Reaction of Human Myoglobin and H2O2

ELECTRON TRANSFER BETWEEN TYROSINE 103 PHENOXYL RADICAL AND CYSTEINE 110 YIELDS A PROTEIN-THIYL RADICAL*

Paul K. Witting and A. Grant MaukDagger

From the Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received for publication, December 26, 2000, and in revised form, February 8, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The sequence of human myoglobin (Mb) is similar to that of other species except for a unique cysteine at position 110 (Cys110). Adding hydrogen peroxide (H2O2) to human Mb affords Trp14-peroxyl, Tyr103-phenoxyl, and Cys110-thiyl radicals and coupling of Cys110-thiyl radicals yields a homodimer through intermolecular disulfide bond formation (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2000) J. Biol. Chem. 275, 20391-20398). Treating a solution of wild type Mb and H2O2 with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) at DMPO:protein <=  10 mol/mol yields DMPO-Cys110 adducts as determined by EPR. At DMPO:protein ratios (25-50 mol/mol), both DMPO-Tyr103 and DMPO-Cys110 adducts were detected, whereas at DMPO:protein >=  100 mol/mol only DMPO-Tyr103 radicals were present. The DMPO-dependent decrease in DMPO-Cys110 was matched by a near 1:1 stoichiometric increase in DMPO-Tyr103. In contrast, reaction of the Y103F human Mb with H2O2 gave no DMPO-Cys110 at DMPO:protein <=  10 mol/mol, and only trace DMPO-Cys110 at DMPO:protein >=  100 mol/mol (i.e. conditions that consistently gave DMPO-Tyr103 in the case of wild type Mb). No detectable homodimer was formed by incubation of the Y103F variant with H2O2. However, the homodimer was detected in a mixture of both the Y103F and C110A variants of human Mb upon treatment with H2O2 (C110A:Y103F:H2O2 2:1:5 mol/mol/mol); the yield of this homodimer increased with increasing ratios of C110A:Y103F. Together, these data suggest that addition of H2O2 to human Mb can produce Cys110-thiyl radicals through an intermolecular electron transfer reaction from Cys110 to a Tyr103-phenoxyl radical.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein-centered free radicals are now recognized as intermediates for an increasing number of enzymes (1). Examples 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 tryptophanyl radical of cytochrome c peroxidase (6). Protein-centered radicals have also been implicated in oxidative stress associated with pathology of inflammatory disease(s) such as atherosclerosis and possibly in aging (7, 8). At least some of the toxic effects of protein radicals, as exemplified by protein-mediated initiation of lipid peroxidation (9-12), involve translocation of radical centers from one amino acid residue to another through both intramolecular (13) and intermolecular (14) mechanisms. Also, it has been proposed that free radical damage in proteins may well occur with a chain process similar to that for lipid peroxidation (15). One example of radical translocation is provided by the protein-centered radical(s) formed upon reaction of mammalian myoglobin with H2O2 (16).

Myoglobin (Mb)1 shows a limited peroxidase activity in the presence of H2O2. Reaction of Mb and H2O2 yields water with the concomitant formation of ferryl (Fe(IV)=O) heme and a porphyrin radical cation (16). At H2O2:protein ratios of <= 5, reaction of ferric or metmyoglobin (metMb) with H2O2 yields both ferryl (Fe(IV)=O) Mb and protein radicals (globin·) (17). Although the ferryl Mb is stable for hours at room temperature and is characterized readily by electronic absorption spectroscopy (17), the identity of the Mb residue(s) that form globin· in the presence of H2O2 has been the subject of some controversy. The precise location of the radical on the protein likely depends to some extent on both the mechanism by which it is formed and the stability of the radical species formed at a particular amino acid residue (14). Globin· have been localized to tyrosine (18, 19) and/or tryptophan residues (20) that effectively stabilize the odd electron through extended delocalization over unsaturated bonds. Additionally, globin· may undergo subsequent chemistry (20) and is capable of oxidizing other biological molecules (21). Importantly, hydroxyl radicals do not appear to be involved in the transfer of oxidative damage to either protein or target molecules (22, 23).

Human Mb is similar in sequence to other mammalian myoglobins. One significant difference, however, is the presence of Cys110 (24). No other known mammalian Mb possesses a reactive thiol group. Recently (25), we investigated the reaction of human Mb and its C110A variant to evaluate the role of the reactive sulfhydryl group in the reaction of this protein with H2O2. Similar to sperm whale and horse heart Mb, reaction of human Mb and H2O2 yielded radicals derived from Trp14 and from tyrosine residues Tyr103 and Tyr146. Our data also indicated that the reaction of human Mb and H2O2 differed from the corresponding reaction of other myoglobin species by formation of Cys110-derived thiyl radical and that bimolecular coupling of thiyl radicals can lead to formation of a Mb homodimer through intermolecular disulfide bond formation. The spin trapping agent DMPO readily reacted with both Tyr103-phenoxyl and Cys110-thiyl radicals where present in reactions of human Mb and H2O2. However, the precise mechanism for the formation of the Cys110-thiyl radical on wild type human Mb was not elucidated. We now report further studies of the mechanism of Cys110-thiyl radical formation in the reaction of human Mb and H2O2 by the combined use of site-directed mutagenesis, gel electrophoresis, and EPR spectroscopy.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Horse heart Mb, iodoacetamide, trypsin (Type III, 10, 200 unit/mg protein), urea, EDTA, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), diethylenetriamine pentaacetic acid (DTPA), ammonium-d-camphor-10-sulfonate, 2-methyl-nitrosopropane (MNP), and 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 (26). MNP was used without further purification. Solutions of MNP (500 mM in acetonitrile) were prepared immediately prior to use. Tryptone and yeast extract were from Becton Dickinson (Sparks, MD). Dithiothreitol and NaCl were obtained from Fisher Scientific (Fair Lawn, NJ). 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 the ascorbate autoxidation analysis (27). Organic solvents and all other chemicals employed were of the highest quality available.

