠O⫺ 2 is generated as an unavoidable by-product of aerobic oxidations by STAR or 䡠SO5⫺ (9), and STAR can form radical adducts (1, 15). More recently, neutrophil peroxidase and Cu,ZnSOD were shown to generate STAR in vitro (16 –19) and 䡠SO4⫺ oxidized and fragmented proteins in vitro (19). Evidence is surprisingly absent for the formation or toxicity of STAR, 䡠SO5⫺, or 䡠SO4⫺ within cells despite the ubiquitous formation of sulfite via reductive sulfate assimilation, and oxidative catabolism of sulfur amino acids or sulfide, and the common exposure of humans to sulfite through diet and atmospheric SO2. STAR formation has been judged relevant, but of uncertain significance, even in plants under photo-oxidative stress (20, 21). Erben-Russ et al. (22) suggested many years ago that the rapid reaction of STAR with antioxidants, such as GSH (23), would effectively detoxify STAR, if STAR ever formed. Sulfite oxidases and sulfite reductases are expected to maintain low intracellular sulfite levels (1, 24) and limit STAR formation. While investigating the reactivity of (bi)sulfite with oxyMb, we observed the oxidation of deoxyMb to metMb by STAR. We now report that the reaction of STAR with ferrous globins is exceptionally rapid with STAR reacting 260- and ⬃1000-fold faster with Ngb and flavoHb, respectively, than with GSH, suggesting a STAR detoxification function. By studying STAR formation, STAR reactivities, and Saccharomyces cerevisiae flavoHb-deficient mutants, we identify targets and modes for STAR toxicity and sulfite stress that help define the STAR detoxification function.

Experimental Procedures Materials—Crocin, Aspergillus niger glucose oxidase (600 units/ml), and all other reagents, unless otherwise specified, were obtained from Sigma-Aldrich. Crocin (catalog no. 17304), with a reported homogenous mass of ⬃999 g (M ⫹ Na⫹) by electrospray-MS, showed an extinction equal to 19,500 ⫾ 500 ⫺1 M cm⫺1 at 432 nm in methanol with additional maxima at 323 and 457 nm indicative of a mixture of the 13-cis- and allS. cerevisiae; Ec, Escherichia coli; Ca, Candida albicans; Re, Ralstonia eutropha.

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䡠SO3ⴚ Scavenging by Globins trans-crocetin di(␤-D-gentibiosyl) esters (25). Bovine erythrocyte Cu,ZnSOD and bovine liver catalase were obtained from Roche Applied Science. Catalase was resuspended in acetate buffer (pH 5.8), concentrated, filter-sterilized, and stored at 4 °C. CO-saturated water stocks were prepared as described (26). Superdex 200 was obtained from GE Healthcare Life Sciences. Human HbA was from John Olson (Department of Biochemistry and Cell Biology, Rice University, Houston, TX). Recombinant rat cyt b5, mouse Ngb, Ec MnSOD, Ec flavoHb, Ca flavoHb, Sc flavoHb, and Re flavoHb were prepared as described (27, 28). Sodium (bi)sulfite, pH 7.2, stock 1.0 M solutions were prepared by adding 1.0 M NaOH to an equal volume of 2.0 M sodium bisulfite and stored frozen at ⫺20 °C. The dinitrosyl iron complex was freshly prepared as described (29). A 1 ⫺ ⫺ M KI solution was incubated under air to produce I3 . I3 concentrations were determined by applying an extinction coefficient of 26.4 mM⫺1 cm⫺1 at 353 nm (30). BactoTM tryptone and BactoTM yeast extract were purchased from Thermo Scientific. For illumination, a high intensity blue light emitting diode (Radio Shack; catalog no. 276-0013), powered at 3.2 V and 20 mA and providing a peak emission at 462 nm with a bandwidth of ⬃50 nm at half-maximal intensity and an illuminance of ⬃20,000 lux at a 2-cm distance, was directed into a 1-ml cuvette at a non-interfering 90° angle to the spectrophotometer light path. OxyMb—Horse heart metMb (30 mg) was reduced with dithionite in a 1-ml volume of 50 mM potassium phosphate buffer, pH 7.8, containing 0.1 mM EDTA. Mb was separated on a Superdex 200 column, concentrated, and assayed for heme (26). Reduced cyt c—Horse heart cyt c(III) (55 mg) was reduced with dithionite, separated on a Superdex 200 column, and concentrated. Cyt c(II) was measured using an extinction coefficient of 27.7 mM⫺1 cm⫺1 at 550 nm (31). Mitochondrial Membranes—Mitochondria (1.2 g of wet weight) were isolated (32) from fresh chicken livers (900 g) purchased from a local market and were stored in 70-mg aliquots at ⫺70 °C. Aliquots were sonicated in 0.5 ml of 50 mM sodium MOPS, pH 7.0, buffer at 4 °C, and membranes were washed twice by centrifuging at 25,000 ⫻ g for 1 min. Membranes were resuspended in 0.5 ml of MOPS buffer and kept on ice. Cyt c oxidase activity was measured by following the oxidation of 20 ␮M cyt c(II) at 550 nm in 0.5 ml of 10 mM sodium MOPS buffer, pH 7.0, at 37 °C and by applying an extinction coefficient of 21.0 mM⫺1 cm⫺1 (31). Cyt c oxidase activity was 0.9 nmol s⫺1 per mg mitochondria or ⬃2.5 ␮M when applying a kcat of 350 s⫺1 (33) and a density of 1 g/ml. STAR Generation—STAR was generated by adding (bi)sulfite-oxidizing catalysts to O2-depleted buffer in 1-ml quartz cuvettes thermostatted at 37 °C. Unless otherwise indicated, buffer was depleted of O2 with glucose oxidase (0.6 unit/ml), 5 mM glucose, and 15 nM catalase (350 units/ml) for 5 min prior to adding (bi)sulfite and crocin. Catalysts were added, and STAR was detected by the oxidative bleaching of crocin (22) with high bisulfite concentrations or O2-depleted conditions, thus favoring oxidation by STAR over 䡠SO5⫺ or 䡠SO4⫺. Initial rates of crocin oxidation were generally followed at 480 nm (E ⫽ 10.3 mM⫺1 cm⫺1), a noninterfering wavelength. Unless indicated otherNOVEMBER 6, 2015 • VOLUME 290 • NUMBER 45

