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J. Biol. Chem., Vol. 282, Issue 10, 7491-7503, March 9, 2007
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1
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From the
Department of Biochemistry and Microbiology, Laval University, Quebec G1K 7P4, Canada, the Department of Chemistry, Brooklyn College of the City University of New York, Brooklyn, New York 11210, the Departments of ||Chemistry and **Biochemistry, Graduate Center of the City University of New York, New York, New York 10016, and the ¶Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, September 26, 2006 , and in revised form, December 14, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq. We propose that Compound I formed transiently is reduced with concomitant oxidation of Trp(G8) to give the detected oxoferryl heme and a radical on Trp(G8) (detected by EPR of the trHbO Tyr(CD1)Phe mutant). In the wild-type protein, the Trp(G8) radical is in turn reduced rapidly by Tyr(CD1). In a second cycle, Trp(G8) may be reoxidized by the ferryl heme to yield ferric heme and two protein radicals. In turn, these migrate to form tyrosyl radicals on Tyr55 and Tyr115, which lead, in the absence of a reducing substrate, to oligomerization of the protein. Steady-state kinetics in the presence of H2O2 and the one-electron donor 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) indicated that trHbO has peroxidase activity, in accord with the presence of typical peroxidase intermediates. These findings suggest an oxidation/reduction function for trHbO and, by analogy, for other Group II trHbs. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The x-ray structure of trHbO reveals the presence of three potentially oxidizable residues, Tyr23(B10), Tyr36(CD1), and Trp88(G8), in the vicinity of the heme, with Tyr(CD1) and Trp(G8) within H-bonding distance from the bound ligand (Fig. 1) (3). Studies of trHbO variants suggest that Tyr(CD1) and Trp(G8) control O2 association and dissociation rates, with Tyr(B10) playing a minor role (1). Interestingly, O2, NO, and CO all combine with deoxyferrous trHbO at similar very slow rates (1). These slow ligand-independent combination rates indicate that the electronic factors that give rise to the large ligand-specific differences in most Mbs and Hbs are not dominant in trHbO. Instead, these observations are most consistent with limited access of ligands to the heme iron. Similarly, the comparable slow rate for the reaction of NO with heme-bound O2 of oxy-trHbO demonstrates that access of small molecules to heme-bound ligands is also limited.
Three observations suggest that trHbO may be designed to perform redox reactions. First, there is the presence of the electron-rich oxidizable residues Tyr(B10), Tyr(CD1), and Trp(G8) in the vicinity of the heme, which is quite unusual for a globin. Second, the crystal structure of the cyanomet derivative of M. tuberculosis trHbO revealed the presence of a presumably posttranslational Tyr(B10)-Tyr(CD1) cross-link in the heme active site of one-half of the molecules (3). The Tyr-Tyr cross-link adds trHbO to a growing list of hemoproteins that have aromatic amino acids covalently modified in their active sites: cytochrome c oxidase (His240-Tyr244), catalase HPII (His392-Tyr415), catalase-1 (Cys356-Tyr379), the catalase-peroxidases (KatG; Met255-Tyr229-Trp107), and a cytochrome c peroxidase mutant (Trp51-Tyr52) (4-12). Third, oxy-trHbO is reduced by dithionite to deoxyferrous trHbO without prior dissociation of the oxygenous ligand (1). This is an unusual reaction for an oxyferrous globin, which has also been reported for trHbO of Bacillus subtilis (13). The ability of dithionite to reduce hemebound O2 without the prior dissociation of the O2 and the presence of oxidizable residues in the immediate heme vicinity points to the possibility that highly sequestered ligands may be especially prone to redox reactions. In this regard, substitutions of Phe(CD1) with either Tyr or Trp in Mb have been shown to promote changes in redox properties (14, 15).