Site-directed Mutagenesis-- Restriction enzymes were from New England Biolabs or Life Technologies, Inc. DNA manipulations were performed using procedures described in Ref. 28. Complementary oligomers were synthesized, and DNA sequence analysis was performed at the UBC Nucleic Acid and Protein Services Unit (University of British Columbia, Canada). Site-directed mutagenesis was performed with the pBluescript II KS (±) vector (Stratagene, La Jolla, CA). DNA was amplified by the polymerase chain reaction with a high fidelity PfuTurbo® DNA polymerase (Stratagene). Point mutations were confirmed by DNA sequence analysis prior to protein expression in bacteria. Once the sequence was confirmed, the BamHI-HindIII fragment from the amplified DNA that also contained the mutant Mb coding was ligated to the BamHI-HindIII fragment from the vector pMb3 (29) to yield the final expression vector. The circular plasmid was then transformed to the appropriate cell line for protein expression.

Preparation of Recombinant Proteins-- Transformed Escherichia coli (strain AR68) containing plasmids for both the wild type recombinant human myoglobin and the C110A variant (29) were obtained from Prof. S. G. Boxer. The DNA bearing the Y103F mutation was obtained by site-directed mutagenesis (above) and was used to transform E. coli DHB10 cells. For transformed bacteria (strain AR68), cells were grown at 28 °C in 10 × 2-liter flasks containing 2YT superbroth (1-liter flask: 16 g/liter tryptone, 10 g/liter yeast extract, and 5 g/liter NaCl) to an optical density (A600 nm) of 1.2-1.5. The expression of recombinant Mbs was induced by immersing each flask into a water bath (55 °C) for 5 min and then transferring the flask to an incubator operating at 42 °C. However, for the case of transformed DHB10 cells, cultures were grown at 28 °C in 10 × 2-1iter flasks containing 2YT superbroth to A600 = ~3-4, and then the heat shock was applied in a similar fashion to that described for transformed AR68 cells. All cultures were subsequently incubated for a further 6-7 h, and the cells were harvested. 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.

Myoglobin expressed by either cell line (isolated as a fusion product) was purified by anion exchange chromatography (Whatman DE52 resin) as described (29). The partially purified fusion protein(s) was reconstituted with excess hemin (heme:protein = ~1.5) (Porphyrin Products, Logan, UT), treated with trypsin (final concentration, ~0.1 µg/ml) to cleave the fusion segment and then further purified as described (29). Under these conditions, recombinant myoglobin was isolated as metMb. When required, proteins were concentrated by centrifugal ultrafiltration (Centriprep-10 concentrators, Millipore). The yield of recombinant protein obtained from E. coli DH10B cells was significantly lower than for the original cell line used to express human Mb; however, we were forced to use the former because of unavailability of the Ar68 cells. As a result of the poor yields (<= 2 mg of protein/liter of culture medium), several protein samples were pooled to obtain sufficient material for our various studies.

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 (~0.5 mM in 50 mM sodium phosphate buffer, pH 7.4) were treated with H2O2 (H2O2:protein = ~1.2-5 mol/mol) in both the presence and absence of the appropriate spin trap (spin trap:protein = 5-150 mol/mol). For spin trapping studies with MNP, the reagent was added from concentrated stock solutions to minimize effects of the organic solvent (final acetonitrile concentration in reaction mixtures, <5% v/v). Analyses of spin adducts were performed using 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 analysis 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 3-5 scans with modulation frequency 100 kHz and sweep time of 84 s. Microwave power, modulation amplitude, and scan range used for each analysis varied appropriately as indicated in the legends to the figures. Hyperfine couplings were obtained by spectral simulation using the simplex algorithm (30) provided in the WINSIM program, which is available at the NIEHS, National Institutes of Health web site. All hyperfine couplings are expressed in units of mT. Simulations were considered acceptable if they produced correlation factors of R > 0.85. Peak areas were determined by integration with standard WINEPR software (Bruker). Yields of the DMPO-Cys110 and DMPO-Tyr103 adducts were determined by peak area comparison with 5 µM TEMPO prepared in 50 mM phosphate buffer, pH 7.4, and measured under similar experimental conditions. DTPA (100 µM) was included in all Mb solutions prior to the addition of H2O2 to minimize the possibility of free transition metal-mediated decomposition of peroxide by Fenton chemistry.

Circular Dichroism Spectroscopy-- CD spectroscopy was performed using a JASCO model J-720 spectropolarimeter calibrated with ammonium-d-camphor-10-sulfonate as described (31). Spectra (190-250 nm) were recorded for samples placed in a water-jacketed, cylindrical quartz cuvette (path length, 0.1 cm), and temperature was controlled with a NESLAB model RT 100 circulating water bath operated under computer control. The cuvette temperature was measured using a NESLAB RS-2 remote sensor interfaced into a computerized data acquisition system. Protein solutions (5-10 µm) were prepared in sodium phosphate buffer (10 mM, pH 7 and 8).

Thermal denaturation curves were determined by monitoring ellipticity at 222 nm as temperature was increased from 25 to 85 °C (50 °C/h). Melting temperatures (Tm) are reported as the means ± S.D. from three measurements. Melting curves were transformed to ASCI text files for export to Scientist software (MicroMath, Salt Lake City, UT), which was used to calculate the first derivative of the curve for more accurate determination of Tm. The irreversibility of myoglobin folding under these conditions precluded determination of thermodynamic parameters consistent with a previous study using horse heart Mb (31).