wise, 50 mM potassium phosphate, pH 7.2, containing 0.5 mM EDTA (buffer A) was used as the buffer. Mitochondrial cyt c oxidase-catalyzed STAR generation was assayed by following crocin (60 ␮M) oxidation in the presence of 200 ␮M O2 in 10 mM sodium MOPS buffer, pH 7.0, containing 0.5 mM EDTA, 5 mM sodium (bi)sulfite, and 10 ␮M cyt c(II) in a 0.5-ml volume at 37 °C. Rates were calculated by applying an extinction coefficient of 15.0 mM⫺1 cm⫺1 at 432 nm and by subtracting rates measured without added cyt c(II). STAR Scavenging—Bimolecular rate constants for STAR oxidation of deoxyMb and deoxyHbA, k⬘globin, were calculated from the inhibition of globin oxidation by GSH, where the percentage of inhibition ⫽ 100 (k⬘GSH[GSH])/(k⬘globin[globin] ⫹ k⬘GSH[GSH]), and k⬘GSH ⫽ 9.6 ⫻ 106 M⫺1 s⫺1 (22). Other bimolecular rate constants were calculated from inhibition of crocin oxidation, where the percentage of inhibition ⫽ 100(k⬘B[B]/ (k⬘crocin[crocin] ⫹ k⬘B[B]), k⬘crocin ⫽ 1.0 ⫻ 109 M⫺1 s⫺1 (22), and B is the competitor. Cyt c(II) or NADH were also used to estimate STAR reaction rate constants for targets with lower reaction rate constants. The extinction coefficients and wavelengths applied for measuring cyt c(II) and NADH oxidation were 21.0 mM⫺1 cm⫺1 at 550 nm (31) and 6.2 mM⫺1 cm⫺1 at 340 nm, respectively. Glucose oxidase (0.6 unit/ml), glucose (5 mM), and catalase (350 units/ml) were used to deplete O2 from reactions as indicated. Rate constants for the reaction of STAR with O2 were estimated from the difference in the rates of crocin (60 ␮M) bleaching in the absence or presence of 200 ␮M O2. Media, Cell Culture, Harvests, and Enzyme Assays—Starter cultures of S. cerevisiae parental strain BY4742 and the isogenic flavoHb-deficient yhb1⌬:KanMX strain 15887 (34) were grown overnight in 3 ml of LB medium (10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride per liter of tap water, pH 7.0) supplemented with 20 mM glucose in 15-ml culture tubes with aeration in a rotary shaker at 250 rpm at 30 °C. Experimental cultures were grown in 50-ml Erlenmeyer flasks in 10 ml of LB medium with specified glucose and were initiated with 1% inocula from overnight cultures. Flasks were agitated at various speeds in an Innova 3100 elliptical rotary shaker water bath at 30 °C (New Brunswick, Edison, NJ) to achieve varying levels of aeration. All cultures were grown under low light. Growth was monitored by the turbidity at 600 nm. Cells were harvested by centrifugation and rapidly frozen in a ⫺20 °C ethanol bath. Extracts were prepared by sonicating cell pellets in Y-Per S reagent (Thermo Scientific-Pierce) containing 2.5 mM sodium citrate and 0.6 mM MnCl2 and centrifuging lysates at 25,000 ⫻ g for 20 s. Aconitase activity was immediately measured at 30 °C with the coupled IDH assay (35). IDH activity was measured by following 2 mM isocitrate-dependent NADP⫹ reduction. Protein was measured at 280 nm with an absorbance of 1 equal to 1 mg/ml. Sulfonation of GSH and NADPH—S-Sulfoglutathione was synthesized by the Cu2⫹-catalyzed oxidation of (bi)sulfite essentially as described (36). NADPH (5 ␮mol) was sulfonated by 5 ␮mol Cu2⫹-catalyzed oxidation of (bi)sulfite (25 ␮mol) in a 1.1-ml volume, and the sulfo-NADP derivative was isolated in ⱖ50% yield. Copper ions were removed by batch Dowex 50w-X8 treatment. Sulfate and sulfite were removed by precipitation with barium acetate. The sulfo-NADP was fractionated JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 2. Cu,ZnSOD and MnSOD reduction by (bi)sulfite. A and B, Cu,ZnSOD (0.5 mM, A) and MnSOD (0.5 mM, B) were reduced with 5 mM (bi)sulfite in the absence (lines 1) or presence (lines 2) of 5 mM GSH or were incubated with 5 mM GSH (lines 3). The reactions were at 37 °C in buffer A depleted of O2 with glucose (5 mM), catalase (350 units/ml) and glucose oxidase (0.6 unit/ml) prior to (bi)sulfite addition.

FIGURE 1. Oxidation of Mb by (bi)sulfite. A–C, oxyMb (10 ␮M heme) was incubated with no additions (A), 3 ␮M Cu,ZnSOD (B), or 3 ␮M Cu,ZnSOD and 5 mM GSH (C) at 37 °C in 40 mM potassium phosphate buffer, pH 7.3. The spectra were recorded before (red lines) and at 2-min intervals after adding 20 mM sodium sulfite (black lines). D and E, OxyMb (5 ␮M) was deoxygenated in buffer A in the absence (D) or presence of 20 ␮M CO (E). Mb spectra were recorded prior to O2 depletion (solid red line), following deoxygenation and the addition of 5 mM (bi)sulfite (blue lines), or immediately after the addition Cu,ZnSOD (3 ␮M) (dashed red lines), and at 10-s intervals thereafter (solid black lines). E, potassium ferricyanide (10 ␮M) was added, and the spectrum was recorded after 20 s (dashed black line). Arrows indicate absorbance increases and decreases. OxyMb was deoxygenated with 0.1 unit/ml glucose oxidase, 350 units/ml catalase, and 5 mM glucose at 37 °C.

on AG1-X8 resin (Bio-Rad) with 0 –2 M ammonium acetate. Barium acetate was added (40 mg/ml), and the barium salt of sulfo-NADP was precipitated with 5 volumes of ethanol at ⫺20 °C, centrifuged, vacuum dried, and resuspended in water. Barium was replaced with ammonium (sulfate), and barium sulfate was removed by centrifugation. Sulfo-NADP and C21H29N7O20P3S, showed an absorbance maximum of 264 nm and shoulder at ⬃288 nm and an accurate molecular weight ⫽ 824.0412 (⬍1 ppm deviation) by MS.5