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-cation radical. Compound I can perform either a two-electron oxidation reaction to regenerate the ferric resting state, as observed in catalases, or a one-electron oxidation reaction (peroxidase activity) to generate Compound II, which consists of oxoferryl heme. Compound II can perform a second one-electron oxidation, which returns the enzyme to its ferric state. In some cases such as yeast cytochrome c peroxidase, fungal lignin peroxidase, and versatile peroxidase, Compound I is unstable, leading to the formation of a protein radical and oxoferryl heme (17-24). We show here that trHbO reacts with H2O2 to give a putative transient Compound I intermediate that is rapidly converted through oxidation of Tyr(CD1) and Trp(G8) to a species with heme in the ferric state and two protein radicals. Optical stopped-flow experiments revealed a transient oxoferryl intermediate, whereas EPR spectroscopy of rapidly frozen samples provided evidence for both tyrosyl and tryptophanyl radicals, the latter clearly identified in the mutant Tyr(CD1)Phe. In absence of a reducing substrate, the protein radicals lead to oligomerization of the protein involving Tyr55 and Tyr115. The steady-state kinetics in the presence of H2O2 and the one-electron donor ABTS indicate that trHbO has peroxidase activity. These findings may provide insights into the function of trHbO and other Group II trHbs.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Chemicals—H2O2 (30%, v/v) was obtained from BDH. The concentration of the stock solution was determined spectro-photometrically at 240 nm (
= 43.5 M-1 cm-1). Horseradish peroxidase (HRP), ferricytochrome c, ABTS, (hexa)amine ruthenium III, sodium ascorbate, NADH and KCN were obtained from Sigma.
Buffer—Except when noted, all solutions were prepared in 50 mM potassium phosphate buffer (pH 7.0) containing 50 µM EDTA.
Optical Absorption Spectroscopy—Optical absorption spectra were recorded using a Cary 3E spectrophotometer (Varian, Inc., Mississauga, Canada) equipped with a temperature-controlled multicell holder. Ferric trHbO samples were prepared in buffer. All spectra were recorded at 5 µM trHbO and 23 °C and analyzed using KaleidaGraph software (Synergy Software).
Determination of H2O2 Concentration—H2O2 concentration was determined using a peroxidase assay. H2O2 consumption was detected by the HRP-catalyzed formation of ABTS oxidation product at 414 nm in a mixture containing 1 mM ABTS, 10 nM HRP, and 2.5 µM trHbO that was reacted or not with H2O2 (1 and 2 eq). H2O2 concentration was calculated from the increase in the absorbance at 414 nm using a molar extinction coefficient of 3.6 x 104 M-1 cm-1 (26). A standard curve was made using known concentrations of H2O2 (0-20 µM).
Resonance Raman Spectroscopy—Protein samples for the resonance Raman experiments were used at a concentration of 50 µM in buffer. The trHbO/H2O2 (1:1) reaction product was obtained by manually mixing ferric trHbO with 0.01 volume of 5mM H2O2. trHbO Compound III was formed by reacting 100 µM ferric protein with 2 mM H2O2 on ice for 2 min. The excess H2O2 was rapidly removed by gel filtration on a P-6DG column equilibrated with buffer, and the protein concentration was then adjusted to 50 µM. The oxyferrous form of trHbO was produced by the reduction of the ferric derivative with sodium ascorbate (1 mM) and the mediator (hexa)amine ruthenium III (5 µM) and was directly transferred in a Raman quartz cell after 10 min. The resonance Raman spectra were obtained as described previously (27). Briefly, the 413-nm line of a krypton ion laser (Innova 302, Coherent Inc., Santa Clara, CA) was used to probe the ferric and H2O2-treated ferric and ferrous oxygenated forms of trHbO. The resonance Raman spectra were calibrated with the lines of indene in the 200-1700 cm-1 range. All measurements were made at room temperature. Cosmic ray lines were removed from the spectra by a routine of WinSpec software (Roper Scientific, Trenton, NJ). Several 5-min spectra were acquired over a 30-min period and analyzed using GRAMS/AI software (Thermo Scientific).