Electronic Absorption Spectroscopy-- Electronic spectra were obtained with a Cary 3E UV/Vis spectrophotometer. Mb solutions were prepared in sodium phosphate buffer (50 mM, pH 7.4), and Mb concentration was determined spectrophotometrically (epsilon 408 nm = 188,000 M-1 cm-1) (32). The pH-linked spectroscopic transitions of metMb were monitored between 280 and 700 nm from pH 6.3 to 10.5, and the corresponding pKa values were determined by fitting absorbance data obtained at 580 nm to the Henderson-Hasselbach equation with the program Scientist (MicroMath).

Protein Cross-linking-- Cross-linking experiments were performed by incubating solutions of either wild type human Mb or mixtures of the C110A and Y103F variants (protein concentration, 0.025-1 mM) with H2O2 (each in 50 mM sodium phosphate buffer, pH 7.4) at a protein:H2O2 ratio of ~5 mol/mol and 37 °C. All samples contained the iron chelator DTPA (100 µM) to minimize the possibility of Fenton-type chemistry. After 30 min, the reaction mixture was diluted with 50 mM sodium phosphate buffer, pH 7.4 (final protein concentration, 25 µM), and each of the carefully matched samples was diluted further with nonreducing loading buffer (protein solution:loading buffer = ~2 v/v). Nonreducing loading buffer was prepared by mixing 7 ml of 0.5 M Tris-Cl containing 0.4% SDS, 3.6 ml of glycerol, 1 g of SDS, and 1.2 mg of bromphenol blue, and finally water was added to 10 ml of final volume. Note that loading buffer did not contain either mercaptoethanol or dithiothreitol because these reagents can reduce the dimeric product (25). Dilute protein samples were then heated at 90-100 °C (5 min) and analyzed by SDS-PAGE (33) after staining with Coomassie Blue.

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

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Electronic Absorption Spectroscopy of the Y103F Variant-- To investigate the relationship between Tyr103-phenoxyl radicals and the formation of the Cys110-thiyl radical, we sought to prepare the Y103F variant of human Mb. It has been reported previously that the point mutation Y103F in recombinant sperm whale myoglobin destabilizes the protein sufficiently that isolation of the variant is precluded (13). For this species of Mb, the destabilizing influence of the Y103F substitution can be offset by introduction of a second point mutation, K102Q (13). In contrast, the expression of the corresponding Y103F variant of human Mb afforded a stable protein despite the presence of Lys102 (24) in the sequence for the human protein. In view of this difference in stability between these mammalian proteins to the alteration at Tyr103, we have further characterized the Y103F variant Mb by electronic absorption and CD spectroscopy and determined the pKa for titration of the water molecule coordinated to the ferric iron of the variant and wild type human proteins.

The electronic spectrum obtained from solutions of the Y103F variant of human Mb exhibited Soret (409 nm) and visible bands (lambda max = 505 and 631 nm) similar to those of the wild type protein (Fig. 1A), consistent with corresponding spectra reported for the wild type and Y103F/K102Q variant of sperm whale Mb (see Fig. 4 in Ref. 13). The CD spectra for wild type human Mb and the Y103F variant of human Mb (10 mM phosphate buffer, pH 8) were also virtually identical (Fig. 1B) and indicated that the two proteins possess similar helical content at pH 7 (not shown). The melting curves for each protein afforded similar Tm values at pH 8: Tm = 77.3 ± 0.5 and 76 ± 1 °C (data represent means ± S.D., n = 3) for wild type Mb and the Y103F variant, respectively (Fig. 1B, inset). Melting curves determined for the two proteins at pH 7 (10 mM phosphate buffer) also afforded similar Tm values: Tm = 80 ± 1 and 78.1 ± 0.2 °C for wild type human and Y103F variant proteins, respectively (not shown). These Tm values were not significantly different independent of the pH of the buffer employed as judged by t test analyses (p 0.05).


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Fig. 1.   Electronic absorbance and CD spectra of wild type and Y103F variant Mb. A, electronic spectra were obtained for wild type human Mb (solid line) and the Y103F variant (broken line) (~5 µM; 50 mM phosphate buffer, pH 7.4). The inset shows the Henderson-Hasselbach plots for wild type Mb (solid line) and the Y103F variant (broken line) of human Mb (50 µM) over the pH range 6.3-10.5. B, CD spectra were obtained with solutions of wild type (solid line) and Y103F (broken line) Mb (10 µM; 10 mM phosphate buffer, pH 8) at 20 °C. The inset shows corresponding first derivative melting curves from the wild type (solid line) and Y103F (broken line) Mb monitored over 25-85 °C. Results represent three or more independent experiments.

On the other hand, the pKa determined for the Y103F variant was significantly lower (p < 0.05) than that of the wild type protein (8.26 ± 0.09 and 8.85 ± 0.03, respectively, mean ± SD, n = 3) (Fig. 1A, inset). This difference in pKa indicates that the Y103F point mutation probably induces some subtle change in the heme binding site that alters the binding of the water ligand. The crystal structure of a related (K45R/C110A) variant of human Mb (24) indicates that Tyr103 resides near the surface of the protein and is the tyrosine residue nearest the heme group as is also the case for sperm whale Mb (34). For the human protein, the point of closest approach is a phenyl carbon that is ~3.44 Å from the edge of the heme group. Conceivably, a minor reorganization of the heme pocket that is not detected by electronic spectroscopy or by visible CD spectroscopy affords a lower pKa for the acid-base transition in the Y103F protein. Regardless of the basis for the difference in this pKa value, it is clear that the thermal stability and the electronic absorbance characteristics of the Y103F variant are comparable with that of the wild type protein.