Results Oxidation of Ferrous Mb by (Bi)sulfite—In air-saturated buffer, (bi)sulfite simultaneously deoxygenated oxyMb (␭max ⫽ 414 nm) forming deoxyMb (␭max ⫽ 434 nm) and oxidized deoxyMb to metMb (␭max ⫽ 409 nm) (Fig. 1A). The addition of Cu,ZnSOD increased the rate and amount of metMb formed (Fig. 1B). MnSOD, Cu2⫹, and Fe2⫹/3⫹ also accelerated metMb formation (data not shown). GSH, a STAR scavenger (22), inhibited metMb formation catalyzed by Cu,ZnSOD (Fig. 1C). In an O2-depleted buffer (ⱕ1 ␮M), deoxyMb (Kd (O2) ⫽ ⬃0.9 ␮M) was similarly oxidized to metMb when Cu,ZnSOD was added to (bi)sulfite (Fig. 1D). Binding of CO to form 5

P. R. Gardner, unpublished results.

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MbFe2⫹CO (␭max ⫽ 422 nm) blocked ferrous heme oxidation (solid black lines) but did not prevent ferrous heme oxidation by ferricyanide (dashed black line) (Fig. 1E). The data suggest STAR generation and ferrous heme binding and oxidation. STAR Generation—In confirmation of the work of Ranguelova et al. (19), (bi)sulfite reduced Cu(II),ZnSOD and also Mn(III)SOD (Fig. 2, A and B, respectively, lines 1). The addition of GSH (5 mM) to scavenge STAR and prevent Cu(I) and Mn(II) oxidation increased the rate and extent of reduction (lines 2). GSH did not reduce Cu(II),ZnSOD or Mn(III)SOD (lines 3). Cu,ZnSOD and MnSOD were directly assayed for catalysis of STAR formation from the bleaching of the water-soluble carotenoid crocin (22). The reaction rate constants are summarized in Table 1. MnSOD catalyzed STAR generation more rapidly than Cu(II),ZnSOD as expected from the faster rate of Mn(III)SOD reduction (Fig. 2, A and B, respectively, compare lines 1). The ⬎1:1 stoichiometry of crocin oxidized to oxidant added suggests a chain reaction under the O2-depleted condition because of residual O2, crocin, and catalyst intermediates or intermediates in sulfate formation. Other oxidants also catalyzed STAR generation (Table 1). Ferricyanide oxidized (bi)sulfite to STAR with a rate constant ⬃5 orders of magnitude smaller than that for the solvent-exposed heme propionate of Mb (37) arguing for a direct oxidation of MbFe2⫹CO by ferricyanide in Fig. 1E. Notably, neither oxyMb nor oxyHb oxidized (bi)sulfite. Photo-excited FMN and cyt c oxidase generated STAR with high rate constants, thus confirming the early data and interpretations of Fridovich and Handler (7, 8). Contrary to an earlier report (6), the flavoenzyme glucose oxidase produced STAR in the presence of glucose and O2. STAR was also generated by I3⫺/I⫺, dinitrosyl iron complex, and HNO2, the latter agents of 䡠NO stress (38). Cu,ZnSOD and FMN/light showed favorable characteristics for use as STAR generators for kinetic competition assays (see below). These characteristics included catalytic amplification (Table 1) with initial rates of crocin bleaching measureable over the course of 20 –30 s. The initial rates were linearly dependent upon Cu,ZnSOD and (bi)sulfite concentrations (Fig. 3, A and B). FMN/light also showed a linear dependence with ⱕ0.4 ␮M VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015

䡠SO3ⴚ Scavenging by Globins TABLE 1 Rate constants for STAR formation by (bi)sulfite oxidizing catalysts The assays were at 37 °C in buffer A containing 5 mM sodium (bi)sulfite, 350 units/ml catalase, and 5 mM glucose. Crocin was included at 15 ␮M for deoxygenated reactions or 60 ␮M for air-saturated reactions. For all reactions, except cyt c oxidase, glucose oxidase, oxyMb, and oxyHbA, buffer was deoxygenated with glucose oxidase prior to adding (bi)sulfite and catalyst. Cu2⫹ catalysis was measured in 50 mM sodium MOPS buffer, pH 7.0. The data are reported for monomeric catalysts and represent the means of n ⱖ 3 measurements with standard deviations of ⬍30%. ND, not detected. Catalyst/Oxidant Cu(II),ZnSOD Mn(III)SOD Cu2⫹ Ferricyanide oxyMb oxyHbA FMN/light Cyt c oxidase Glucose oxidase I3⫺ (in 5 mM KI) DNIC HNO2 (pKa ⫽ 3.4)

Concentration

kⴕapparent

␮M

⫺1 ⫺1 M s

ⱕ12 ⱕ15 ⱕ4 ⱕ50 10 10 ⱕ0.4 ⱕ0.014 ⱕ0.5 ⱕ0.6 ⱕ3 ⱕ5

5.1 10.9 41 10.7 ND ND 4,700 460 43 410 31 4.1

Total crocin oxidized/catalyst 4.7 2.0 9.9 3.9 41 22 2.4

FIGURE 3. Cu,ZnSOD and FMN/light-catalyzed STAR generation. Initial rates of crocin oxidation were measured as a function of [Cu,ZnSOD] (dimer) with 5 mM (bi)sulfite (A), [(bi)sulfite] with 3 ␮M Cu,ZnSOD (B), [FMN] with 5 mM (bi)sulfite (C), or [(bi)sulfite] with 0.2 ␮M FMN (D). The rates were measured following Cu,ZnSOD addition (A and B) or illumination (C and D). The reactions were at 37 °C in 1-ml cuvettes in 1 ml of O2-depleted buffer A containing 15 ␮M crocin.

FMN (Fig. 3C), and the photo-excited FMN showed saturation by relatively low concentrations of (bi)sulfite (Fig. 3D). Oxidation of deoxyMb and deoxyHb by STAR—The reaction of STAR with deoxyMb was measured in the absence of O2 and O2-derived radicals (i.e. 䡠SO5⫺ and 䡠SO4⫺). FMN/light and (bi)sulfite were used to deplete O2 from buffers, reduce tracecontaminating metMb and produce a homogeneous deoxyMb spectrum (Fig. 4A). Under these conditions, light-generated FMNH䡠 reduced metMb, and STAR, faster than STAR oxidized deoxyMb. STAR, generated by adding MnSOD under low lux, oxidized deoxy Mb by ⬃70% after 60 s, and GSH inhibited NOVEMBER 6, 2015 • VOLUME 290 • NUMBER 45