Titration with Ferrocytochrome c—A stock solution of ferrocytochrome c was prepared by reducing ferricytochrome c with 10 mM ascorbate. The progress of the reduction reaction was followed by optical absorption spectroscopy in the visible region. Excess ascorbate was then removed by gel filtration on a Bio-Gel P-6DG column (10 ml) equilibrated with buffer. The concentration of the stock solution was determined spectrophotometrically at 550 nm (550 = 27.6 mM-1 cm-1) (28). The stock solution was kept frozen in liquid nitrogen until used. To determine the number of oxidizing equivalents in the product formed by the reaction of ferric trHbO with 1 eq of H2O2, increasing amounts of ferrocytochrome c were premixed with 5 µM ferric trHbO in 50 mM potassium phosphate buffer (pH 7.0). The reactions were started by the addition of H2O2 toa5 µM final concentration and followed at 550 nm. The extinction coefficient 
550 (ferrocytochrome minus ferricytochrome c = 19.6 mM-1 cm-1) was used to calculate the yield of oxidized cytochrome c (28). This experiment was performed twice with trHbO and once with HRP. The graph presented in Fig. 3 represents one set of data obtained with each of the two proteins. A control experiment in which 5 µM ferrocytochrome c was mixed with 5 µM H2O2 showed that <5% of the ferrocytochrome c was oxidized in the absence of trHbO.
Oligomerization and SDS-PAGE of Protein Samples—Cross-linking experiments were performed in buffer at 23 °C for 5 min. Ferric trHbO (20 µM) was reacted with 0, 0.25, 0.5, 1, 5, 10, 20, and 40 eq of H2O2 in a total volume of 75 µl. Following incubation, excess H2O2 and other low molecular weight substances were removed by gel filtration on a Microspin P-6 column (Bio-Rad) equilibrated with 30 mM Tris-Cl buffer (pH 6.8). SDS-PAGE sample buffer was added to the protein samples and heated at 65 °C for 5 min prior to loading onto 15% polyacrylamide gels. The gels were stained with Coomassie Brilliant Blue (ICN). The effect of NADH, ascorbate, mannitol, and KCN was checked by including these agents separately (1 mM) in the reaction mixture before the addition of H2O2 (H2O2/trHbO molar ratio of 5).
Separation and Quantification of trHbO Cross-linked Products by Gel Filtration Chromatography—Protein samples (100 µM) in buffer were exposed to 0, 1, and 3 molar eq of H2O2 for 5 min at 23 °C. Following incubation, excess H2O2 was removed by gel filtration on a Microspin P-6 column equilibrated with buffer containing 300 mM KCl. Protein samples were subjected to size exclusion chromatography at 23 °C on a Superdex 75 HR 10/30 column equilibrated in buffer containing 300 mM KCl. Gel filtration standards (Bio-Rad catalog no. 151-1901) were used to calibrate the column. The standards were dissolved in 100 µl of buffer containing 300 mM KCl. Elution of the protein was followed at 280 nm. The area under the peaks was determined and used to estimate the proportion of cross-linked proteins.
Measurement of Catalytic Oxidation Activities—The peroxidase catalytic pathway of trHbO and horse heart myoglobin was investigated by stopped-flow spectrophotometry (SX. 18MV stopped-flow spectrophotometer, Applied Photophysics Ltd., Leatherhead, UK) at 23 °C in buffer. At least two experiments were performed for each experimental point. Steady-state kinetic constants for the oxidation of ABTS were obtained by measuring the initial rates while varying the H2O2 concentration. Vmax and Km values were determined by fitting data with the Levenberg-Marquardt robust method (SoftZymics, Inc., Princeton, NJ). The formation rate of the ABTS oxidation product was determined from the increase in the absorbance at 414 nm using a molar extinction coefficient of 3.6 x 104 M-1 cm-1 (26). The reaction mixture contained 50 nM protein, 1 mM ABTS, and 0.1-100 mM H2O2.
Kinetics Study of the Formation of trHbO Higher Oxidizing Intermediates—Stopped-flow experiments were carried out with the SX.18MV stopped-flow spectrophotometer equipped with a photodiode-array detector. The integration time was 2.5 ms. Ferric proteins (5 µM) were reacted with 1 molar eq of H2O2. 1600 spectra were collected on time scales ranging from 4 to 524 s. Singular value decomposition and global analysis were performed using the ProK program (Applied Photophysics Ltd.). Kinetics constants obtained from fitting had uncertainties of 
5%. The results shown in Figs. 7, 8, 9 and 10 are representative of at least two experiments.