Spin Trapping of Globin· in Wild Type Mb and the C110A and Y103F Variant Proteins-- We next investigated the formation of globin· in the reactions of wild type human Mb and both the C110A and Y103F variants with H2O2 using the spin traps DMPO and MNP. Addition of H2O2 to solutions of wild type human Mb containing DMPO at DMPO:protein <=  10 mol/mol consistently produced a product with an EPR spectrum comprised of a broad four-line signal (Fig. 2A, solid line). This broad signal was simulated well (Fig. 2A, broken line) with hyperfine data previously reported for the trapping of thiyl radicals on human Mb at Cys110 (DMPO-Cys110) (25). The treatment of the C110A variant of human Mb with peroxide in the presence of DMPO under identical conditions, however, consistently gave a more complex EPR spectrum (Fig. 2B, solid line). This complex signal was also detected in the corresponding reaction of horse heart Mb and H2O2 (Fig. 2C) where the radical has been assigned previously as resulting from the DMPO adduct of a Tyr103-phenoxyl radical (DMPO-Tyr103) (20). Thus, the DMPO adduct derived from the reaction of C110A variant of human Mb and H2O2 was readily simulated (20) with the reported coupling values (Fig. 2B, broken line), and the globin· was assigned as a DMPO-Tyr103 adduct. These data support the conclusion that Cys110-thiyl radicals are thermodynamically stable end products following reaction of the wild type protein with hydrogen peroxide, whereas for Mb lacking Cys110, the Tyr103-phenoxyl radical is the most stable globin· produced.


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Fig. 2.   Trapping globin radicals on wild type human Mb, C110A Mb and horse heart Mb with DMPO. Mb (0.5 mM protein in 50 mM sodium phosphate buffer, pH 7.4) was mixed with H2O2 (five equivalents) and DMPO (2.5 mM). After 2 min at 20 °C, EPR spectra were recorded for wild type Mb (A, solid line); C110A variant Mb (B, solid line); and horse heart Mb (C). EPR simulations were performed with WINSIM software (30) and afforded good fits to the data, r = 0.86 and 0.94 for the C110A and wild type proteins (A and B, broken lines), respectively. EPR parameters: microwave power, 5 mW; modulation amplitude, 0.1 mT; modulation frequency, 100 kHz; sweep, 84 s. All protein solutions contained DTPA (100 µM). Data represent three or more experiments using different Mb preparations.

In marked contrast to the analogous reaction with wild type human Mb, no radical adducts were detected upon addition of H2O2 to a solutions containing the Y103F variant and DMPO (Y103F:H2O2:DMPO 1:5:<= 10 mol/mol/mol) (Fig. 3A). Furthermore, at the highest trap efficiency investigated (DMPO:H2O2:Y103F 1:5:~100 mol/mol/mol), only a weak four-line EPR spectrum was obtained, and this spectrum exhibited hyperfine coupling identical to that for the DMPO adduct derived from trapping DMPO-Cys110 radicals (Fig. 3B). Under these conditions, the corresponding reaction of wild type human Mb and H2O2 gave both DMPO-Cys110 and DMPO-Tyr103 adducts (e.g., see Fig. 2). Overall, the concentrations of DMPO-Cys110 radicals measured from the wild type and Y103F variant proteins varied by ~130-fold as determined by double integration of the respective DMPO-Cys110 EPR signals from the two proteins. Collectively, these data independently confirm the alteration at Tyr103 in the Y013F variant.


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Fig. 3.   Reaction of the Y103F variant Mb and H2O2 in the presence of DMPO does not yield significant concentrations of radical adducts. The Y103F variant (0.5 mM protein in 50 mM sodium phosphate buffer, pH 7.4) was mixed with H2O2 (five equivalents) in the presence of DMPO. After 2 min at 20 °C, EPR spectra were recorded for samples with DMPO:protein ratios of ~5 mol/mol (A) and ~100 mol/mol (B). EPR parameters were as for Fig. 2. The EPR spectra are plotted with a 4-fold lower intensity range on the vertical axis than spectra shown in Fig. 2. DTPA (100 µM) was present in all reaction mixtures. Results represent three or more independent experiments with different Mb preparations.

Radical adducts of protein-derived tyrosine phenoxyl radicals have also been reported to form with the nitroso spin trap MNP (20, 35, 36). Therefore, to verify that replacement of Tyr103 resulted in the loss of the Tyr103-phenoxyl radical, MNP was employed in reactions of both the wild type and Y103F variant Mb with H2O2 (Mb:H2O2:MNP 1:5:20 mol/mol/mol). For the case of wild type human Mb, reaction mixtures treated with MNP afforded a radical with a three-line EPR spectrum after 2 min at 20 °C (Fig. 4A). The signal was relatively broad with the outer most line at lower g value broadened almost to base line. The broad nature of the EPR signal is consistent with a radical exhibiting restricted rotational motion on the EPR time scale and indicative of MNP trapping a radical on a large molecule (i.e. a protein). No EPR signal was detected in the absence of protein, MNP, or H2O2 (Fig. 4, B-D, respectively).


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Fig. 4.   Trapping globin radicals on wild type and Y103F human Mb proteins with MNP. Wild type Mb (0.5 mM protein in 50 mM sodium phosphate buffer, pH 7.4) was mixed with H2O2 (five equivalents) and MNP (20 mM). After 2 min at 20 °C, EPR spectra were recorded for the complete system (A); same as in A but in the absence of protein (B); same as in A but in the absence of MNP (C); same as in A but in the absence of H2O2 (D); and same as in A after incubation with "Pronase" (1000 units/ml, E). F, treatment of the Y103F Mb under identical conditions as for A did not yield a detectable MNP radical adduct. EPR parameters were as for Fig. 2. Results represent three or more independent experiments using pooled protein.

