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Originally published In Press as doi:10.1074/jbc.M000373200 on April 21, 2000

J. Biol. Chem., Vol. 275, Issue 27, 20391-20398, July 7, 2000
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Reaction of Human Myoglobin and H2O2

INVOLVEMENT OF A THIYL RADICAL PRODUCED AT CYSTEINE 110*

Paul K. WittingDagger , D. J. Douglas§, and A. Grant MaukDagger

From the Departments of Dagger  Biochemistry and Molecular Biology and § Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received for publication, January 18, 2000, and in revised form, April 17, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H2O2 (protein/H2O2 = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO·) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g ~ 2.036) was detected after decay of Trp-OO· that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios <= 1:10 mol/mol, a DMPO adduct exhibiting broad absorptions was detected. Hyperfine couplings of this radical indicated a DMPO-thiyl radical. Incubation of wild-type human Mb with thiol-blocking reagents prior to reaction with peroxide inhibited DMPO adduct formation, whereas at protein/DMPO ratios >= 1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H2O2 demonstrated the formation of a homodimer (mass of 34,107 ± 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H2O2 differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein-centered free radicals are now well recognized as normal intermediates for an increasing number of enzymes (1). Examples of such radical intermediates include the thiyl (2) and tyrosyl (3) radical centers of ribonucleotide reductase, the tyrosyl radical of prostaglandin H synthase (4), the glycyl radical of pyruvate formate-lyase (5), and the tryptophan radical of cytochrome c peroxidase (6). On the other hand, detrimental protein-centered radicals have been implicated in oxidative stress associated with pathology of inflammatory disease(s) and possibly in aging (7, 8). At least some of the toxic effects of protein radicals, as exemplified by initiation of lipid peroxidation (9-12), involve both intramolecular (13) and intermolecular (14) translocation of radical centers from one amino acid residue to another. One particularly well studied example of radical translocation is provided by the protein-centered radical formed by myoglobin upon reaction with hydrogen peroxide.

Myoglobin (Mb)1 has a limited ability to reduce H2O2 to water with the concomitant formation of ferryl (Fe(IV)=O) heme. At protein/H2O2 ratios <= 1:5 mol/mol, reaction of metmyoglobin with H2O2 yields both ferryl (Fe(IV)=O) Mb and protein radicals (globin·). Although the ferryl Mb is stable for hours at room temperature (15), the identity of the Mb residue(s) that form radicals in the presence of H2O2 has been the subject of some controversy. Globin· radicals have been localized to tyrosine (16, 17) and/or tryptophan residues (18). Additionally, globin· radicals may undergo subsequent chemistry (18) and are capable of oxidizing a variety of biological molecules (19). Hydroxyl radicals, however, do not appear to be involved in the transfer of oxidative damage (20, 21).

Human Mb is similar in sequence to other mammalian myoglobins. One significant difference, however, is the presence of Cys110. No other species of known mammalian Mb possesses a cysteine residue (22). As Mb is released under some pathological situations such as ischemia/reperfusion of the heart (23) and H2O2 is produced continuously in vivo (24), the reaction of human Mb and H2O2 may afford a physiologically relevant oxidant. Although the reactions of horse heart Mb and sperm whale Mb with H2O2 have been studied extensively, the corresponding reaction of human Mb has not been investigated previously. In this work, we have studied the reaction of human Mb and its C110A variant to evaluate the possible role of the reactive thiol group in the reaction of this protein with hydrogen peroxide through the combined use of EPR spectroscopy and electrospray mass spectrometry.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Horse heart Mb, reduced glutathione, iodoacetamide, bovine serum albumin (BSA), trypsin (type III; 10,200 unit/mg of protein), bovine liver catalase (40,000 unit/mg of protein), urea, ascorbate, EDTA, TEMPO, DTPA, 2,2-dithiodipyridine (DTP), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were obtained from Sigma. DMPO was purified by stirring solutions (1 M in 50 mM phosphate buffer, pH 7.4) with activated charcoal (100 mg/ml) in the dark. After 30 min, the solution was filtered, and portions were stored at -80 °C prior to use (25). Tryptone and yeast extract were from Becton Dickinson (Sparks, MD). Dithiothreitol (DTT) and NaCl were obtained from Fisher. H2O2 was from Bio-Rad. Buffers were prepared from either glass-distilled water or glass-distilled water purified further by passage through a Barnstead Nanopure system. All buffers were stored over Chelex 100® (Bio-Rad) at 4 °C for at least 24 h to remove contaminating transition metals as verified by ascorbate autoxidation analysis (26). Organic solvents and all other chemicals employed were of the highest quality available.

Preparation of Recombinant Proteins-- Transformed bacteria (strain AR68) containing plasmids for both wild-type recombinant human myoglobin and the C110A variant (27) were obtained from Prof. Steven G. Boxer. The cells were grown at 28 °C in 10 × 2-liter flasks containing 2YT Superbroth (1 liter/flask: Tryptone (16 g/liter), yeast extract (10 g/liter), and NaCl (5 g/liter)) to A600 nm = 1.2. The expression of recombinant myoglobins was induced by immersing each flask in a water bath (55 °C) for 5 min and then transferring the flask to an incubator operating at 42 °C. Cultures were incubated for a further 6-7 h, and the cells were harvested. Myoglobin (isolated as a fusion product) was purified by anion-exchange chromatography (Whatman DE52 resin) as described (27). Rapidly increasing the culture temperature in this manner was essential as simply increasing incubation temperature from 28 to 42 °C failed to induce protein expression. The partially purified fusion protein was reconstituted with excess hemin (heme/protein ~ 1:1.5; Porphyrin Products, Logan, UT), treated with trypsin (to cleave the fusion segment) and then further purified as described (27). Under these conditions, recombinant myoglobin was isolated as metmyoglobin. When required, proteins were concentrated by centrifugal ultrafiltration (Centriprep-10 concentrators, Millipore Corp.).