EPR Spectroscopy—X-band EPR spectra were recorded using a Bruker E500 EPR spectrometer with data acquisition and manipulation performed using XeprView and WinEPR software (Bruker Daltonics Inc., Billerica, MA). Low temperature spectra were recorded using an Oxford Instruments Spectrostat continuous-flow cryostat and ITC503 temperature controller for temperatures from 4 to 20 K. A finger Dewar flask inserted into the microwave cavity was used for recording spectra at liquid nitrogen temperature (77 K). The spectra of ferric proteins (200 µM) were recorded in 50 mM Tris-HCl buffer (pH 7.5) at 4 K. For spectra of intermediates, ferric proteins (167 µM final concentration) were reacted with 3 molar eq of hydrogen peroxide, both in 50 mM Tris buffer (pH 7.5) for 10 s and then rapidly frozen in precision bore EPR tubes immersed in liquid nitrogen. The samples were examined at 77 K (microwave frequency of 9.3910 GHz), 4 K, and 20 K (microwave frequency of 9.4940 GHz). Simulation of EPR data was performed using SimFonia software (Bruker Daltonics Inc.).
Electron transfer coupling factors were calculated by PATHWAYS analysis (29) and the HARLEM program based on coordinates from the wild-type trHbO crystal structure (Protein Data Bank code 1NGK [PDB] ) (30). This analysis provides the optimal donor-acceptor electron transfer pathway between tryptophan or tyrosine residues and the heme or between particular residues.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3 and 2)of the heme iron (32). As shown in supplemental Fig. S1A, the resonance Raman spectra of untreated trHbO and H2O2-treated trHbO are nearly identical, consistent with the UV-visible spectroscopic observations above, suggesting that the heme iron is in the ferric state after incubation with peroxide. To demonstrate that the reaction with hydrogen peroxide generates a new oxidation state and to evaluate how many oxidizing equivalents are stored, we titrated the protein product of the reaction of trHbO and 1 eq of H2O2 with the one-electron donor ferrocytochrome c. We chose ferrocytochrome c as reductant for two reasons: 1) it does not reduce ferric trHbO to ferrous trHbO, which avoids possible side reactions involving molecular O2; and 2) it does not react with H2O2 under the conditions used. When 5 µM ferric trHbO was incubated simultaneously with 1 eq of H2O2 and ferrocytochrome c, a maximum of 10 µM ferrocytochrome c could be oxidized to ferricytochrome c (Fig. 3). Thus, both oxidizing equivalents from H2O2 were retained in the trHbO product, although, as shown above, the iron was found in the ferric state when observed after manual mixing with peroxide. Titration of HRP under these conditions indicated that the expected 2 oxidizing eq were retained in the product.
Most peroxidases and many Hbs and Mbs form Compound III at high H2O2 concentration (33-36). This species is similar to oxy-Mb. As shown in Fig. 2A, mixing ferric trHbO with 100 molar eq of H2O2 gave a stable species with an absorption spectrum identical to that of oxy-trHbO. To confirm the identity of the trHbO species, we used resonance Raman spectroscopy. As shown in supplemental Fig. S1B, the spectrum of H2O2-treated trHbO is nearly identical to that of oxy-trHbO, with 4, 3, and 2 mode lines at 1379, 1506, and 1582 cm-1, respectively, indicating a reaction with heme iron, but no modification or break-down of the macrocycle. These results demonstrate that, in common with many Hbs and peroxidases, trHbO forms Compound III readily at high H2O2 concentrations.