Addition of the nonspecific protease "Pronase" (1000 units/ml) to reaction mixtures containing the MNP adduct resulted in the digestion of the protein into small peptide fragments. Subsequent analyses of the digest by EPR yielded a spectrum with a sharp triplet signal (Fig. 4E). Hyperfine couplings were obtained by spectral simulation to high correlations (r = 0.97, not shown) using aN = 14.3 ± 0.1 mT, abeta H = 10.6 ± 0.2 mT, and agamma H = 1.2 ± 0.2 mT. These hyperfine couplings are identical to that determined previously (20, 35) for MNP adducts of Tyr103-phenoxyl radicals on horse heart Mb. On this basis, the radical detected here with wild type human Mb was also assigned as the MNP adduct of the Tyr103-phenoxyl radical. In contrast, the corresponding reaction of the Y103F variant of Mb and H2O2 failed to produce a detectable MNP adduct under identical reaction conditions (Fig. 4F). Higher mol ratios of MNP:protein (<= 100 mol/mol) also failed to yield a detectable EPR signal (not shown). These latter results independently verify that no tyrosyl radical that is accessible to water-soluble spin traps forms upon reaction of the Y103F Mb and peroxide.

DMPO Concentration-dependent Changes in the Distribution of DMPO-globin· Adducts-- At higher DMPO trapping efficiencies, both DMPO-Cys110 and DMPO-Tyr103 radical adducts could be detected in the reaction of wild type human Mb and peroxide (25). These results are consistent with the idea that these different globin· may be related by an electron transfer reaction. To investigate the putative electron transfer between Cys110 and Tyr103, we tested the effect of systematically increasing DMPO:protein ratios on the distribution of globin· adducts derived from the reaction of wild type human Mb and H2O2.

In agreement with our previous study (25), treatment of the globin· formed by reaction of wild type Mb and H2O2 with DMPO (over DMPO:protein ranging 25-150 mol/mol) resulted in distinct changes in both the type and the yield of DMPO adduct detected by EPR spectroscopy (Figs 5). These changes are attributed to the increasingly efficient trapping of the Tyr103-phenoxyl radical (25). Computer simulation of the data obtained at various mol ratios of DMPO: Mb (e.g., see Fig. 2B, broken lines) coupled with double integration (37, 38) afforded the relative mol fraction of DMPO-Cys110 or DMPO-Tyr103 adducts in the composite EPR spectrum for a given DMPO concentration (Fig. 6). Maximum concentrations of DMPO-Cys110 and DMPO-Tyr103 were determined under experimental conditions that provided the EPR signal of each in the absence of the other to define a ratio for (DMPO-Tyr103)max/(DMPO-Cys110)max of 1.18 ± 0.25 (means ± S.D., n = 3). Therefore, the DMPO-dependent change in product distribution is consistent with a near 1:1 stoichiometric relationship between DMPO-Tyr103 and DMPO-Cys110 radicals and is further support for the idea that the thiyl radical is formed by transfer of an electron from Cys110 to the Tyr103-phenoxyl radical.


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Fig. 5.   Trapping of DMPO-Tyr103 and DMPO-Cys110 adducts from reactions of wild type human Mb and H2O2. Wild type Mb (0.5 mM protein in 50 mM sodium phosphate buffer, pH 7.4) was mixed with H2O2 (five equivalents) and increasing concentration of DMPO. After 2 min of reaction at 20 °C, EPR spectra were recorded for samples with DMPO:protein ratios of 5 mol/mol (A); 20 mol/mol (B); 50 mol/mol (C); and 100 mol/mol (D). EPR parameters were as for Fig. 2. DTPA (100 µm) was present in all reaction mixtures. Results represent three or more experiments with different preparations of human Mb.


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Fig. 6.   DMPO-dependent changes to the relative mol fraction of DMPO-Cys110 and DMPO-Tyr103 in composite EPR spectra from the reaction of wild type Mb and H2O2. Relative contributions of the DMPO-Cys110 (squares) and DMPO-Tyr103 (circles) adducts were determined from composite spectra by simulation (30) and double integration and are plotted as the relative mol fraction as a function of the DMPO:protein ratio. Peroxide was maintained at H2O2:protein ~5 mol/mol, and wild type Mb was 0.5 mM for all DMPO concentrations investigated. The absolute concentrations of DMPO adduct were determined to be 0.26 ± 0.02 (mean ± S.D.; three independent measurements) and 0.22 ± 0.03 spins/mol Mb protein for the DMPO-Tyr103 and DMPO-Cys110 adducts, respectively.

SDS-PAGE Analyses of Reactions of Wild type and Y103F Variant Proteins and H2O2-- The evidence obtained from spin trapping studies (above) were consistent with the transfer of an electron from Cys110 to a Tyr103-phenoxyl radical that is formed as a result of the reaction of wild type human Mb and H2O2. This electron transfer reaction may be intramolecular or intermolecular or both. To evaluate this issue, we studied the dependence of intermolecular disulfide bond formation that results from the reaction of wild type Mb with peroxide on the concentration of protein. In this experiment, wild type human Mb (25-1000 µM) was incubated at 37 °C in either the absence or presence of H2O2 (at a constant ratio of H2O2:protein ~5:1 mol/mol). After 30 min (a time previously determined to be optimal for formation of the disulfide dimer (25)), reaction mixtures were diluted with 50 mM phosphate buffer, pH 7.4, so that the final total protein concentrations of all the samples were carefully matched prior to gel electrophoresis. Subsequent SDS-PAGE analyses of the various reaction mixtures showed that two protein bands were present in samples treated with H2O2 for each of the protein concentrations investigated (Fig. 7). Comparison with molecular mass markers indicated that these species have masses of ~34 and 17 kDa, so these bands were assigned as the dimeric product and monomeric wild type proteins, respectively (25). Overall, the intensity of the band corresponding to the dimeric product increased in samples that initially contained greater protein concentration (Fig. 7, lanes 2-6). No dimer product was detected in the absence of H2O2 (Fig. 7, lane 7).