FIGURE 4. STAR oxidation of deoxyMb and GSH competition. A, deoxyMb (10 ␮M heme) was exposed to STAR generated by adding MnSOD (12 ␮M), and the spectra were recorded at 5-s intervals. B, deoxyMb was exposed to STAR in the presence of increasing GSH concentrations, and the percentage of inhibition of deoxyMb oxidation was calculated from the absorbances at 434 nm after 10 s exposures (n ⫽ 3; ⫾ S.D.). The reactions were in 1.0 ml of buffer A containing 5 mM (bi)sulfite and 1 ␮M FMN in 1-ml cuvettes at 37 °C. Buffer was deoxygenated with a 1 min pre-exposure to light as described under “Experimental Procedures.” OxyMb was added to deoxygenated buffer, fully deoxygenated and reduced with 5 min of illumination, and exposed to MnSOD with only low (ⱕ500 lux) and intermittent (0.1 s) light exposure from the diode array spectrophotometer. Arrows indicate absorbance changes.

the reaction with 50% inhibition observed at ⬃40 ␮M GSH (Fig. 4B). STAR also rapidly oxidized deoxyHbA, but only by ⬃10% (Fig. 5) and in a low salt phosphate-(bi)sulfite buffer. Additional STAR exposures caused further rapid deoxyHbA oxidation; however, each subsequent STAR exposure oxidized only ⬃10% of the deoxyHbA (inset). For deoxyHbA, 50 and 200 ␮M GSH inhibited oxidation by 30.0 ⫾ 2.5 and 61.3 ⫾ 5.0% (n ⫽ 3 ⫾ S.D.), respectively. Similar low oxidation yields were observed with deoxyHbA formed by depleting O2 with glucose oxidase (data not shown). By applying a rate constant of 9.6 ⫻ 106 M⫺1 s⫺1 for the reaction of STAR with GSH (22), rate constants of 3.8 ⫻ 107 M⫺1 s⫺1 and 1.2 ⫻ 108 M⫺1 s⫺1 are estimated for reaction of STAR with deoxyMb and deoxyHbA, respectively. STAR Scavenging by Ngb—A mitochondrial NADH-cyt b5 reductase/cyt b5 system (32, 39) was reconstituted for reduction of metNgb under anoxic conditions. Mitochondrial membrane NADH-cyt b5 reductase efficiently reduced cyt b5 (Fig. 6A, inset) and reduced cyt b5 reduced metNgb at a constant rate of 1.5 ␮M min⫺1 with an estimated bimolecular rate constant of ⬃103 M⫺1 s⫺1 and achieved ⱖ 85% metNgb reduction at equilibrium (Fig. 6B, inset). DeoxyNgb was tested for its capacity to inhibit STAR-mediated crocin oxidation (Fig. 6C), and the percentage of inhibition of initial rates was calculated (Fig. 6D). Using the bimolecular rate constant of 1.0 ⫻ 109 M⫺1 s⫺1 for the reaction of STAR and crocin (22) and correcting for fractional metNgb reduction, an average reaction rate constant of 2.6 ⫻ 109 M⫺1 s⫺1 is estimated. STAR Scavenging by flavoHb—The STAR scavenging activity of reduced microbial flavoHb was measured from initial rates of STAR-mediated crocin oxidation in the presence of NADH. Inhibition was measured with increasing concentrations of Ec flavoHb (Fig. 7A, lines 1–7), and the percentage of inhibition of crocin oxidation was calculated (Fig. 7B, line 1). The effects of [Ca flavoHb] on crocin oxidation are also shown (line 2). Re flavoHb and Sc flavoHb were also tested at 1 ␮M. Reaction rate constants of 2.1 ⫻ 1010, 7.5 ⫻ 109, 1.9 ⫻ 1010, and 7.5 ⫻ 109 M⫺1 JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 5. STAR oxidation of deoxyHbA. DeoxyHbA (10 ␮M heme) spectra were recorded at 5-s intervals following three successive additions of MnSOD (12 ␮M) to generate STAR (arrows). Inset, the percentage of deoxyHb oxidized to metHb was calculated from the change in absorbance at 430 nm with the successive additions of MnSOD and STAR generation (arrows). The reactions were in 1 ml of a 20-fold dilution of buffer A, with 5 mM sodium bisulfite, and 1 ␮M FMN. Reaction buffer was deoxygenated with 1 min of preillumination as described under “Experimental Procedures.” OxyHb was added to deoxygenated buffer, fully reduced and deoxygenated using an additional 1 min of illumination, and exposed to MnSOD with only a low and intermittent lux from the spectrophotometer.

FIGURE 6. STAR scavenging by cyt b5-reduced Ngb. A, reduction of soluble rat cyt b5 (10 ␮M heme) by cyt b5 reductase containing mitochondrial membranes (1.4 mg/ml) and NADH (25 ␮M) was followed by recording spectra at 30-s intervals (dotted lines) to equilibrium (solid line). Cyt b5 reduction as measured at 423 nm is plotted (inset). B, MetNgb (5 ␮M heme) was added to the reduced cyt b5, and spectra were recorded at 30-s intervals (dotted lines) to equilibrium (solid line). Reduction of Ngb as detected at 423 nm is plotted (inset). C, 40 ␮M crocin and 3 ␮M Cu,ZnSOD were added to reactions containing reduced cyt b5 (10 ␮M) and 0, 5, 10, 15, or 20 ␮M of cyt b5-reduced Ngb (lines 1–5, respectively), and crocin oxidation was monitored at 480 nm. STAR generation was initiated by adding 5 mM (bi)sulfite at 0 s. Extrapolations of initial rates are shown as dashed lines. D, the percentage of inhibition of crocin oxidation was calculated from initial rates (n ⫽ 3; ⫾ S.D.). The reactions were at 37 °C in 1.0 ml of 10 mM sodium MOPS buffer, pH 7.0, depleted of O2 with 5 mM glucose, glucose oxidase (0.6 unit), and catalase (350 units).