Oligomerization of H2O2-treated trHbO
The preceding results led us to hypothesize that 2 oxidizing eq, initially resident on the heme and its ligand, may have been transferred to amino acid residues near the heme to form radicals. These primary protein-based radicals may have then led, via internal electron transfer, to the formation of surface-exposed amino acid radical(s) with subsequent cross-linking of the protein. Such reactions have been observed in several hemeproteins, including sperm whale Mb, when reacted with H2O2 in the absence of an exogenous electron donor (37-43). In most cases reported to date, the process involves the formation of tyrosine-tyrosine cross-links. In trHbO, there are six tyrosines, Tyr6, Tyr23(B10), Tyr36(CD1), Tyr55, Tyr62, and Tyr115 (Fig. 1), potentially available for conversion to tyrosyl radicals and for quenching by radical combination. Of these, Tyr6 and Tyr55 are exposed to the solvent. Accordingly, we reacted ferric trHbO with increasing amounts of H2O2 and analyzed the samples by SDS-PAGE to detect the formation of cross-linked products. As shown in Fig. 4A, trHbO dimers were detected with as little as 0.25 molar eq of H2O2. The amount of the cross-linked dimers increased with the H2O2 concentration, reaching a maximum with 10 molar eq of H2O2. Above 5 molar eq of H2O2, small amounts of trimer and tetramer were observed, suggesting the existence of more than one surface-exposed radical site. It should be noted that the amount of trimers and higher oligomers varied from one experiment to the other.
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0.6%) at 1:1, increased to 5% at 3:1, consistent with the presence of at least two surface-exposed protein-based radicals (supplemental Fig. S2). To determine whether trHbO oligomerization requires reaction of H2O2 at the heme, we repeated this experiment with the cyanomet derivative of trHbO. Blocking the heme by bound cyanide inhibited the oligomerization reaction, confirming that the initial reaction of trHbO with H2O2 must involve the heme group and, accordingly, that H2O2 did not directly oxidize surface-exposed residues (Fig. 4B).
In another set of experiments, we examined the effects of two reductants, NADH and ascorbate, on the oligomerization of trHbO. As shown in Fig. 4B, the addition of excess NADH or ascorbate to the reaction mixture completely inhibited the formation of cross-linked products. These observations suggest either that both agents reduced an initial product in which the 2 oxidizing eq resided on the peroxide-treated protein or that they both directly reduced the surface-exposed radicals.
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Identification of the Residue(s) Involved in Protein Oligomerization
To identify the site(s) of dimerization, we produced six mutants in which a single tyrosine was replaced with phenylalanine. Each of these mutants was incubated with 5 eq of H2O2 and analyzed by SDS-PAGE. As shown in Fig. 5, all of the mutants formed cross-linked products to about the same extent as did wild-type trHbO. Interestingly, in both the Y55F and Y115F variants, the slower and faster migrating species were no longer observed. Because Tyr55 and Tyr115 are close to each other in the folded protein, with Tyr55 exposed to solvent, we produced the double mutant Y55F/Y115F and checked for the formation of cross-linked products. As demonstrated in Fig. 5, Y55F/Y115F no longer formed dimers.
Peroxidase Activity of trHbO
We examined whether trHbO possesses peroxidase activity. For this, the one-electron oxidation of ABTS to its corresponding radical cation was followed at 414 nm using H2O2 as an oxidant of trHbO. Fig. 6A shows the progress curves in the presence of 1 mM H2O2 at two protein concentrations. The absorbance increase trailed off in a nonlinear manner before 10% of ABTS was depleted, presumably because of enzyme inactivation. Selwyn's test (46) confirmed that the enzyme was indeed inactivated during the course of the reaction (supplemental Fig. S3, A and B). Only initial velocities could be determined. The apparent rate of the reaction of trHbO was obtained from the slope of the plot of initial velocity versus H2O2 concentration (Fig. 6B). The linear relationship that was found indicates an apparent first-order dependence on H2O2 concentration of (1.35 ± 0.025) x 103 M-1 s-1. For comparison, the reaction of H2O2 with horse heart Mb was also examined. Mb displayed a hyperbolic dependence on the concentration of H2O2 (Fig. 6C). The apparent kcat and Km values for H2O2 are 0.45 ± 0.008 s-1 and 1.06 ± 0.10 mM, respectively. The kcat value (turnover number at maximal velocity) for trHbO could not be determined, but the turnover number calculated at 40 mM H2O2(50 s-1) indicates that trHbO can achieve a much higher number of catalytic events per unit of time compared with Mb.