EPR Spectroscopy-- X-band EPR spectra (293 or 77 K) were obtained with a Bruker ESP 300e spectrometer equipped with a Hewlett-Packard microwave frequency counter. Mb solutions (~1 mM in 50 mM sodium phosphate buffer, pH 7.4) were treated with H2O2 at molar ratios of 1:1.2-5 in both the presence and absence of DMPO (protein/DMPO = 1:5-100 mol/mol). For low temperature studies, a sample of the reaction mixture (250 µl) was placed into a standard 3-mm quartz cell (Wilmad, Buena, NJ), snap-frozen in liquid nitrogen, and transferred to a finger Dewar insert (77 K) for EPR analyses. Analyses of DMPO spin-trapped adducts were performed with samples (50 µl) of the reaction mixture transferred into capillary tubes with a glass pipette. The capillary was then placed into a quartz EPR tube, and the tube was transferred to the cavity for EPR analyses at 293 K. The limit of detection of a stable nitroxide (TEMPO) under identical conditions was determined to be ~50 nM. Unless indicated otherwise, the time between removal of the sample, transfer to the appropriate cell, and tuning the spectrometer was consistently <30 s. EPR spectra were obtained as an average of three to five scans with a modulation frequency of 100 kHz and a sweep time of 84 s. Microwave power, modulation amplitude, and scan range used for each analysis varied appropriately as indicated in the figure legends. Hyperfine couplings were obtained by spectral simulation using the simplex algorithm (28) provided in the WINSIM program (NIEHS, National Institutes of Health). All hyperfine couplings are expressed in units of milliteslas. Simulations were considered acceptable if they produced correlation factors of R > 0.85. Peak area was estimated by integration using standard WINEPR software. Where possible, peak areas were standardized against a freshly prepared solution of TEMPO (5 µM in 50 mM phosphate buffer, pH 7.4) measured under identical spectrometer conditions; concentrations are expressed as moles of spins/mol of Mb protein. DTPA (100 µM) was included in all Mb solutions prior to addition of H2O2 to minimize the possibility of free transition metal-mediated decomposition of peroxide by Fenton chemistry.

For the analyses of power saturation data, a plot of log(I/P0.5) versus log P, where I represents intensity and P represents the microwave power, was chosen, as this results in a line that is parallel to the abscissa so long as the signal is not saturated and that slopes downward with increasing saturation. The saturation data (29, 30) were fitted to the relationship I proportional to  (P0.5)/(1 + P/P1/2)0.5b, where b ranges from 1 (for an inhomogeneous case) to 2 (for a homogeneous case). The curve of the fitted line tends to a slope of 0.5b under conditions of power saturation, and P1/2 is the half-saturation power obtained from the intersection of the two straight line segments of the line of best fit.

Electrospray Ionization Mass Spectrometry-- Electrospray ionization mass spectrometry (ESI/MS) was performed with a triple quadrupole mass spectrometer described in detail elsewhere (31). Samples of myoglobin/H2O2 reaction mixtures were diluted to a final concentration of 10 µM in heme (using methanol/water (1:10, v/v)) and were infused continuously (flow rate of 1 µl/min) into the ion source at 20 °C. Experiments were performed without an internal standard. Multiply charged gas-phase proteins were generated by pneumatically assisted ESI. Depending on the voltage difference between the ion sampling orifice and skimmer, the heme-protein interactions can be disrupted, and free heme can be completely dissociated from the heme-protein complex (31). For this study, the voltage difference between the orifice and skimmer was set at +100-110 V, such that dissociation of heme from the heme-protein complex could occur, and mass peaks were obtained largely for the apoprotein (apoMb) form of the protein. This dissociation of heme from the native protein was necessary to reduce the complexity of species analyzed where a homodimer was obtained (see below), although some contamination from intact protein (or holoprotein) was detected. Estimates of mass for holoMb, however, were performed with a voltage difference of 50-60 V to preserve the heme-protein complex and to afford charge-state distributions largely for the holoMb form of the protein. The ESI/MS system was calibrated with solutions of CsI, and horse heart Mb (mass of the apoprotein predicted from the amino acid sequence of 16,950 atomic mass units) was employed as a standard to test the mass accuracy of the system prior to use. Mass values were obtained by standard fitting analyses of the various m/z distributions using BioMultiView software (Perkin-Elmer Sciex Instruments, Foster City, CA). Mass determinations were performed for two or more independent protein preparations on different days. Mass values reported here refer to the means ± S.D. from five or more analyses.