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FIGURE 7. STAR scavenging by flavoHb and CO inhibition. A, effects of 0, 0.16, 0.31, 0.63, 1.3, 2.5, and 5.0 ␮M Ec flavoHb (heme) (colored lines 1–7, respectively) on STAR-mediated crocin oxidation. The data are staggered and extrapolated (dashed black lines) for presentation. B, percentage of inhibition of crocin oxidation by [Ec flavoHb] (line 1) and [Ca flavoHb] (line 2). C, crocin oxidation was measured in the absence (line 1) or presence of 1.0 ␮M Ec flavoHb alone (line 2) or with 10 ␮M CO (line 3) or without Cu,ZnSOD addition (line 4). Duplicate data are interpolated. The reactions were at 37 °C in 1.0 ml of buffer A containing 5 mM glucose and depleted of O2 with 0.6 unit of glucose oxidase and 350 units of catalase prior to adding 50 ␮M NADH, 30 ␮M crocin, 5 mM (bi)sulfite, flavoHb, and initiating STAR formation with 3 ␮M Cu,ZnSOD.

s⫺1 were estimated for the Ec, Ca, Re, and Sc flavoHb, respectively. 1 ␮M Ec flavoHb, a concentration that inhibited crocin oxidation by ⬃50% (Fig. 7C, compare lines 1 and 2), was tested for CO sensitivity. 10 ␮M CO decreased the inhibition of crocin oxidation (compare line 3 with line 2). Reactions of STAR with Biomolecules—The rate constants for reactions of STAR with biomolecules, determined with suitable competitors, are summarized in Table 2. The values were measured for GSH, ethanol, L-Trp, ascorbate, and O2 for comparison with published values. The values for several amino acids, NAD(P)H, and phenolic molecules including the antioxidant epigallocatechin-3-gallate were measured to further elucidate STAR reactivity. L-Cys rapidly reduced Cu(II),ZnSOD (data not shown), and a rate constant was determined only for STAR generated with photo-excited FMN. Differences in the values estimated using Cu,ZnSOD and FMN/light as STAR generators may be due to specific interfering reactions. For example, FMNH䡠 formed via photoreduction reduces O2 to 䡠O2⫺, and 䡠O⫺ 2 can diminish or enhance the apparent O2 rate constant by acting as a facile STAR reactant or STAR generator (9, 11). STAR Toxicity Detoxification in Yeast—Evidence for damage to critical targets and scavenger protection within cells is essential for demonstrating a detoxification function (35, 38, 40). S. cerevisiae, an organism bearing mitochondria that produce sulfite (41, 42) but lack the sulfite-detoxifying sulfite oxidase (43) and that harbor the STAR-scavenging flavoHb (Yhb1p) VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015

䡠SO3ⴚ Scavenging by Globins TABLE 2 Rate constants for reactions of STAR with biomolecules Unless otherwise indicated, measurements were made in buffer A at 37 °C as described under “Experimental Procedures.” Crocin was generally used at 15 ␮M; when O2 was the competing reactant, 60 ␮M was used. Measured k⬘ox values are the means of n ⱖ 3 with standard deviations of ⬍30%. Reactant Crocin GSH

Reactant concentration

kⴕox

␮M

⫺1 ⫺1 M s

ⱕ2,000 ⱕ2,000

Ethanol

106 106

Ascorbate

ⱕ1,000 200 5,000 200 200 ⱕ900 140–660 ⱕ400 400 400 400 ⱕ10 ⱕ2,000 500

O2

NADH NADPH Cyt c(II) L-Cys L-Trp L-Met L-His

Epi EGCG

1,000 5,000 ⱕ600 ⱕ600 ⱕ200 ⱕ200

1.0 ⫻ 109 8.9 ⫻ 106 9.6 ⫻ 106 9.6 ⫻ 106 2.3 ⫻ 103 2.2 ⫻ 103 ⱕ 2 ⫻ 103 7.4 ⫻ 107 1.9 ⫻ 108 0.9–1.3 ⫻ 107 1.0 ⫻ 108 4.3 ⫻ 107 1.5 ⫻ 109 1.1–2.3 ⫻ 109 1.9 ⫻ 107 4.4 ⫻ 107 2.2 ⫻ 107 1.9 ⫻ 107 4.4 ⫻ 106 1.7 ⫻ 107 2.4 ⫻ 104 ⬃8 ⫻ 104 9.6 ⫻ 103 6.4 ⫻ 102 2.8 ⫻ 107 1.9 ⫻ 107 3.2 ⫻ 108 8.9 ⫻ 107

(44), was employed to explore the capacity of a globin for STAR scavenging detoxification in a cellular milieu rich in STARreactive GSH, thiols, NAD(P)H, and varied O2. We were unable to observe selective growth inhibition of a Yhb1p-deficient yeast mutant relative to its isogenic parent by adding (bi)sulfite to cultures (data not shown) perhaps because of multiple mechanisms for sulfite removal (e.g. cytosolic sulfite reductase and sulfite efflux pumps) and toxicity (e.g. sulfitolysis, nucleophilic additions, and extracellular radical chains). Nevertheless, the ⌬yhb1 strain was observed to grow slower and to lower densities than its isogenic parent following glucose depletion and the shift to oxidative metabolism (Fig. 8A, red square; compare dashed lines with solid lines), suggesting STAR-mediated damage to mitochondria. Lower culture aeration and a lower [O2] (black square, partially relieved the growth inhibition of the mutant (dashed line), demonstrating an O2-dependent toxicity. In support of sulfite/STAR involvement, supplementation of cultures with sulfide (white square), a metabolic precursor to mitochondrial sulfite (41, 42), further impaired the growth of the Yhb1p-deficient mutant (dashed line) but not the parent (solid line). Neither aeration (black circle) nor sulfide (white circle) affected the growth of the Yhb1p-deficient strain, as observed after 7 h of glucose-limiting growth (red square), when sufficient glucose was provided for fermentation (compare dashed lines with solid lines), further demonstrating critical damage by STAR, or its metabolites, to oxidative metabolism. In a limited search, two critical sites of damage to oxidative metabolism were identified. At moderate culture aeration achieved at 200 rpm (Fig. 8B, top panel), the yhb1⌬ strain expressed ⬃50% lower aconitase activity than the parent strain (Fig. 8B, top panel), whereas NADP⫹-IDH activity, measured as NOVEMBER 6, 2015 • VOLUME 290 • NUMBER 45

STAR generator

Competitor

Pulse radiolysis Cu,ZnSOD FMN/light pulse radiolysis Cu,ZnSOD FMN/light flash photolysis Cu,ZnSOD FMN/light pulse radiolysis Cu,ZnSOD FMN/light pulse radiolysis pulse radiolysis Cu,ZnSOD FMN/light Cu,ZnSOD FMN/light Cu,ZnSOD FMN/light Cu,ZnSOD pulse radiolysis Cu,ZnSOD Cu,ZnSOD Cu,ZnSOD FMN/light Cu,ZnSOD FMN/light

None Crocin Crocin Crocin Crocin Crocin None Crocin Crocin None Crocin Crocin Ascorbate None Crocin Crocin Crocin Crocin NADH Crocin Cyt c(II) None Cyt c(II) Cyt c(II) Crocin Crocin Crocin Crocin