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Fig. 7.   Reaction of wild type human Mb and H2O2 affords a cross-linked dimer that increases in yield with increasing protein concentration. Human Mb (25-1000 µM) was treated with H2O2 (at H2O2:protein 5 mol/mol for all protein concentrations investigated). After 30 min at 37 °C all reaction mixtures were diluted to 25 µM in protein using 50 mM phosphate buffer, pH 7.4, and the carefully matched samples were analyzed by SDS-PAGE. Lane 1, protein standard; lanes 2-6, human Mb initially at 25 (lane 2), 50 (lane 3), 250 (lane 4), 500 (lane 5), and 1000 µM (lane 6), respectively. Lane 7 shows control human Mb incubated in the absence of H2O2.

In contrast to the reaction of wild type human Mb and H2O2, reaction of the Y103F variant and H2O2 failed to yield a dimeric product even after incubation with peroxide for up to 60 min at 37 °C (Fig. 8A). These data are consistent with a critical role for Tyr103 in the formation of Cys110-thiyl radicals (i.e. one of the intermediate radicals that is formed prior to bimolecular disulfide bond formation). If the reactions leading to formation of the Cys110-thiyl radical were solely intermolecular processes, then such a mechanism would be expected to result in dependence on the concentration of target protein. Therefore, we considered that the protein-dependent increase in dimeric product (Fig. 7) was indicative of an intermolecular electron transfer event that yielded the Cys110-thiyl radical. To test whether Cys110-thiyl radical formation involved intermolecular electron transfer to reduce the phenoxyl radical at Tyr103, we investigated the reaction of a mixture of Y103F (lacking Tyr103 but with Cys110) and C110A (lacking Cys110 but containing Tyr103) variant proteins with H2O2 (at C110A:Y103F:H2O2 ~2:1:5 mol/mol/mol). Despite the inability of either protein to form an intermolecular disulfide bond upon treatment with H2O2 individually (see Fig. 8A and also Fig. 7B in Ref. 25), treatment of the mixture of variants with H2O2 afforded the dimeric product (Fig. 8B). Overall, the yield of dimer obtained from the mixture of C110A/Y103F Mb was similar to that from the wild type protein treated with peroxide under identical conditions. Interestingly, the yield of dimeric product could be increased in independent studies by changing the ratio of C110A:Y103F. For example, increasing the content of C110A from 0.5 to 3 mol equivalents resulted in an increased intensity of the protein bands assigned as the dimeric product. These latter data indicate that the concentration of Tyr103-phenoxyl radical in the C110A variant was important in generating the Cys110-thiyl radical in the Y103F variant and, in turn, in determining the overall yield of disulfide homodimer product (not shown).


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Fig. 8.   Treatment of Y103F Mb with H2O2 does not produce a disulfide cross-linked product unless C110A Mb is also present in the reaction mixture. Y103F Mb was incubated with H2O2 (H2O2:total protein = ~5 mol/mol) in either the absence (A) or presence (B) of C110A Mb at C110A:Y103F ~2 mol/mol. A, lane 1, mass standard; lanes 2 and 3, authentic wild type protein before and after incubation with H2O2, respectively; lanes 4-8, Y103F variant and H2O2 after incubation at 37 °C for periods of 0 (lane 4), 5 (lane 5), 15 (lane 6), 30 (lane 7) and 60 min (lane 8), respectively. B, lane 1, mass standard; lane 2, protein before H2O2 addition; lanes 3-6, reaction mixture after incubation at 37 °C for 5 (lane 3), 15 (lane 4), 30 (lane 5), and 60 min (lane 6), respectively. Lane 7 shows authentic disulfide product from reaction of wild type human Mb and peroxide. Note the protein standard used in B differs from that in A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Formation of ferryl-heme and a protein-centered radical (globin·) upon reaction of metMb with H2O2 is well established (see the Introduction). Recently (25), we investigated the reaction of human Mb and its C110A variant to evaluate the role of the unique sulfhydryl group in the reaction of this protein with H2O2 and demonstrated by low temperature EPR spectroscopy that the human protein undergoes radical formation at Trp14, Tyr103, and Cys110 in this reaction. The spin trapping studies that are reported here using both the C110A and the Y103F variants of human Mb independently confirm the assignment of the Tyr103-phenoxyl and Cys110-thiyl radicals on the wild type human protein. Additionally, comparison of the reactions of C110A and Y103F Mb with H2O2 afforded a useful means of evaluating the precise mechanism of Cys110-thiyl radical generation in the reaction of peroxide with wild type human Mb.

Identifying Key Residues Involved in globin· Formation on Wild type Human Mb-- Spin trapping studies with the spin trap DMPO showed that the Cys110-thiyl radical is trapped exclusively using relatively low DMPO:Mb ratios (<10 mol/mol), indicating that this radical is thermodynamically favored under these conditions (i.e. Cys110 acts as a radical sink). Conversely, our observations that the Tyr103-phenoxyl radical is detected in the absence of all other DMPO-globin adducts at high DMPO trapping efficiency (DMPO:protein >100 mol/mol), where the rates of secondary reactions are overcome by the kinetics of the trapping reaction, supports the argument that a Tyr103-derived globin· is formed before a radical center is generated at position Cys110. The observation of a near 1:1 stoichiometry for the yield of DMPO-Tyr103 and DMPO-Cys110 radical adducts with increasing mol ratios of DMPO: Mb indicates that electron transfer occurs from Cys110 to Tyr103.