Electronic Absorption Spectra-- Electronic absorption spectra were obtained with a Cary 3E UV/visible spectrophotometer. Mb concentrations were determined for solutions prepared in 50 mM sodium phosphate buffer, pH 7.4, assuming epsilon 408 nm ~ 188,000 M-1 cm-1. Reactive thiol content was determined by incubating protein samples (1-5 µM in 50 mM sodium phosphate buffer, pH 7.4) with either DTP or DTNB (1:10 molar ratio of protein to reagent). Absorbance was then measured at 325 or 446 nm, respectively (32, 33), and concentrations of free thiol were estimated by comparison with GSH standards. For hemeproteins, the absorbance of the DTNB adduct (421 nm) is nearly coincident with the heme absorption at 408 nm. To avoid this interference, TNB- anion formation was measured at 446 nm and used to estimate protein thiol content (34). In some experiments, wild-type human Mb was incubated with iodoacetamide (protein/thiol-blocking reagent = 1:10 mol/mol) prior to addition of DTP or DTNB. The stability of mixed disulfides (i.e. from the reaction of reduced GSH with DTNB or DTP) to reduction by ascorbate was evaluated at 25 °C at variable concentrations (0.1-1 molar eq) of ascorbate by monitoring the spectrum between 250 and 500 nm following addition of ascorbate.

Protein Cross-linking-- Cross-linking experiments were performed by incubating solutions of either wild-type human Mb or the C110A variant (65 µM in 50 mM sodium phosphate buffer, pH 7.4) with H2O2 (325 µM final concentration) and DTPA (100 µM) at 37 °C. Samples of the reaction mixture were removed periodically and diluted immediately with the same buffer with or without added DTT. In some cases, wild-type human Mb was incubated with iodoacetamide prior to addition of H2O2. Samples were then heated at 100 °C for 5 min and analyzed by SDS-PAGE (35) after staining with Coomassie Blue.

Statistical Analyses-- Statistics were performed with Student's t test available in Microsoft Excel, and significant difference was accepted at p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reactive Thiol Content of Recombinant and Native Proteins-- The reactive thiol content of wild-type human Mb and the C110A variant and that of horse heart Mb and BSA are shown in Table I. BSA was found to possess a single reactive thiol that was accessible to both DTP and DTNB, consistent with previous reports (36, 37). As expected from their amino acid sequences, wild-type human Mb also exhibited a single reactive thiol, whereas horse heart Mb and the C110A variant of the human protein contained no free thiol irrespective of treatment (Table I). Treatment of wild-type human Mb with iodoacetamide prior to free thiol determination decreased the thiol content of this protein significantly as expected following carboxymethylation of Cys110, indicating that the thiol had been effectively blocked by this treatment.

                              
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Table I
Free thiol content of human myoglobins and bovine serum albumin
Data represent the means ± S.D. of at least three separate determinations. All reactions were monitored until steady state was achieved, and then absorbance was measured. Absorbance was measured at 325 and 446 nm for adducts of DTP and DTNB, respectively (see "Experimental Procedures").

EPR Spectroscopy of Myoglobins Reacted with H2O2-- EPR spectra (77 K) for various mammalian myoglobins reacted with H2O2 at a protein/H2O2 molar ratio of 1:1.2 and frozen and recorded immediately after mixing are shown in Fig. 1. For horse heart Mb, the C110A variant of human Mb, and the wild-type human protein, weak EPR signals were detected at g ~ 2.036, 2.013, and 2.006, indicating that the initial globin· formed under these reaction conditions was the same for each protein. Upon incubation of the reaction mixture for wild-type human Mb at 77 K, the signal at g ~ 2.036 decayed to the base line within 5 min. Subsequently, a second signal appeared at an identical g-value. This latter species was stable at 77 K for at least 15 min (Fig. 2). During this time, the signal at g ~ 2.011 both increased in intensity and broadened in appearance, such that the features noted earlier were no longer distinguishable. The decay of the g ~ 2.036 signal and the subsequent broadening of the g ~ 2.011 signal were also observed for horse heart Mb and the human C110A variant; however, the signal at g ~ 2.036 did not reappear in either case, even after 30 min of incubation at 77 K (data not shown). The latter observation is consistent with that described for the decay of Trp-OO· in horse heart Mb (38).


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  		Fig. 1.   Trp-OO· detected by EPR spectroscopy (77 K) immediately after mixing human Mb with 1.2 eq of H2O2. Proteins were frozen in liquid nitrogen immediately after addition of peroxide and transferred to a finger Dewar insert to record spectra (77 K). A, horse heart Mb; B, human Mb C110A variant; C, wild-type human Mb. Where indicated (arrows), g-values correspond to gx ~ 2.036, gy ~ 2.013, and gz ~ 2.006. Spectra represent averages of three scans and were obtained under the following conditions: microwave power, 5 mW; modulation amplitude, 0.1 milliteslas; modulation frequency, 100 kHz; and sweep time, 84 s. All proteins were resuspended in 50 mM sodium phosphate buffer, pH 7.4, containing DTPA (100 µM). Data represent three or more experiments with different Mb preparations. mT, milliteslas.