Kinetic Studies of the Reaction of H2O2 with Wild-type trHbO and Its Heme-distal Mutants Tyr(B10)Phe, Tyr(CD1)Phe, and Trp(G8)Phe
We showed above that the reaction of trHbO with an equimolar amount of H2O2 leads to the formation of an intermediate product with a ferric heme and bearing 2 oxidizing eq on the protein. To capture the optical spectra of the species intermediate in the formation of this product, the reaction was analyzed using a stopped-flow spectrophotometer equipped with a photodiode array detector. The role of the distal residues Tyr(B10), Tyr(CD1), and Trp(G8) in the formation of the intermediate was investigated. For this, the ferric proteins were mixed with 1 eq of H2O2, and 1600 spectra were collected on increasing time scales. For clarity, only data collected over 524 s are presented (supplemental Figs. S4A-S7A). Singular value decomposition and global analysis allowed fitting of the kinetic data of all proteins to the model A 
B C. The calculated rates and the wavelength maxima of the intermediate species are shown in Tables 1 and 2. As detailed below, a new intermediate species (SP-427) with maxima in difference spectra at 427, 528, and 561 nm was detected as a transient intermediate. The concentration of SP-427 was much enhanced in the reaction of the mutant lacking Tyr(CD1), and SP-427 was not detected in the reaction of the mutant lacking Trp(G8). A maximum at 600 nm in the difference spectra is evident with all of the proteins except the mutant lacking Tyr(B10). The models allowed prediction of the formation and decay of each species for the reaction, as shown in supplemental Figs. S4B-S7B.
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Tyr(CD1)Phe Mutant—The spectral changes of the Tyr(CD1)Phe mutant were more significant. Spectrum A (corresponding to the initial form) shows a high and sharp Soret band at 405 nm and a well developed CT1 band at 634 nm, indicating that the Tyr(CD1)Phe mutant is enriched in six-coordinated high spin heme compared with the wild-type protein (Fig. 9A) (47). Spectrum B shows a Soret band at 410 nm with a broad absorbance centered at 530 nm. Spectrum C is very similar to that of the initial form. The difference spectrum shown in Fig. 9B (spectrum B minus spectrum A of Fig. 9A) shows the same SP-427 peaks as observed in the wild-type protein except that the difference in absorbance is 6-fold higher. These data suggest that Tyr(CD1) is involved in the formation and/or decay of SP-427.
Trp(G8)Phe Mutant—Fig. 10A presents the singular value decomposition and global analysis of the reaction of the ferric Trp(G8)Phe mutant with H2O2. Spectrum B is representative of a six-coordinated high spin complex with a more intense Soret band at 405 nm and a CT1 band at 630 nm (47). Spectrum C is similar to species B except for a small decrease in intensity at the Soret band. The difference spectrum shown in Fig. 10B (spectrum B minus spectrum A of Fig. 10A) is different from that of SP-427 and instead indicates the disappearance of a low spin species evidenced by the troughs at 420, 548, and 582 nm. The Trp(G8)Phe mutant retains 2 oxidizing eq of H2O2 following the reaction with H2O2 as determined by titration with reduced ferrocytochrome c (data not shown). These results indicate that SP-427 is too short-lived to be detected or is not formed. They also show that the mutant still forms radicals and that an intermediate is therefore formed and subsequently reduced.
EPR Spectroscopy of trHbO and Its Distal Variants before and after Reaction with H2O2
Ferric Protein—The low temperature (4 K) EPR spectra of trHbO and its mutants have features typical of six-coordinated high spin heme with nearly axial signals (g 
= 5.80 and 2) as well as significant amounts of one or more 6-coordinated low spin ferric heme signals with g1 = 2.66-2.82, g2 = 2.16-2.20, and g3 = 1.69-1.79 (supplemental Table S1). The low spin signals are similar to those reported previously for 6-coordinated low spin species with a hydroxy ligand to the heme (31, 48-50). The small differences in the g-values of the six-coordinated low spin species in the mutants compared with the wild-type protein are likely due to small structural differences in the distal side residues within the heme pocket.
