Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m210383200
ISSN1083-351X
AutoresTsuyoshi Egawa, Shiro Yoshioka, Satoshi Takahashi, Hiroshi Hori, Shingo Nagano, Hideo Shimada, Koichiro Ishimori, Isao Morishima, Makoto Suematsu, Yuzuru Ishimura,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at ≥423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O–O–H]–) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pK a ( pKaapp ) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKaapp paralleled that in pKa of the acid-alkaline transition ( pKaAB ) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O–O bond cleavage in heme peroxidases. The reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at ≥423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O–O–H]–) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pK a ( pKaapp ) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKaapp paralleled that in pKa of the acid-alkaline transition ( pKaAB ) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O–O bond cleavage in heme peroxidases. A large number of heme enzymes catalyze heterolysis of hydrogen peroxide (H2O2) 1The abbreviations used are: H2O2, hydrogen peroxide; metMb, metmyoglobin; ferryl Mb, ferryl myoglobin; CcP, cytochrome c peroxidase; HRP, horseradish peroxidase; LiP, lignin peroxidase; SVD, singular value decomposition. and utilize H2O2 as a source of oxidizing equivalents for biological oxidative reactions. The enzymes include a family of heme peroxidases from plants, yeast, fungi, and mammals (1Schonbaum G.R. Chance B. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 363-408Google Scholar, 2Yonetani T. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 345-361Google Scholar, 3Dunford H.B. 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High valent intermediates such as compounds I and II, which possess an oxoferryl porphyrin π-cation radical (Por+-Fe(IV)[O]2–, where Por+ represents porphyrin π-cation radical) and an oxyferryl heme (Por-Fe(IV)[O]2–), respectively, are known to be created upon the reaction of the heme enzymes with H2O2 and to carry out various oxidation reactions (1Schonbaum G.R. Chance B. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 363-408Google Scholar, 2Yonetani T. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 345-361Google Scholar, 3Dunford H.B. Stillman J.S. Coord. Chem. Rev. 1976; 19: 187-251Crossref Scopus (946) Google Scholar, 4Hewson W.D. Hager L.P. Dolphin D. The Porphyrins. Vol. 7. Academic Press, New York1979: 295-332Crossref Google Scholar). Compound I is the first detectable intermediate in the reactions of the heme enzymes previously studied, and the subsequent one-electron reduction of compound I by the substrate generates compound II (1Schonbaum G.R. Chance B. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 363-408Google Scholar, 2Yonetani T. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 345-361Google Scholar, 3Dunford H.B. Stillman J.S. Coord. Chem. Rev. 1976; 19: 187-251Crossref Scopus (946) Google Scholar, 4Hewson W.D. Hager L.P. Dolphin D. The Porphyrins. Vol. 7. Academic Press, New York1979: 295-332Crossref Google Scholar). In some enzymes such as cytochrome c peroxidase (CcP), the oxidizing equivalent on the π-cation heme of compound I is rapidly transferred to an amino acid residue in the protein moiety, creating another type of compound I (CcP-type compound I), which oxidizes the substrates (2Yonetani T. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 345-361Google Scholar). The chemical and enzymatic properties of the reaction between ferric heme and H2O2 have attracted considerable research interests to understand the reaction mechanisms of the heme enzymes. As an approach to the above-mentioned research interest, investigations on the reaction between myoglobin (Mb) and H2O2 have long been made by several groups (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 12George P. Irvine D.H. Biochem. J. 1952; 52: 511-517Crossref PubMed Scopus (202) Google Scholar, 13King N.K. Looney F.D. Winfield M.E. Biochim. Biophys. Acta. 1967; 133: 65-82Crossref Scopus (77) Google Scholar, 14Yonetani T. Schleyer H. J. Biol. Chem. 1967; 242: 1974-1979Abstract Full Text PDF PubMed Google Scholar, 15Ortiz de Montellano P.R. Catalano C.E. J. Biol. Chem. 1985; 260: 9265-9271Abstract Full Text PDF PubMed Google Scholar, 16Grisham M.B. 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J. 1952; 52: 511-517Crossref PubMed Scopus (202) Google Scholar, 13King N.K. Looney F.D. Winfield M.E. Biochim. Biophys. Acta. 1967; 133: 65-82Crossref Scopus (77) Google Scholar, 14Yonetani T. Schleyer H. J. Biol. Chem. 1967; 242: 1974-1979Abstract Full Text PDF PubMed Google Scholar). Both analogs can oxidize organic compounds (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Ortiz de Montellano P.R. Catalano C.E. J. Biol. Chem. 1985; 260: 9265-9271Abstract Full Text PDF PubMed Google Scholar, 16Grisham M.B. J. Free Radicals Biol. Med. 1985; 1: 227-232Crossref PubMed Scopus (96) Google Scholar, 17Galaris D. Sevanian A. Cadenas E. Hochstein P. Arch. Biochem. Biophys. 1990; 281: 163-169Crossref PubMed Scopus (80) Google Scholar, 18Adachi S. Nagano S. Ishimori K. Watanabe Y. Morishima I. Egawa T. Kitagawa T. Biochemistry. 