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  		Fig. 2.   Changes in EPR spectral features of globin· following the reaction of wild-type human Mb and H2O2. Reaction conditions and EPR parameters were as described for Fig. 1. Where indicated (arrows), g-values correspond to gx ~ 2.036, gy ~ 2.013, and gz ~ 2.006. Spectra were obtained after 0 (A), 5 (B), 10 (C), and 30 (D) min. The EPR signal at g = 2.036 decayed to below the detection limit over 0-5 min and was detected again after a further 10 min at 77 K. The maximum concentrations of radicals detected were 0.26 ± 0.01 and 0.44 ± 0.01 mol of spins/mol of human Mb for signals at g ~ 2.036 and 2.011, respectively, whereas the concentration of radicals measured at g ~ 2.036 after 15 min was determined to be 0.20 ± 0.01 mol of spins/mol of human Mb. Data represent three or more experiments with different Mb preparations. mT, milliteslas.

Spin Trapping of Protein Thiyl Radicals in Wild-type Human Mb with DMPO-- To obtain further information concerning the identity of the protein radicals generated by the reaction of wild-type human Mb with H2O2, the spin trap DMPO was added to the reaction mixture prior to addition of peroxide. After 2 min at 20 °C, reaction mixtures treated with DMPO afforded a radical with a four-line EPR spectrum (Fig. 3A). The signal was relatively broad, with the outer most line at a lower g-value broadened almost to the base line. The broad nature of the EPR signal is consistent with a radical exhibiting restricted rotational motion on the EPR time scale and is indicative of DMPO trapping a radical on a large molecule (i.e. a protein). No EPR signal was detected in the absence of protein, DMPO, or H2O2 (Fig. 3, C-E, respectively). Treatment of the wild-type protein with iodoacetamide prior to addition of peroxide inhibited the formation of the radical adduct (Fig. 3F). Simulation of the EPR signal from the DMPO adduct with WINSIM software (Fig. 3B) indicated hyperfine couplings from single nitrogen (AN) and hydrogen (AH) atoms. Consistent with a previous report on DMPO trapping of protein thiyl radicals (39), addition of iodoacetamide after formation of the DMPO adduct did not significantly affect the EPR signal of the radical adduct on wild-type recombinant human Mb (data not shown). Hyperfine couplings obtained from these simulations together with reported values for relevant DMPO adducts are summarized in Table II. The couplings obtained from the quartet signal in this work are similar to those previously reported for the glutathione-derived thiyl radical (Table II). Importantly, hyperfine coupling of the free electron to the beta -hydrogen in the DMPO adduct detected here is also similar to that reported for DMPO-thiyl radicals derived from both BSA and cytochrome c oxidase (Table II). The magnitude of the hydrogen coupling is typical of a heteroatom-centered radical (40). These data, together with the EPR spectra of the reaction mixtures reported above, suggest that a thiyl radical is produced on wild-type human Mb upon reaction with hydrogen peroxide.


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  		Fig. 3.   DMPO spin trapping indicates the presence of a thiyl protein radical on human Mb. Human Mb (1-1.2 mM in 50 mM sodium phosphate buffer, pH 7.4) was mixed with H2O2 (5 eq) in the presence of DMPO (10 mM) and DTPA (100 µM). A, after 2 min at 20 °C, a sample was transferred to a capillary to record the EPR spectrum. B, simulation of the signal with WINSIM software (28) afforded a good fit (R = 0.88). C-E, no EPR signal was detected in the absence of human Mb, H2O2, or DMPO, respectively. F, modification of human Mb with iodoacetamide prior to addition of peroxide inhibited DMPO adduct formation. EPR parameters were the same as described for Fig. 1, except that microwave power was 20 mW and spectra represent the average of five scans. The concentration of radical adduct detected was determined to be 0.32 ± 0.01 mol of spins/mol of human Mb protein. The results shown represent three or more independent experiments with different preparations of human Mb. mT, milliteslas.

                              
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Table II
EPR hyperfine coupling constants for various DMPO adducts
Hyperfine coupling constants are given as data from a single literature source or mean of literature values (where applicable) or were derived from this study. All values are given in milliteslas. Hyperfine values determined in this study were optimized by simulation using WINSIM software (28), accepting correlation coefficients of R > 0.85. Data shown represent the means ± S.D. of three independent studies using different preparations of wild-type human Mb. CcO, cytochrome c oxidase.

DMPO adducts derived from tyrosine phenoxyl radicals have been reported for other mammalian myoglobins (18, 19). Therefore, we next investigated whether tyrosyl radicals were also generated on the surface of human Mb. One possibility was that the broad EPR signal detected from the mixture of DMPO, wild-type human Mb, and H2O2 resulted from DMPO trapping a mixture of radicals with closely overlapping EPR signals. As different diamagnetic centers can relax at different rates, it may be possible to separate overlapping signals by measuring spectra as a function of the power and at lower temperatures (41). Thus, we examined the effect of microwave power on the EPR signal from samples of frozen reaction mixture. Cooling the DMPO adduct to 77 K caused the quartet signal to collapse to a broad three-line spectrum similar to a simple nitroxide radical (Fig. 4). Power saturation studies performed on the reaction mixture at 77 K resulted only in a uniform decrease in the peak intensity of the signal obtained from the DMPO adduct. Importantly, other spectral features remained unaffected, i.e. the broad triplet feature was consistently detected (e.g. Fig. 4, A and B), and hyperfine couplings and ratios of peak area remained constant (data not shown) at all the power settings (0.01-200 mW) investigated. These observations are consistent with trapping of a single radical species by DMPO on the wild-type protein under the experimental conditions investigated. A line of best fit to the saturation data was obtained with b = 1.8 and P1/2 = 1.6 mW (Fig. 4, inset).