trHbO Reacted with H2O2—Resting (ferric) protein was mixed with a small excess (3-fold) of hydrogen peroxide and frozen after 10 s of incubation. These conditions are expected first to produce hypervalent heme iron and then to generate amino acid-based radicals according to the observations reported above. EPR spectra were recorded at 4, 20, and 77 K in attempts to identify the intermediates and/or radical species. A signal at g = 2.004 with a line width of 21 G was detected at 77 K in all samples (Fig. 11A) with no or poorly resolved hyperfine splitting. In addition, a small proportion of ferric iron was still present as evidenced by g = 6 signals visible in the low field region of spectra recorded at 4 K (data not shown). Preliminary simulation of the g = 2.004 signal for wild-type trHbO suggested that it arises from a tyrosyl radical, as hyperfine interactions for two nonequivalent 
-methylene protons with coupling constants of 12 and 1 G, similar to protein-based tyrosyl radical species formed in other enzymes (50-52), gave an adequate fit to the data. The hyperfine coupling parameters in tyrosyl radicals depend on the orientation of the phenolic ring plane relative to the position of -methylene protons (24, 53). Examination of the three-dimensional crystal structure of trHbO suggested that Tyr(B10), Tyr(CD1), or Tyr115 could give rise to tyrosyl radicals with EPR signals similar to those observed here. However, no further analysis or detailed simulation was performed because of the heterogeneous nature of the structures of Tyr(B10) and Tyr(CD1), which are covalently linked in some subunits of the enzyme according to the three-dimensional crystal structure (3). Such linkage would remove the contribution of some ring protons from the total hyperfine coupling in the spectra of the radicals. Furthermore, neutral tryptophanyl radicals can give signals that may overlap with tyrosyl radical signals in X-band EPR spectra. Rapid freeze-quench EPR experiments were also performed to examine radical formation at time scales <1 s, but the low intensity of the signals did not provide useful information (data not shown).
EPR spectra were also recorded at liquid helium temperatures (on the same samples as described above) in an attempt to identify other heme or radical intermediates formed in the protein upon treatment with peroxide. No new features were found at 20 K for any of the samples (Fig. 11B, upper spectrum shown as an example), whereas at 4 K, the Tyr(CD1)Phe mutant exhibited a new axial signal with effective g-values of g|| = 2.026 and g|| = 2.005 (Fig. 11B, lower spectrum). This spectrum is very similar to that of the exchange-coupled tryptophanyl -cation radical species found in cytochrome c peroxidase Compound I (54, 55). Therefore, the signal found at low temperature in this mutant is reasonably assigned to the same type of tryptophan radical exchange-coupled to oxoferryl heme (S = 1). A small component due to the signal from a tyrosyl radical is present with diminished intensity because of power saturation at this temperature (20-milliwatt microwave power) (Fig. 11B, lower spectrum). No evidence for a classical oxoferryl heme -cation radical (classical peroxidase Compound I) was found in the EPR spectra of any of the proteins, consistent with rapid electron transfer(s) from the nearby amino acids that quench this intermediate. Although an EPR signal arising from a Trp cation radical coupled to oxoferryl heme as in cytochrome c peroxidase Compound I could be present along with a tyrosyl radical in the same protein molecule (according to the fact that 2 oxidizing eq are stored), its contribution at X-band is not apparent in the spectra of the wild-type protein at 77 and 4 K.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3-fold higher than that of horse heart Mb but 100-1000-fold lower than that reported for class III peroxidases such as HRP (16, 58-60). However, the turnover rate for ABTS peroxidation (50 s-1 at 40 mM H2O2) is much faster than that in horse heart Mb (kcat = 0.45 s-1). The latter observation suggests that the active site of trHbO may have evolved to perform oxidation reactions. In this regard, dehaloperoxidase from the polychaete worm Amphitrite ornata provides strong evidence for a peroxidase having evolved from an oxygen carrier (61-63). Dehaloperoxidase catalyzes the dehalogenation of halometabolites in the presence of H2O2, allowing A. ornata to inhabit sediments contaminated by halometabolites.