Reference Ref. 22 Ref. 22 Ref. 10 Ref. 12 and 23 Ref. 12 Ref. 14 and 56

Ref. 86

FIGURE 8. Protective effects of flavoHb (Yhb1p) expression in S. cerevisiae. A, parental (solid lines) and Yhb1p-deficient (broken lines) yeast cultures were grown in LB medium containing 5 mM glucose (squares) or 20 mM glucose (black circles) with moderate aeration (200 rpm) (red squares) or low aeration (100 rpm) (black squares) and growth was followed by turbidity at 600 nm. Sodium sulfide (30 ␮M) was added at the arrow for low glucose (white squares) and high glucose (white circles) growth with low aeration. 1% inocula from overnight cultures were used to initiate cultures. The data are representative of two or more trials. B, aconitase and IDH activities were measured in parental (black bars) and yhb1⌬ (red bars) yeast harvested immediately after 30 h of growth at the indicated agitation rate (solid bars) or following 20 min of stasis (open bars). The error bars represent the standard error of the mean of eight independent cell harvests and assays.

a control, was unaffected (Fig. 8B, bottom panel). Further, aconitase was activated by ⬃50% following 20 min of culture stasis and respiratory-induced anoxia (open bars), thus demonstrating the accumulation of a large fraction of reactivatable [3Fe4S]-containing aconitase in the ⌬yhb1 strain, similar to that observed with 䡠O2⫺ stress (35, 45). Aconitase activity differences JOURNAL OF BIOLOGICAL CHEMISTRY

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䡠SO3ⴚ Scavenging by Globins The product of the rapid reaction of STAR and NADPH, sulfo-NADP (see below), competitively inhibited the yeast NADP⫹-IDH activity with respect to NADP⫹ as measured during the initial 2 min of catalysis (Fig. 9B). The extract activity showed an apparent Km (NADP⫹) of ⬃5 ␮M and was inhibitable to ⬃50% with an estimated Ki (sulfo-NADP) of ⬃10 ␮M, suggesting a sensitivity of either the mitochondrial, or the cytosolic, NADP⫹-IDH isozyme to sulfo-NADP. Following 20 min of catalysis, inhibition by sulfo-NADP (Fig. 9C, line 1) was not reversed by adding a competitive excess of NADP⫹ (line 2), thus revealing a progressive or suicide inactivation of IDH by sulfo-NADP and supporting a role for STAR in the loss of IDH activity within yeast.

Discussion Reduced globins reacted rapidly with STAR. Reaction rate constants ranged over nearly 3 orders of magnitude from 3.8 ⫻ 107 M⫺1 s⫺1 for deoxyMb to a remarkable 2.1 ⫻ 1010 M⫺1 s⫺1 for Ec flavoHb, a value exceeding that for 䡠NO dioxygenation by ⬃9-fold (47, 48). We can use the conventional hemispherical formula to approximate the diffusion limit for the reaction, k ox ⫽ 2␲␣DSTAR,37共N/1000兲

FIGURE 9. Effects of STAR-derived sulfonates on aconitase and IDH. A, aconitase activity was measured in the presence of the indicated concentrations of S-sulfoglutathione (line 1) or GSH (line 2) after 20 min. B, IDH activity was determined from initial rates with 100, 20, 10, or 5 ␮M NADP⫹, lines 1– 4, respectively, and the indicated concentrations of sulfo-NADP as described under “Experimental Procedures.” C, IDH activity was measured with 50 ␮M NADP⫹ and the indicated concentrations of sulfo-NADP. Activity was determined from initial rates (line 1) and following the addition of 200 ␮M NADP⫹ after 20 min of catalysis (line 2). The error bars represent the standard deviation from the mean for three independent measurements.

were less, or negligible, in cells grown under hypoxic conditions (ⱕ 150 rpm). Unexpectedly, however, respiratory-induced anoxia caused aconitase activity losses in cells grown under hypoxia, and greater losses were observed in yhb1⌬ cells. Moreover, IDH also showed activity losses following hypoxic growth and respiratory-induced anoxia (Fig. 8B, bottom panel), albeit less marked (ⱕ 30%), suggesting hypoxic mechanisms for STAR toxicity. Sensitivity of Aconitase and IDH to STAR and Sulfonates— STAR, generated by MnSOD or Cu2⫹-catalyzed (bi)sulfite oxidation, did not directly inactivate aconitase in the coupled NADP⫹-IDH assay (data not shown). Nor was aconitase inactivated by S-sulfoglutathione, a product of STAR and GSH (see below), but rather aconitase was activated (Fig. 9A, line 1). In contrast, GSH caused progressive aconitase inactivation (line 2) presumably because of enhanced O2 sensitivity. Similar effects of GSH on aconitase stability have been reported for 䡠NO (46). The data suggest indirect mechanisms for STAR-mediated damage to aconitase within the yhb1⌬ strain involving 䡠O2⫺ (35, 45).

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(Eq. 1)

where ␣ equals the ⬃14 Å radius of a positively charged cavity for STAR capture near the heme formed by Lys-11, Arg-134, Arg-184, Lys-373, Lys-381, His-386, His-393, and Lys-394 (Protein Data Bank code 1GVH) plus the radius of STAR (⬃1.4 Å), DSTAR,37 is the diffusion coefficient for STAR at 37 °C, N is Avogadro’s constant, and the small flavoHb diffusion constant is ignored. Using 1.54 ⫻ 10⫺7 cm for ␣ and Dnitrate,37 ⫽ 2.5 ⫻ 10⫺5 cm2 s⫺1 for NO3⫺ (49), a ⬃0.2 Å smaller isomorphic trigonal planar anion, in place of an unknown value for STAR, we calculate k⬘ox ⫽ 1.5 ⫻ 1010 M⫺1 s⫺1, a value not too far from the value measured. As pointed out by Chou and Jiang (50) and Chou and Zhou (51), the equation ignores diffusion along the protein surface from ⬎180° and electrostatic forces. In flavoHb, the cavity is complex with projections and crevices and a net charge of approximately ⫹4. The cavity, or short channel, may also serve in the efflux of the product SO32⫺ and the 䡠NO dioxygenation product NO3⫺ (38, 52). CO inhibited STAR reduction by deoxyMb and Ec flavoHb (Figs. 1E and 7C). STAR, with a dismutation rate constant of ⬃5.5 ⫻ 108 M⫺1 s⫺1 (1, 10), and high reactivity with crocin, did not accumulate to sufficient levels to outcompete 10 –20 ␮M CO with respective kon values of 0.5–2 ⫻ 106 M⫺1 s⫺1 for ferrous Mb and Ec flavoHb (47, 48). In further support of binding reduction of STAR by the ferrous iron, deoxyHbA in a tense conformation with subunits bearing predominantly out of plane iron (53, 54) largely resisted oxidation by STAR (Fig. 5). Only the smaller fraction of deoxyHbA subunits with an inplane iron, similar to that in Mb, appear accessible to STAR binding reduction. O2 binding also apparently inhibited the STAR reaction; heme oxidation and STAR scavenging was only observed with the deoxy forms. Together, the results suggest a mechanism for globin-catalyzed STAR reduction (Scheme 1) in which ferric heme is reduced by 1e⫺ (Reaction 1), ferrous heme transiently interacts with STAR (Reaction 2), reduces STAR to VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015