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  		Fig. 4.   Power saturation of the DMPO adduct at 77 K indicates that a single radical species is present on human Mb that decays uniformly with increasing microwave power. Samples of DMPO adducts were prepared as described for Fig. 3 and frozen, and spectra were recorded at 77 K. The spectra shown indicate a broad three-line signal obtained at 0.1 (A) and 100 (B) mW, respectively. The inset shows the power saturation profile for the nitroxide radical. mT, milliteslas.

Next, we investigated the effect of increasing the concentration of DMPO relative to protein on thiyl radical adduct formation while maintaining H2O2 at a protein/H2O2 molar ratio of 1:5. Addition of 5 eq of DMPO to a mixture of wild-type human Mb resulted in the detection of a quartet EPR signal (data not shown) identical to that obtained with a protein/DMPO ratio of 1:10 mol/mol. In contrast, increasing the concentration of DMPO to a protein/DMPO ratio of 1:25 mol/mol resulted in a complex mixture of overlapping EPR signals (Fig. 5A). This mixture of radicals appeared to include the EPR signal of the DMPO-thiyl radical adduct and that of a second species (c.f. Figs. 3A and 5A). Further increasing the concentration of DMPO to a protein/DMPO ratio of 1:100 mol/mol gave the EPR signal shown in Fig. 5B (solid line). Simulation of this EPR spectrum (Fig. 5B, dashed line) indicated a radical with hyperfine couplings to single nitrogen and hydrogen atoms (AN = 1.39 ± 0.01 and AH = 0.79 ± 0.01 milliteslas, respectively; mean ± S.D., n = 3). These hyperfine values are identical to data reported previously for the DMPO adduct of tyrosine phenoxyl radicals generated on sperm whale and horse heart Mb (18, 19) and were therefore assigned as the adduct of the tyrosine phenoxyl radical of Tyr103.


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  		Fig. 5.   Tyrosine phenoxyl-derived radical adducts are formed upon reaction of recombinant human Mb and H2O2 at high protein/DMPO ratios. Human Mb (1-1.2 mM in 50 mM sodium phosphate buffer, pH 7.4) was mixed with H2O2 (5 eq) and increasing concentrations of DMPO. After 2 min at 20 °C, samples were transferred to a capillary, and EPR spectra were recorded with DMPO present at protein/DMPO = 1:25 mol/mol (A) and protein/DMPO = 1:100 (B, solid line). Simulations of the EPR signal at the highest dose (B, dashed line) were performed using WINSIM software (28) and afforded a good fit to the experimental data (R = 0.86). EPR parameters were as described for Fig. 3. The maximum concentration of the DMPO-tyrosine adduct was determined to be 0.35 ± 0.03 mol of spins/mol of human Mb. DTPA was present in all reaction mixtures at a final concentration of 100 µM. The results shown represent three or more independent experiments with different preparations of human Mb. mT, milliteslas.

Effect of Ascorbate on DMPO Adduct Formation-- Addition of ascorbate (0.5-1 mol/mol of protein) to wild-type human Mb prior to addition of H2O2 resulted in the immediate formation of an ascorbyl radical detectable by EPR spectroscopy as judged by a characteristic doublet signal with AH ~ 0.18 milliteslas (42, 43). No other g ~ 2 radical was detected under these conditions, even in the presence of excess H2O2, i.e. at protein/H2O2 molar ratios ~1:5 (data not shown). These data indicate that ascorbate effectively reduces globin· accumulated on wild-type human Mb.

Addition of both ascorbate and DMPO prior to H2O2 also inhibited formation of the DMPO-radical adduct (data not shown), consistent with the possibility that ascorbate reacts with globin· to regenerate the protein before DMPO can trap the protein radical. However, ascorbate and other reductants could reduce the nitroxide moiety of the DMPO adduct to its corresponding EPR-silent hydroxylamine (44). To assess the possible involvement of simple nitroxide reduction subsequent to DMPO trapping, the effect of adding varying amounts of ascorbate to the reaction mixture of human Mb and H2O2 before and after addition of DMPO was evaluated (Fig. 6). Ascorbate added prior to the spin trap (Fig. 6, squares) rapidly decreased the intensity of the DMPO-trapped radical. In contrast, addition of ascorbate after the formation of the DMPO adduct (Fig. 6, circles) resulted in a relatively slow decay of the EPR signal from the DMPO-trapped radical. Together, these data support the conclusion that ascorbate readily competes with other reactants for globin· formed by the reaction of wild-type human Mb and H2O2.


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  		Fig. 6.   Ascorbate decreases DMPO adduct formation in a concentration-dependent fashion. A mixture of human Mb (1-1.2 mM in 50 mM sodium phosphate buffer, pH 7.4) and H2O2 (5 molar eq) was divided into two aliquots after reaction for 2 min at 20 °C. The first aliquot () was treated with DMPO (10 mM) and then catalase (1000 units). The second (black-square) was treated with catalase alone. After 30 s, ascorbate was added to both samples (final concentrations indicated), and DMPO (10 mM) was added where appropriate. Finally, the EPR spectrum of each mixture was recorded at 20 °C. Decay of the DMPO adduct was assessed as a percentage decrease in the low-field peak that was unaffected by the peak response of the overlapping ascorbyl radical. EPR parameters were the same as described for Fig. 3. Results are reported as the means ± S.D. (n = 3) for different preparations of human Mb. Asterisks indicate significant difference from the corresponding sample treated with ascorbate before the DMPO adduct was allowed to form (p < 0.05).