Mechanism of Electron Transfer Between Tyr103 and Cys110 Residues in Human Mb-- Having established that tyrosyl phenoxyl and thiyl radicals form sequentially on the globin portion of wild type Mb and that these radicals are generated through an electron transfer process, we next determined whether this process occurs through an intra- or intermolecular mechanism. Because the three-dimensional structure of wild type human Mb has not been reported, the best alternative structure that is available for estimating distances between structural elements of this protein is the structure of the K45R/C110A variant of this protein (24). In the structure of this variant determined at 2.8 Å resolution, Tyr103 is the tyrosine residue nearest the heme group, and the shortest distance separating these two group is ~3.4 Å. This distance is similar to the corresponding distance in both sperm whale (13, 34) and horse heart (39) Mb. In the human protein, however, the sulfhydryl group of Cys110 is expected to be ~9-10 Å from the heme edge, and the distance between the Cys110 and Tyr103 residues is ~12 Å (24).

Formation of Tyr103-phenoxyl radicals prior to formation of Cys110-thiyl radicals is consistent with the proximity of Tyr103 to the heme group. The relatively large distance separating the Tyr103 and Cys110 residues decreases the efficiency of intramolecular electron transfer between these residues, although long range intramolecular through-bond processes are known to occur in heme-proteins and therefore cannot be excluded (40). Interestingly, tyrosyl phenoxyl radicals (generated from reaction of horseradish peroxidase, H2O2, and free tyrosine) have been demonstrated to oxidize thiols to thiyl radicals by an intermolecular mechanism (41). Therefore, it is not inconceivable that relatively rapid intermolecular electron transfer from Cys110 (on one protein molecule) to Tyr103-phenoxyl radical (on a second protein molecule) results in the formation of the thiyl radical at Cys110 of human Mb. To evaluate this possibility, we studied formation of the dimeric product from reactions of human Mb and peroxide using SDS-PAGE.

The accumulation of the disulfide homodimer in reactions of human metMb and H2O2 follows the simplified mechanism outlined in Reactions 1-R4.
<UP>metMb</UP>+<UP>H<SUB>2</SUB>O<SUB>2</SUB> → </UP>[<UP>Globin-Fe</UP>(<UP>IV</UP>)<UP>&z.dbnd6;O</UP>]<SUP><UP>⋅</UP><UP>+</UP></SUP>+<UP>H<SUB>2</SUB>O</UP>

[<UP>Globin-Fe</UP>(<UP>IV</UP>)<UP>&z.dbnd6;O</UP>]<SUP><UP>⋅</UP><UP>+</UP></SUP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>et1</UP></SUB></UL></LIM><UP> Mb-Tyr<SUP>103</SUP>-phenoxyl radical</UP>

<UP>Mb-Tyr<SUP>103</SUP>-phenoxyl radical</UP>+<UP>Mb-Cys<SUP>110</SUP></UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>et2</UP></SUB></UL></LIM>

<UP>Mb-Tyr<SUP>103</SUP></UP>+<UP>Mb-Cys<SUP>110</SUP>-thiyl radical</UP>

<UP>2</UP>[<UP>Mb-Cys<SUP>110</SUP>-thiyl radical</UP>]<UP> → Mb-Cys-S-S-Cys-Mb</UP>

<UP><SC>Reactions</SC> 1–4</UP>
Human metMb reacts with hydrogen peroxide to yield a ferryl heme (Fe(IV)-oxo) porphyrin radical cation ((Globin-Fe(IV)=O)·+) that is related to the compound I formed by peroxidases (Reaction 1). Formation of Tyr103-phenoxyl radicals may then result from an intramolecular electron transfer reaction (rate constant ket1) in which the porphyrin radical cation is reduced by Tyr103 as shown in Reaction 2 (13). The resulting Tyr103-phenoxyl radical is the initial radical trapped by DMPO on wild type human Mb at high DMPO:protein ratios. Subsequently, a second electron transfer reaction (rate constant ket2) affords the Cys110-thiyl radical and regenerates the Mb protein (Reaction 3). Finally, Cys110-thiyl radicals react in a bimolecular reaction to generate the disulfide homodimer (Reaction 4).

Varying the protein concentration may affect the yield of the homodimer in two ways. First, if formation of the Cys110-thiyl radical involves an intermolecular process(es), then the yield of this radical and the disulfide-linked homodimer should be directly dependent on protein concentration. That is, the yield of the homodimer is related to the efficiency of the intermolecular electron transfer from the sulfhydryl group at Cys110 to the tyrosyl phenoxyl radical at Tyr103 (Reaction 4). At higher protein concentration, this intermolecular process would occur with greater efficiency to generate a higher concentration of Cys110-thiyl radicals that in turn generates greater concentrations of homodimer. Overall, the rate of accumulation of the homodimer is proportional to both the concentration of Cys110-thiyl radical and the absolute concentration of protein as described by the process shown (Reaction 4). Alternatively, if the mechanism for Cys110-thiyl radical formation involves intramolecular reduction of the Tyr103-phenoxyl radical by Cys110, then the yield of Cys110-thiyl radical and the overall protein-standardized yield of homodimer would be independent of protein concentration.

Therefore, the increased yield of disulfide dimer observed with increasing concentration of human Mb strongly supports the argument that Cys110-thiyl radical formation (Reaction 3) involves an intermolecular electron transfer reaction. That the disulfide dimer is detected in a reaction mixture of the Y103F and C110A variants and H2O2 confirms that Cys110-thiyl radicals are generated on the Y103F variant by reaction with Tyr103-phenoxyl radicals on a molecule of the C110A variant and is further evidence to support the occurrence of an intermolecular electron transfer reaction. Furthermore, the dependence of homodimer yield on the initial concentration of C110A in the mixed variant reactions with peroxide (data not shown) emphasizes the role of the Tyr103-phenoxyl radical in generating Cys110-thiyl radicals by an intermolecular reaction. That is, the concentration of Tyr103-phenoxyl radical in the mixture is dependent on the starting concentration of C110A variant, and increasing this concentration results in more efficient generation of the Cys110-thiyl radical on the Y103F variant present in the reaction mixture that in turn yields higher concentrations of disulfide dimer.