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6-fold higher in Tyr(CD1)Phe than in the wild-type protein, implying that Tyr(CD1) is involved in the decay of SP-427. EPR analyses performed at liquid helium temperatures revealed a transient tryptophanyl radical signal, similar to the cytochrome c peroxidase Compound I species, in the Tyr(CD1) Phe mutant reacted with H2O2. The absence of this signal in the wild-type protein is consistent with Tyr(CD1) being intimately involved in the rapid quenching of the tryptophanyl radical. The proximity of Trp(G8) to the heme makes it a likely candidate for the tryptophanyl radical cation. There is an additional tryptophan (Trp56) in trHbO, but the latter is located 14 Å from the heme iron. SP-427 could not be detected in the Trp(G8)Phe mutant, which underscores the participation of Trp(G8) in the formation of SP-427. However, a tyrosyl radical is still found in the EPR spectra of this mutant, which indicates that, in addition to electron transfer involving Trp(G8) and a Tyr residue, there is also direct oxidation of Tyr residue(s) without any Trp intermediary. Interestingly, the crystal structures of both yeast ferric cytochrome c peroxidase and the cyanomet derivative of trHbO show a distal Trp residue in close proximity to the heme. In both proteins, the indole ring is oriented parallel to the heme plane, and the nitrogen of the indole is positioned within H-bonding distance of their respective ligands to iron (3, 64). However, in contrast to Trp(G8) in trHbO, Trp51 in cytochrome c peroxidase does not form a radical in the reaction with H2O2, pointing to different functions for these two residues (23).
We propose that Trp(G8) is very rapidly oxidized in trHbO by the porphyrin radical cation of a postulated Compound I (Fig. 12, Reaction 1) to generate oxoferryl heme and a Trp(G8)-centered radical (Reaction 2). The Trp(G8) radical is in turn rapidly reduced by Tyr(CD1) (as only the tyrosine radical appears in the EPR spectra of the wild-type protein) (Reaction 3). Although speculative, Trp(G8) may then be reoxidized, this time by the oxoferryl heme, leaving a ferric heme and two protein radicals (Reaction 4). The Trp(G8) radical must again propagate to other residues, as the EPR spectra of the wild-type protein does not reveal the Trp radical cation found in the Tyr(CD1) mutant. Direct oxidation of other residues (by oxoferryl heme) could also lead to the formation of other tyrosyl radicals.
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Conclusion—The presence of Tyr(B10), Tyr(CD1), and Trp(G8) makes the active site of trHbO unique within the globin family. trHbO does not form a stable Compound I and/or II upon reaction with H2O2. This is in contrast to heme peroxidases, which form a stable Compound I and/or II plus a protein radical upon reaction with H2O2. The reaction of H2O2 with trHbO involves formation of a transient oxoferryl species (SP-427) with a short-lived radical likely on Trp(G8) that is reduced by Tyr(CD1). Overall, our data indicate that trHbs constitute a new enzymatic system to study how hemeproteins perform oxidation reactions.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S7 and Tables S1 and S2.
1 Present address: Dept. of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446.
2 To whom correspondence should be addressed: Dept. of Biochemistry and Microbiology, Pavillon Marchand, Rm. 3145, Laval University, Quebec G1K 7P4, Canada. Tel.: 418-656-2131 (ext. 5581); Fax: 418-656-7176; E-mail: mguertin{at}bcm.ulaval.ca.
3 The abbreviations used are: trHbO, truncated hemoglobin O; Mb, myoglobin; ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); HRP, horseradish peroxidase.
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) and HRP (
) at a concentration of 5 µM were incubated with increasing amounts of ferrocytochrome c to the final concentrations indicated on the abscissa, followed by the addition of 1 molar eq of H2O2. Because the initial protein concentrations were each 5 µM, the addition of cytochrome c to a final concentration of 10 µM corresponds to molar ratios of cytochrome c to trHbO or HRP of 2:1. The extinction coefficient 