䡠SO3ⴚ Scavenging by Globins

SCHEME 1

sulfite (Reaction 3), and releases sulfite (Reaction 4). In the catalytic cycle, O2 or CO can bind the ferrous heme and inhibit the STAR reductase activity. Indeed, the linear rates of crocin bleaching inhibition observed with low [flavoHb] (Fig. 7) demonstrate enzymatic turnover. We suppose that turnover would be limited by heme reduction (Reaction 1) as described for nitric-oxide dioxygenase catalysis (48, 52). The rate constants reported in Table 1 describe methods for generating STAR but also provide a basis for modeling STAR generation within cells. Low Km (⬃20 ␮M) and large kcat/Km (⬃5 ⫻ 106 M⫺1 s⫺1) values reported for sulfite oxidase (55) suggest a low steady-state [sulfite] and low rate of STAR formation in most cells under normal conditions. For example, we can estimate that the ⬃2.5 ␮M of cyt c oxidase measured in chicken liver mitochondria reacting with 20 ␮M (bi)sulfite with a bimolecular rate constant of 460 M⫺1 s⫺1 (Table 1), or ⬃10,000-fold less efficiently than sulfite oxidase, would generate STAR at a relatively low rate of ⬃1.4 ␮M min⫺1. However, the availability of co-substrate cyt c(III), molybdenum cofactor (43), and inhibitory anions (e.g. chloride, Ki ⫽ 4 mM, and phosphate) (55), and sulfite biosynthesis or influx rates must be considered when approximating the sulfite removal capacity of sulfite oxidase and steady-state sulfite levels. Moreover, in any cell or organelle, the sum of STAR sources requires computation. Rate constants for reactions of STAR with several molecules were measured to validate the kinetic competition methods and to evaluate the STAR reactivity of globins. Rate constants determined for GSH, ethanol, and L-Trp were similar to those previously reported (Table 2). However, ascorbate and O2 values differed beyond that expected for small pH and temperature differences and require resolution. The ⬃10-fold smaller ascorbate rate constant (12, 23) may be partly resolved by accounting for the back reaction, namely ascorbyl radical reduction by (bi)sulfite. The 10 –50-fold lower O2 value we report is particularly significant because the STAR scavenging detoxification capacity of a globin becomes feasible at physiological [O2]. The larger rate constant for O2 was initially derived from models of multiple reactions and data fitting including the smaller ascorbate rate constant and with other cited possible complications (12) that may have produced an overestimate. Subsequent O2 rate constants determined using large ␥ ray doses to generate ⬎10⫺4 M STAR and to measure O2-enhanced STAR decay rates (14, 56) may have also produced overestimates. Thus, 䡠O2⫺ generation from e⫺(aq) and O2 in a favorable competition with N2O (57), and the rapid reaction of 䡠O2⫺ with STAR (9) (k11 ⫽ ⬃4 ⫻ 1010 M⫺1 s⫺1 (11)) would have rapidly depleted O2 (58) and dominated the O2-dependent component of STAR decay. In NOVEMBER 6, 2015 • VOLUME 290 • NUMBER 45

addition to the data in Table 2, we can estimate a rate constant of ⬃4 ⫻ 108 M⫺1 s⫺1 for the back reaction of STAR with the Cu(I),ZnSOD monomer from the net rate of Cu(II),ZnSOD reduction by (bi)sulfite where the theoretical rate at 0.5 mM Cu(II),ZnSOD is 25.5 ␮M s⫺1 using a k⬘apparent of 5.1 M⫺1 s⫺1 (Table 1) and where rates of 0.05 ␮M s⫺1 and 8.3 ␮M s⫺1 are measured in the absence and presence of 5 mM GSH, respectively (Fig. 2A). STAR is a moderately strong oxidant (Fig. 10A) and can form radical adducts (1). A consensus STAR reduction potential of ⫹0.73 V was reported for neutral pH (59). Fig. 10B shows a summary plot of the log of rate constants versus reduction potential differences for reactions of STAR. The hypothetical dashed blue line represents the linear relationship expected for barrier-free electron transfer and highlights the large ⌬E0⬘ required for a bona fide diffusion-limited scavenger. FlavoHb with a reported heme reduction potential of ⫺0.125 V (60) showed a 4 – 8-fold larger rate constant than Ngb at ⫺0.129 V (61), which may be due to hindrance of STAR access by the distal His E7 Fe2⫹ ligand. The analysis also reveals an ascorbate rate constant more consistent with a calculated ⌬E0⬘ ⫽ ⫹0.448 V. On the other hand, the analysis (Fig. 10B) shows that STAR reacts too fast with GSH, L-Cys, NAD(P)H, and crocin for an electron transfer. We suppose that the reactions with thiols and NAD(P)H proceed via inner sphere bonding forming a sulfo adduct and reducing anion radical (Reaction 1) (15) that is oxidized by O2 to form a sulfonate (Reaction 2). Indeed, STAR conjugates with GSH or NADPH in the copper-catalyzed synthesis of S-sulfoglutathione (36) and a sulfo-NADP,5 respectively. Conversely, bonding of STAR to the polyunsaturated crocin molecule generates a sulfo adduct and an oxidizing radical (Reaction 3). Reduction by sulfite would generate another STAR (Reaction 4) and may partly account for the ⬎1:1 stoichiometry of crocin oxidized measured for Cu(II),ZnSOD, Mn(III)SOD, Cu2⫹, ferricyanide, and dinitrosyl iron complex under the O2-depleted conditions of the STAR generation assay (Table 1). A zero sum STAR reaction is consistent with the negligible effect of crocin on the O2-depleting free radical chain oxidation of sulfite (data not shown). RH ⫹ 䡠SO 3⫺ ¡ R䡠SO 3 H ⫺ R䡠SO 3 H ⫺ ⫹ O 2 ¡ 䡠RSO 3⫺ ⫹ O 2⫺ ⫹ H ⫹ R ⫹ 䡠SO 3⫺ ¡ 䡠RSO 3⫺ 䡠RSO 3⫺ ⫹ SO 32⫺ ⫹ H ⫹ ¡ RHSO 3⫺ ⫹ 䡠SO 3⫺ R EACTIONS 1– 4 While the kinetic data argue for STAR formation and toxicity and STAR scavenging by globins within cells, yeast offered a tractable cell model for exploring STAR formation, toxicity and the capacity of a globin for STAR detoxification. S. cerevisiae express an 䡠NO-inducible flavoHb, Yhb1p (62), with an established nitric-oxide dioxygenase activity (34, 47) and 䡠NO detoxification function (44, 63) and an undefined protective activity against O2 and oxidative stress (44, 64, 65). Within yeast, Yhb1p localizes to the cytosol and mitochondria (44) at ⬃5 ␮M. JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 10. Redox potentials and STAR reaction rate constants. A, bisulfite and sulfite are oxidized to STAR. STAR is reduced to sulfite through an electron transfer or to bisulfite via an H-atom abstraction. B, STAR rate constants (k⬘ox) determined here (䡺) or previously reported (F) are plotted on a log scale against redox potential differences (⌬E0⬘) where a STAR reduction potential of ⫹0.73 V is applied. Multiple rate constant estimates are grouped (thin dashed lines). The hypothetical dependence of log k⬘ox on ⌬E0⬘ is shown by the dashed blue line. The values highlighted by the red box exceed the expected values. The reduction potentials (relative to NHE) applied are: NAD radical (NAD(P)H), ⫹1.38 (71); L-methionine radical (Met), ⫹1.19 (72); L-histidine radical (His), ⫹1.17 (73); L-tryptophanyl radical (Trp), ⫹1.05 (74); 1-hydroxyethyl radical (EtOH), ⫹0.98 (75); L-glutathionyl radical (GSH), ⫹0.92 (76); L-cysteinyl radical (Cys), ⫹0.92 (77); crocin radical, ⫹0.74 (78); epinephrine radical (Epi), ⫹0.345 (79); ascorbyl radical (Asc), ⫹0.282 (80); cyt c(III) (cyt c), ⫹0.254 (81); L-epigallocatechin-3-gallate radical (EGCG), ⫹0.43 (82); metHbA (HbA), ⫹0.144 (83); Cu(II),ZnSOD (SOD), ⫹0.102 (84); metMb (Mb), ⫹0.046 (85); Ec, Re, Sc, and Ca flavoHbs (flavoHb(s)), ⫺0.125 (60); and metNgb (Ngb), ⫺0.129 (61).