Alternate Mechanisms for Cys110-thiyl Radical Formation in Human Mb-- Direct oxidation of the sulfhydryl group at Cys110 in the wild type protein by peroxide might produce Cys110-thiyl radicals (i.e. a one-electron oxidation product), but thiol oxidation in this manner conflicts with the expectation that H2O2 acts as a potent two-electron oxidant. Furthermore, as evidence against this potential mechanism of direct Cys110-thiyl radical generation, incubation of Y103F (a protein that contains Cys110) with H2O2 gave only trace concentrations of DMPO-Cys110 and no detectable disulfide product (Figs. 3 and 8A, respectively). Interestingly, cysteine residues within a protein may be oxidized to the sulfenic acid (RS-OH) by added peroxide, and this reaction may be reversed in the presence of suitable reductants (42, 43). Conversion of cysteine to the corresponding sulfenic acid in this manner does not involve thiyl radical formation. In addition to Tyr103-phenoxyl radicals, Trp14-derived peroxyl radicals are also formed on wild type human Mb (25) and other mammalian Mbs (20). Trp14-peroxyl radicals are reportedly (20, 44) capable of reacting with a wide range of substrates (including other free amino acids). Therefore, another possible mechanism that may result in Cys110-thiyl radical generation on wild type human Mb is through reduction of Trp14-derived radicals by the sulfhydryl at Cys110 (again via either an intra- or intermolecular pathway). However, any potential electron transfer between Trp14 and Cys110 can be ruled out in reactions of wild type human Mb and H2O2 by way of a similar argument against direct H2O2-mediated oxidation of Cys110. That is, despite the presence of Trp14 and Cys110 in the Y103F variant, reaction of this protein with H2O2 failed to yield either a significant concentration of DMPO-Cys110 or detectable quantities of disulfide-coupled Mb homodimer. Thus, it is unlikely that Cys110 can reduce a Trp14-centered radical.

Another means by which protein thiol groups may be oxidized is through direct oxidation of the sulfhydryl by compound I in a manner similar to peroxidase-catalyzed mono-oxygenase reactions of the P450 class of heme-proteins (45). However, by analogy to the reaction of compound I of Mb and organic sulfides to yield sulfoxides (46, 47), oxygenation reactions involve direct oxygen transfer that does not generate thiyl radicals. Yet another oxidant remaining in reaction mixtures of Mb and H2O2 that is also capable of oxidizing a variety substrates (48, 49) is the ferryl (Fe(IV)=O) heme center. For example reaction mixtures of horse heart Mb and H2O2 reportedly show a lipoxygenase-like activity that leads to peroxidation of low molecular mass polyunsaturated lipids (49). However, a substrate that is oxidized directly by ferryl (Fe(IV)=O) heme is limited by the requirement that it must gain sufficient proximity to the heme binding site to permit reaction with the activated oxygen and then leave the heme cavity as an oxygenated product (49). Although a thiyl radical would not be expected to form in this manner, it it conceivable that a sulfoxide group could be produced in this two-electron oxidation process. Additionally, if electron transfer occurred between the edge of ferryl heme and Cys110, then treatment of the Y103F variant with H2O2 would be expected to yield significant amounts of DMPO-Cys110 and detectable homodimer, which is not the case.

One final alternate explanation for our data is that the rate of the reaction leading to the formation of the homodimer is dependent on the half-life of the Cys110-thiyl radical. At lower protein concentrations, Cys110-thiyl radicals may decompose before taking part in the bimolecular termination reaction described by reaction (4), whereas at higher protein concentration this may not be the case. This dependence is difficult to substantiate, and pathways for the decay of Cys110-thiyl radicals (other than Reaction 4) have not been identified. Therefore, the dependence of homodimer formation on the half-life of Cys110-thiyl radicals remains a plausible explanation for our data.

Importantly, we reiterate that hydroxyl radicals do not appear to be involved in the transfer of oxidative damage to the protein (22, 23) at least under the experimental conditions employed here. In any case, the risk of hydroxyl radical formation in the current study has been prevented by the use of the iron chelator DTPA throughout.

In summary, we have investigated the reactions of wild type human Mb and both the C110A and Y103F variants to evaluate the mechanism of Cys110-thiyl radical formation. Our studies outlined in this work indicate that the mechanism leading to Cys110-thiyl radical formation involves intermolecular electron transfer. In contrast, alternate mechanisms of thiol oxidation including direct oxygenation of the protein-thiol do not lead to thiyl radical production. Overall, we have combined use of site-directed mutagenesis, SDS-PAGE, and EPR spectroscopy to demonstrate that the Cys110-thiyl radical can result from an intermolecular electron transfer reaction in which the Tyr103-phenoxyl radical is reduced by the sulfhydryl group at Cys110.

    ACKNOWLEDGEMENTS

We thank Professor Steven G. Boxer and Dr. Eun Sun Park for providing the plasmids used to express wild type and the C110A variant human proteins and Dr. Michael J. Davies for stimulating discussions on probing mechanisms of protein electron transfer.

    FOOTNOTES

* This work was supported by Grant O 98S 0008 from the National Heart Foundation of Australia (to P. K. W.) and Medical Research Council of Canada Grant MT-7182 (to A. G. M.).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.

Dagger To whom correspondence be addressed: Dept. of Biochemistry and Molecular Biology, 2146 Health Sciences Mall, University of British Columbia, Vancouver, V6T 1Z3, Canada.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011707200

    ABBREVIATIONS

The abbreviations used are: Mb, myoglobin; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DMPO-Cys110, DMPO adduct of cysteine 110; DMPO-Tyr103, DMPO adduct of tyrosine 103; DTPA, diethylenetriaminepentaacetic acid; globin·, protein radicals; H2O2, hydrogen peroxide; MNP, 2-methyl-nitrosopropane; mT, millitesla; metMb, metmyoglobin; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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