Characterization of a Disulfide-linked Mb Dimer from the Reaction of Wild-type Human Mb and H2O2-- Homodimers of both native and recombinant sperm whale myoglobins can form through a dityrosine linkage involving Tyr151 following reaction with H2O2 (14, 16). Unlike sperm whale Mb, wild-type human Mb does not possess a tyrosine residue at position 151 (14, 27). Nevertheless, reaction of this protein and H2O2 (1:5 molar ratio) consistently produced a cross-linked dimeric product as detected by SDS-PAGE analyses of the reaction mixture (Fig. 7). The concentration of the dimeric product increased with time and reached a maximum after ~30 min of reaction at 37 °C (Fig. 7A). No dimer was detected in the absence of H2O2, and treatment of the reaction mixture with DTT eliminated the dimeric product (Fig. 7A). In contrast, incubation of the C110A variant (or the wild-type protein after modification with iodoacetamide) with H2O2 under identical conditions afforded no dimeric product (Fig. 7B). Together, these data indicate that Cys110 is required for dimer formation and that the dimer probably involves intermolecular disulfide bond formation.


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  		Fig. 7.   Reaction of wild-type human Mb and H2O2 (37 °C) produces a cross-linked dimer that can be identified by SDS-PAGE. A: lane 1, standard protein mixture; lane 2, human Mb; lanes 3-6, human Mb reacted with H2O2 after 5, 15, 30, and 60 min, respectively; lane 7, human Mb reacted with peroxide for 30 min and then reacted with DTT. B: lane 1, standard protein mixture; lane 2, human Mb; lane 3, human Mb reacted with H2O2; lane 4, the human Mb C110A variant reacted with peroxide; lane 5, human Mb reacted with iodoacetamide prior to addition of peroxide. The results reported represent three independent measurements with different preparations of human Mb.

It has been proposed that high concentrations of ascorbyl radical produced by ascorbate oxidation may result in the reduction of the disulfide bond (45). To evaluate this possibility, we determined the stability of the disulfide dimer to ascorbate (10-500 µM concentrations) after treatment of the reaction mixture with catalase to remove excess H2O2. Under the conditions investigated, the dimer remained stable as judged by a lack of decrease in intensity of the dimer by SDS-PAGE (data not shown). Consistent with this result, mixed disulfide dimers formed from reaction of reduced glutathione with DTP or DTNB were also unaffected by addition of 0.1-1 molar eq of ascorbate (data not shown).

ESI/MS Analyses of the Disulfide Dimer-- Estimates for the mass of wild-type human Mb were determined by ESI/MS under ionization conditions that produce either apoMb or holoMb. The mass determined for human apoMb was 17,053 ± 1 atomic mass units (mean ± S.D., n = 5), which compares with a mass of 17,053 atomic mass units predicted from the amino acid sequence. The mass of human holoMb was found to be 17,670 ± 2 atomic mass units, which compares with the predicted mass of 17,669 atomic mass units. To confirm that the dimer was derived from the cross-linking of wild-type human Mb, mass spectrometry was performed on reaction mixtures of the protein and H2O2 (Fig. 8). The m/z distribution of dilute samples of the reaction mixture indicated the formation of a species other than apo- or holoMb in the presence of excess H2O2 (Fig. 8A). ESI/MS accumulated with an expanded m/z scale clearly reveals peaks corresponding to apoMb (m/z (charge in parentheses) 1421 (12+), 1550 (11+), 1705 (10+), 1894 (9+), and 2131 (8+)) and holoMb (m/z 1473 (12+), 1606 (11+), 1767 (10+), and 1963 (9+)). In addition, molecular ions were observed at m/z 1480 (23+), 1624 (21+), 1795 (19+), and 2006 (17+) that were assigned as mass charge states from an apoMb homodimer. Note that the apoMb homodimer is also expected to exhibit even m/z charge states that overlap with those for monomeric apoMb (e.g. the molecular ion at m/z 1421 can arise from either an apoMb monomer with a 12+ charge or an apoMb homodimer with a 24+ charge). Importantly, however, the charge states listed above for the apoMb homodimer (Fig. 8B, diamonds) are unique to the dimeric product. Under experimental conditions similar to those that give rise to molecular ions for apoMb alone, no other peaks corresponding to the apoMb homodimer were detected (e.g. Fig. 8C), indicating that formation of the dimer was not an artifact of mass analyses.


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  		Fig. 8.   Mass spectra of the products formed by reaction of wild-type human Mb and H2O2 confirm the formation of a Mb homodimer. Mass spectra of the products formed by reaction of human Mb and H2O2 exhibit m/z charge states corresponding to the human apoMb monomer () and the human holoMb monomer (black-square) and dimer (black-diamond ). A, spectra measured over the range 500 < m/z ratio < 2500 and B, in expanded scale. C, after addition of DTT, monomeric human apoMb was detected under identical spectrometer conditions. The results represent at least eight independent experiments with different preparations of protein. Arrows indicate poorly resolved m/z charge states from dimer species containing one or two heme prosthetic groups.