Although yeast have the capacity to detoxify sulfite via a cytoplasmic sulfite reductase and an inducible sulfite efflux pump that is co-regulated with Yhb1p (24, 62), yeast uniquely lack the molybdenum cofactor (43) and a mitochondrial sulfite oxidase and are thus expected to experience elevated mitochondrial (bi)sulfite, increased STAR formation, and STAR-mediated mitochondrial damage as a consequence of basal mitochondrial sulfur amino acid and sulfide metabolism to sulfite, which in most yeast strains amounts to significant sulfite formation (66). Indeed, nonfermentative mitochondria-dependent growth of the Yhb1p-deficient mutant strain was impaired, and metabolic loading of mitochondria with sulfite, through sulfide supplementation, selectively decreased mutant growth (Fig. 8A). Formation of 䡠SO5⫺ from the reaction of STAR with O2 and formation of 䡠O2⫺ (9) (Reactions 1 and 2), HSO5⫺, and 䡠SO4⫺ from the reactions of STAR, 䡠SO5⫺, and HSO5⫺ with cellular reductants provide one mode for STAR toxicity that can explain the greater growth inhibition of yhb1⌬ cells exposed to a greater [O2]. Although appearing as a small and indirect measure of STAR toxicity, the 25–50% inactive fraction of aconitase measured in yhb1⌬ cells indicates large 5–10-fold increases in mitochondrial 䡠O2⫺ levels (35, 45) and severe oxidative stress. Formation of sulfonates in reactions of STAR with NAD(P)H (Reactions 1 and 2) and inhibition inactivation of NADP⫹-IDH and other dehydrogenases by sulfo-NAD(P) during hypoxia and other low NAD(P)⫹ conditions provide an additional mode for STAR toxicity and increased 䡠O2⫺ generation. The data in Table 2 and a rate equivalence of k⬘ox (Ngb)[Ngb] [STAR] to k⬘ox (GSH)[GSH][STAR] or k⬘ox (ascorbate)[ascorbate] [STAR] allow us to estimate a STAR scavenging capacity for the ⬃100 ␮M Ngb expressed in the light-susceptible retinal rod cell inner segment and mitochondria (67, 68) well beyond that of physiological [GSH] and equal to ⬃2 mM ascorbate. Similar approximations can be made for all globins but estimates must consider globin oxidation and reduction rates and O2 competi-

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tion (Scheme 1). For Ngb, reducing systems (e.g. cyt b5) would appear adequate, and O2 inhibition would appear minimal because ⬍12% of ferrous Ngb binds O2 at the low retinal O2 concentrations (69, 70). Further investigations of STAR generation, STAR reactivity, and STAR metabolite (e.g. sulfoNAD(P), S-sulfoglutathione, and S-sulfocysteines) formation and interactions will be required to fully understand STAR toxicity and the STAR detoxification function of globins. Author Contributions—P. R. G. coordinated the experiments, collected data, and wrote the paper. D. P. G. designed experiments for measuring STAR formation and scavenging and collected data for Figs. 2, 3, and 10 and Tables 1 and 2. A. P. G. prepared mitochondrial membranes, designed and performed experiments describing cyt c oxidase activities and Ngb reduction, and acquired data for Tables 1 and 2 and Figs. 6 and 10. All authors reviewed the results and approved the final version of the manuscript. Acknowledgments—We are indebted to Drs. Thorsten Burmester and Thomas Hankeln for providing the Ngb expression plasmid. We thank Prof. John S. Olson for providing human oxyHbA and Ca flavoHb. We gratefully acknowledge Dr. Hao Zhu and Prof. Austen Riggs for supplying the Sc and Re flavoHbs. We thank Jeremy Keirsey and Dr. ´ rpa´d Somogyi for expert assistance with mass spectrometry. We A thank Prof. Irwin Fridovich for discussion and critical comments and dedicate this work to him on the occasion of his 86th birthday.

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