1993; 32: 241-252Crossref PubMed Scopus (229) Google Scholar, 19Matsui T. Ozaki S. Watanabe Y. J. Biol. Chem. 1997; 272: 32735-32738Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The latter analog is termed ferryl Mb, whereas the former one, which was found recently (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), is named myoglobin compound I. All the basic reactions catalyzed by heme enzymes, i.e. formation of compound I, intramolecular electron transfer from an aromatic amino acid residue to the heme of compound I, and oxidation of organic or inorganic compounds by the high valent species, can be observed in Mb (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Ortiz de Montellano P.R. Catalano C.E. J. Biol. Chem. 1985; 260: 9265-9271Abstract Full Text PDF PubMed Google Scholar, 16Grisham M.B. J. Free Radicals Biol. Med. 1985; 1: 227-232Crossref PubMed Scopus (96) Google Scholar, 17Galaris D. Sevanian A. Cadenas E. Hochstein P. Arch. Biochem. Biophys. 1990; 281: 163-169Crossref PubMed Scopus (80) Google Scholar, 18Adachi S. Nagano S. Ishimori K. Watanabe Y. Morishima I. Egawa T. Kitagawa T. Biochemistry. 1993; 32: 241-252Crossref PubMed Scopus (229) Google Scholar, 19Matsui T. Ozaki S. Watanabe Y. J. Biol. Chem. 1997; 272: 32735-32738Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In this context, the reaction between H2O2 and metMb has been considered as a model system to understand the activation mechanisms of H2O2 by heme enzymes (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Ortiz de Montellano P.R. Catalano C.E. J. Biol. Chem. 1985; 260: 9265-9271Abstract Full Text PDF PubMed Google Scholar, 18Adachi S. Nagano S. Ishimori K. Watanabe Y. Morishima I. Egawa T. Kitagawa T. Biochemistry. 1993; 32: 241-252Crossref PubMed Scopus (229) Google Scholar, 19Matsui T. Ozaki S. Watanabe Y. J. Biol. Chem. 1997; 272: 32735-32738Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Chemical properties of the high valent species and mechanisms of reaction steps after formation of compound I have been studied extensively for peroxidases (1Schonbaum G.R. Chance B. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 363-408Google Scholar, 2Yonetani T. Boyer P.D. The Enzymes. Vol. 13. Academic Press, New York1976: 345-361Google Scholar, 3Dunford H.B. Stillman J.S. Coord. Chem. Rev. 1976; 19: 187-251Crossref Scopus (946) Google Scholar, 4Hewson W.D. Hager L.P. Dolphin D. The Porphyrins. Vol. 7. Academic Press, New York1979: 295-332Crossref Google Scholar, 5Agner K. Acta Chem. Scand. 1941; 2: 1-62Google Scholar, 6Harrison J.E. Schultz J. J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar) and also for Mb (11Egawa T. 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J. 2003; 84: 1998-2004Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In order to address the above problem, and also to give insights into the roles of amino acid residues that form the heme pockets of heme enzymes, we followed the reaction between H2O2 and metMb in detail. By using stopped flow-rapid scan, singular value decomposition (SVD), and rapid freezing-EPR techniques, we succeeded in detecting a hydroperoxy anion compound (Fe(III)[O–O–H]–) that is closely related to one of the postulated intermediates in the peroxidase reactions. Analyses of population changes of the hydroperoxy compound and compound I as a function of pH clearly demonstrated the involvement of the distal histidine in the reaction. Based on the present findings, we postulated mechanisms for the reaction between metMb and H2O2, and we also discussed the reaction mechanisms of the heme peroxidases. Materials—Sperm whale and horse heart metMbs were purchased from Sigma. Human metMb and its T67N mutant were expressed in Escherichia coli and were purified as described elsewhere (18Adachi S. Nagano S. Ishimori K. Watanabe Y. Morishima I. Egawa T. Kitagawa T. Biochemistry. 1993; 32: 241-252Crossref PubMed Scopus (229) Google Scholar, 41Nagano, S. (1985) Roles of the Active Site Residues in Catalytic Activities of Heme Enzymes, Ph.D. thesis, Kyoto University, Kyoto, JapanGoogle Scholar). Each metMb dissolved in a potassium phosphate buffer was treated with a small amount of potassium ferricyanide as described previously (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) to eliminate the reduced form of Mb remaining in the commercial products or in the preparations extracted from E. coli. The oxidized proteins were extensively dialyzed and further purified by CM-cellulose column chromatography (11Egawa T. Shimada H. Ishimura Y. J. Biol. 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Biochemistry. 