Other minor peaks were detected in the mass analyses of the reaction mixture (Fig. 8B, arrows). These smaller peaks can be attributed to a homodimer with one or two heme prosthetic groups bound. However, as these peaks were poorly resolved, they were not used to characterize the dimeric product. Consistent with the corresponding result from SDS-PAGE analyses (see above), treatment of the reaction mixture with DTT decreased the number of peaks in this region to those corresponding to m/z charge states of apoMb alone (Fig. 8C). The mass of the apoMb homodimer was determined to be 34,107 ± 5 atomic mass units (mean ± S.D., n = 8), which agrees with the mass of 34,104 atomic mass units calculated from the amino acid sequence.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

When metmyoglobin reacts with H2O2, the heme center is oxidized to ferryl heme, and a protein-centered radical (globin·) is formed. The radical can be detected by EPR spectroscopy at 77 K. The g-values obtained here for globin· detected immediately after reaction of horse heart Mb with H2O2 are identical to those previously assigned as the radical produced from reaction of a neutral tryptophan radical (derived from tryptophan at position 14) and dioxygen (18). As the reaction of both wild-type and C110A variant human myoglobins with H2O2 (both of which also possess Trp14) initially produced a radical with g-values identical to those observed for the horse heart protein, the radicals detected for both recombinant human proteins immediately after peroxide addition are assigned as Trp-OO·. The broadening of the g ~ 2.011 signal upon decay of the Trp-OO· EPR signal to below the detection limit is consistent with the formation of a tyrosine phenoxyl radical on the various species of myoglobin (13, 17). Thus, the relatively long-lived species with a broad EPR signal at g ~ 2.011, obtained from incubation of wild-type and variant human myoglobins with peroxide, is assigned as a tyrosyl radical.

The second EPR signal detected at g ~ 2.036 after the decay of Trp-OO· on wild-type human Mb has not been assigned previously. Protein thiyl radicals exhibit broad EPR signals that extend over the range g ~ 2.03-2.30 (46). The observation that the second radical detected after decay of Trp-OO· is shifted to g = 2.036 is consistent with the formation of a sulfur-centered radical. Importantly, DMPO spin trapping studies support the notion that a thiyl radical is formed. These data, together with the observation that a dimer, sensitive to reducing agents, is also formed in reactions of wild-type human Mb and H2O2, suggest that in addition to tryptophan- and tyrosine-derived radicals, for wild-type human Mb, globin· may also include a thiyl radical formed by oxidation of Cys110. The observations that after decay of Trp-OO·, mixtures of the human Mb C110A mutant or horse heart Mb and H2O2 failed to produce a second radical in the range g ~ 2.00-2.03 and that no dimer was detected in the absence of Cys110 or when cysteine was alkylated strongly support this conclusion.

As H2O2 is only in slight excess in the studies performed at 77 K (protein/H2O2 = 1:1.2 mol/mol), it is unlikely that the various radicals detected are formed by the direct oxidation of specific amino acids by added peroxide. In addition, as hydroxyl radicals are not formed from the peroxidatic action of Mb on peroxides (20, 21), it is unlikely that the thiyl radical is produced from hydroxyl radical-mediated cysteine oxidation. It is possible, however, that radical transfer can occur between amino acids in proteins by an intra- or intermolecular mechanism. For example, long-range electron transfer between tryptophan and tyrosine has been reported (reviewed in Ref. 47). In addition, it has been demonstrated that the Trp-OO· radical of horse heart Mb is capable of reacting with tyrosyl residues on other, target proteins (38). Therefore, in wild-type human Mb, it is possible that radical transfer from tryptophan (either the neutral indole or corresponding peroxyl radical) to cysteine or from tyrosine to cysteine may occur either via intra- or intermolecular mechanisms, giving rise to the thiyl radical.

The tyrosine phenoxyl radical is the most thermodynamically favored product of all the radicals detected in this study (38) and hence can be detected readily by EPR spectroscopy (e.g. Fig. 2). Studies of tyrosine-deficient horse heart Mb variants have indicated that oxidation of Tyr146 and Tyr103 gives rise to the phenoxyl radical detected by EPR (14). Although tyrosine radicals derived from the oxidation of Tyr146 are thought to be short-lived and inaccessible to DMPO, a DMPO adduct has been localized to Tyr103 in horse heart Mb (18). Also, Tyr103 is involved in the dimerization of sperm whale Mb, consistent with its location near the surface of the protein (18, 48). Therefore, the specific trapping of thiyl radicals alone when wild-type human Mb and peroxide react in the presence of low trapping efficiency would appear difficult to explain, particularly as Tyr146 and Tyr103 are also present in the human protein.