1994; 33: 2198-2209Crossref PubMed Scopus (79) Google Scholar). In the present investigation, the rapid scan spectroscopic system allowed us to record absorption data at up to 512 points for both wavelength and time, and hence 512 × 512 data points were obtained upon each run of the stopped flow experiments. We collected data from 19 experimental runs and averaged them. Among the data thus obtained, those at every 1.62 nm from 322 to 525 nm (126 data points) and at every 3 ms from 6 to 195 ms (64 spectral scans) were selected. This time range was selected in order to cover most of the reaction period under the present experimental conditions. The data set of 126 × 64 points thus obtained was subjected to the SVD calculation. The computer program used for the calculation was described elsewhere (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 42Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar). Kinetic Parameters in Three Component Reactions—Concentrations of chemical species in three component sequential reaction represented by Reaction 1 are by Equations 1 and 2, R→k1I→k2PReaction1(Eq. 1) [R]=[R]0exp(-k1t)(Eq. 2) [I]=[R]0k1k2-k1{exp(-k1t)-exp(-k2t)}(Eq. 3) where k 1 and k 2 are rate constants of the reaction, and [R]0 is the initial concentration of the reactant (R) (49Szabó Z.G. Bamford C.H. Tipper C.F.H. Comprehensive Chemical Kinetics. Vol. 2. Elsevier Science Publishing Co., Inc., New, York1969: 1-31Google Scholar). Integrating Equations 1 and 2 with respect to time (t) from 0 to ∞, we obtained equations for total accumulated amounts of R and I in Reaction 1, and their ratio is given by Equation 3, [I]total/[R]total=k1/k2(Eq. 4) On the other hand, the equation used for determining maximum accumulation time (t max; time at which concentration reaches the maximum value) of intermediate I in Reaction 1 is found in Ref. 49Szabó Z.G. Bamford C.H. Tipper C.F.H. Comprehensive Chemical Kinetics. Vol. 2. Elsevier Science Publishing Co., Inc., New, York1969: 1-31Google Scholar as shown in Equation 4, tmax=1k2-k1lnk2k1(Eq. 5) Rapid Freezing-EPR Spectroscopy—Samples for EPR measurements were prepared by using a recently developed rapid freezing apparatus that was composed of a rapid mixing chamber and a freeze-quench device. The details of this apparatus are described elsewhere (40Tanaka M. Matsuura K. Yoshioka S. Takahashi S. Ishimori K. Hori H. Morishima I. Biophys. J. 2003; 84: 1998-2004Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We mixed metMb (1 mm) and H2O2 (200 mm) solutions in a 1:1 ratio and rapidly froze the mixed solution at 77 K to obtain the frozen flakes. The frozen flakes were collected and transferred into an EPR tube, and the EPR spectra of the samples were recorded at 15 K. The freezing dead time (time period from mixing to complete quenching) of our apparatus is 200 μs at minimum and is changeable by arranging conditions such as mixing flow rate and distance between the mixing and freeze-quench devices (40Tanaka M. Matsuura K. Yoshioka S. Takahashi S. Ishimori K. Hori H. Morishima I. Biophys. J. 2003; 84: 1998-2004Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Methods to calibrate the dead time were described already (40Tanaka M. Matsuura K. Yoshioka S. Takahashi S. Ishimori K. Hori H. Morishima I. Biophys. J. 2003; 84: 1998-2004Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In the present experiments, the freezing dead time was fixed at ∼3 ms. EPR spectra were measured by a Varian E-12 spectrometer equipped with an Oxford ESR-900 liquid helium cryostat. Measurements were carried out at the X-band (9.22 GHz) microwave frequency. Changes in Absorption Spectra during the Reaction between metMb and H2O2— Fig. 2A shows spectral changes observed during the reaction of 5 μm sperm whale metMb with 50 mm H2O2 in 200 mm potassium phosphate buffer, pH 6.0. The spectral changes shown here well reproduce those we reported previously for the same reaction (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). As we already indicated, formation of myoglobin compound I is detectable under the above experimental conditions (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Although these spectral changes might be viewed as an apparently single process of conversion from metMb (λmax, 409 nm) to ferryl Mb (λmax, 422 nm), the time profile of the absorption at an isosbestic point (418 nm) between the spectra of metMb and ferryl Mb shows formation of an intermediate (Fig. 2, inset a). The temporary decrease in the time profile is caused by the accumulation of compound I in the reaction system (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This accumulation of compound I can be observed more clearly in D2O (at pD 6.0) (11Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the present study, we show that the kinetics of the above reaction depends on the pH. Fig. 2B shows the absorption changes of the reaction at pH 8.5 in 200 mm glycylglycine-NaOH buffer. The reaction conditions other than pH and the buffer were identical to those used for Fig. 2A at pH 6.0. Although the gross features of the spectral changes at pH 8.5 were quite similar to those at pH 6.0, the absorption at 418 nm was almost unchanged during the entire reaction period (Fig. 2b). This result indicates that compound I scarcely accumulates at pH 8.5. Detection of a New Spectral Species in the Reaction at pH 8.5—To uncover the difference in the reactions at pH 6.0 and 8.5, we analyzed the above spectral data by SVD. In general, SVD calculations transform an absorption data matrix (i.e. a set of time-resolved absorption spectra) into a product of three matrices, U, S, and V t , where the ith column of the matrix U is termed ith basis spectrum
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