Overall, the trapping of only thiyl radicals under conditions of low DMPO concentrations (protein/DMPO <=  1:10 mol/mol) may be rationalized by two pathways: (i) Cys110 of human Mb may be oxidized directly at the surface of the protein by H2O2 even at low molar excess of H2O2, or (ii) electron transfer (either by intra- or intermolecular processes) from the Trp-OO·, Tyr103, and/or Tyr146 phenoxyl radical(s) results in thiyl radical formation (i.e. the Cys110 thiol acts as a radical sink). It is not inconceivable that a relatively rapid intra- or intermolecular electron transfer from Tyr103 to Cys110 results in the formation of the thiyl radical (see discussion above). For example, tyrosyl phenoxyl radicals have been demonstrated to oxidize free thiols (49). If such a rapid electron transfer process were to occur, then this would explain the inability of relatively low concentrations of DMPO to trap the corresponding Tyr103 phenoxyl radical. In contrast, direct oxidation of the thiol by hydrogen peroxide is more likely to give a sulfoxide without the formation of a thiyl radical. Our experiments with higher concentrations of DMPO (protein/DMPO >=  1:25 mol/mol) indicate that tyrosine phenoxyl radicals can be trapped by DMPO on wild-type human Mb by increasing the concentration of the spin trap and are consistent with the direct detection of the tyrosine phenoxyl radical by EPR (Fig. 2). As the DMPO-tyrosyl adduct is detected only under conditions of high trapping efficiency, the tyrosyl radical is probably generated prior to the formation of the thiyl radical at Cys110. Furthermore, as low protein/DMPO ratios exclusively yield the DMPO-thiyl adduct, it seems likely that the formation of the thiyl radical involves an electron transfer reaction (either by intra- or intermolecular processes) involving the radical produced at Tyr103. Only when sufficiently high concentrations of DMPO are used can the trap compete with this electron transfer process to produce the DMPO-tyrosyl adduct. Further studies on selected tyrosine and tryptophan mutations of human Mb will confirm the precise mechanism(s) leading to the formation of thiyl radicals on human Mb.

Our studies on addition of ascorbate to reaction mixtures of wild-type human Mb and peroxide indicate that this antioxidant can compete with other reactive targets (such as DMPO) for the thiyl radical formed by wild-type human Mb. Thiyl radicals are known to oxidize a wide range of biomolecules (50) and therefore are potentially deleterious in vivo. At physiological concentrations, ascorbate readily reacts with the thiyl radical at the surface of the protein to form an ascorbyl radical and presumably to generate the thiol. The presence of ascorbate and other reductants can evidently modulate the activity of the globin· formed by wild-type human Mb and may offer protection against this form of oxidant. However, under conditions that deplete ascorbate, e.g. acute inflammatory events such as ischemia reperfusion (51), thiyl radical formation and subsequent dimerization of human Mb can reasonably be expected to occur. Interestingly, although ascorbate can inhibit thiyl radical formation, it is unable to reduce the intermolecular disulfide linkage and to convert the dimer to the monomer after it is formed. This observation is consistent with a previous report that neither ascorbate nor the ascorbyl radical is able to reduce a disulfide bond (52). Furthermore, as disulfides generally exhibit low reduction potentials (Ered0 ~ -1500 mV) (53), it is unlikely that common biological reductants will be able to reduce disulfide bonds, at least in the absence of free transition metals. Consequently, it seems likely that once a Mb dimer forms in vivo, it may be sufficiently stable in human plasma to be detected as a marker of oxidative stress associated with reperfusion injury to the heart.

In summary, to assess the role(s) of Cys110 in human myoglobin in the free radical chemistry of the protein, we have investigated the reaction of wild-type human Mb and the C110A variant with H2O2. Reaction of the wild-type protein and H2O2 produces a mixture of protein-centered radical species from which a relatively stable thiyl radical has been observed for the first time. The novel thiyl radical of human Mb appears to be the major radical species formed at the surface of the protein after the decay of Trp-OO·, which is the first radical species detected directly by EPR after addition of H2O2. A dimeric Mb product is formed by reaction of wild-type human Mb and H2O2 that is not observed with the C110A variant of this protein, indicating that the dimer results from intermolecular disulfide bond formation. This conclusion has been confirmed by analysis of the reaction products by mass spectrometry. Overall, Cys110, which is unique to human myoglobin, significantly influences the free radical chemistry of the globin-centered radical species derived from the reaction of wild-type human Mb and peroxides. Future studies are required to determine whether the thiyl radical is produced by intra- or intermolecular electron transfer from Tyr103,Trp14 or by direct oxidation of the thiol. Overall, these observations suggest the possibility that the dimer of human myoglobin formed by the action of peroxide may serve as an in vivo marker for the involvement of myoglobin in the oxidative stress associated with ischemia reperfusion injury.

    ACKNOWLEDGEMENTS

We thank Dr. Bruce Collings for many helpful discussions and Professor Steven G. Boxer for providing the plasmids used to express wild-type human Mb and the C110A variant.

    FOOTNOTES

* This work was supported by Grant O 98S 0008 from the National Heart Foundation of Australia (to P. K. W.), Grant MT-7182 from the Medical Research Council of Canada (to A. G. M.), and an NSERC-SCIEX Industrial Chair (to D. J. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence be addressed: Dept. of Biochemistry and Molecular Biology, Copp Bldg., 2146 Health Sciences Mall, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. E-mail: mauk@ interchg.ubc.ca.

Published, JBC Papers in Press, April 21, 2000, DOI 10.1074/jbc.M000373200

    ABBREVIATIONS

The abbreviations used are: Mb, myoglobin; BSA, bovine serum albumin; DTPA, diethylenetriaminepentaacetic acid; DTP, 2,2-dithiopyridine; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DTT, dithiothreitol; ESI/MS, electrospray ionization mass spectrometry; PAGE, polyacrylamide gel electrophoresis; mW, milliwatts.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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