Kinetic Characterization of Compound I Formation in the Thermostable Cytochrome P450 CYP119
2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês
10.1074/jbc.c100745200
ISSN1083-351X
AutoresDavid Kellner, Shao-Ching Hung, Kara E. Weiss, Stephen G. Sligar,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoThe kinetics of formation and breakdown of the putative active oxygenating intermediate in cytochrome P450, a ferryl-oxo-(π) porphyrin cation radical (Compound I), have been analyzed in the reaction of a thermostable P450, CYP119, withmeta-chloroperoxybenzoic acid (m-CPBA). Upon rapid mixing of m-CPBA with the ferric form of CYP119, an intermediate with spectral features characteristic of a ferryl-oxo-(π) porphyrin cation radical was clearly observed and identified by the absorption maxima at 370, 610, and 690 nm. The rate constant for the formation of Compound I was 3.20 (±0.3) × 105m−1 s−1 at pH 7.0, 4 °C, and this rate decreased with increasing pH. Compound I of CYP119 decomposed back to the ferric form with a first order rate constant of 29.4 ± 3.4 s−1, which increased with increasing pH. These findings form the first kinetic analysis of Compound I formation and decay in the reaction of m-CPBA with ferric P450. The kinetics of formation and breakdown of the putative active oxygenating intermediate in cytochrome P450, a ferryl-oxo-(π) porphyrin cation radical (Compound I), have been analyzed in the reaction of a thermostable P450, CYP119, withmeta-chloroperoxybenzoic acid (m-CPBA). Upon rapid mixing of m-CPBA with the ferric form of CYP119, an intermediate with spectral features characteristic of a ferryl-oxo-(π) porphyrin cation radical was clearly observed and identified by the absorption maxima at 370, 610, and 690 nm. The rate constant for the formation of Compound I was 3.20 (±0.3) × 105m−1 s−1 at pH 7.0, 4 °C, and this rate decreased with increasing pH. Compound I of CYP119 decomposed back to the ferric form with a first order rate constant of 29.4 ± 3.4 s−1, which increased with increasing pH. These findings form the first kinetic analysis of Compound I formation and decay in the reaction of m-CPBA with ferric P450. horseradish peroxidase chloroperoxidase meta-chloroperoxybenzoic acid density functional theory P450 enzymes are ubiquitous in nature and carry out a wide range of important reactions including the activation of carbon centers for catabolism, steroid metabolism, and detoxification of xenobiotics (1Guengerich F.P. Chem. Res. Toxicol. 2001; 14: 611-650Crossref PubMed Scopus (1393) Google Scholar). Due to the important physiological functions of P450 enzymes, the reaction intermediates of P450 chemistry have been a subject of investigation for many years. Several discreet steps occur in the hydroxylation chemistry of the cytochrome P450s including: substrate binding, first electron transfer to the P450 from a physiological redox partner, oxygen binding (to form oxy-P450), a second electron transfer event (to form a reduced oxy or peroxo state), proton transfer to the distal oxygen (forming hydroperoxo), and an oxygen scission event producing a putative high valent iron-oxo species that subsequently generates hydroxylated product (2Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1404-1415Crossref Scopus (408) Google Scholar). The high valent iron-oxo has long been thought to be analogous to the activated iron species characterized in other oxidative enzymes (horseradish peroxidase (HRP),1 catalase, and chloroperoxidase (CPO)) and referred to as Compound I, a ferryl-oxo-(π) porphyrin cation radical (3Palaniappan V. Terner J. J. Biol. Chem. 1989; 264: 16046-16053Abstract Full Text PDF PubMed Google Scholar, 4Benecky M.J. Frew J.E. Scowen N. Jones P. Hoffman B.M. Biochemistry. 1993; 32: 11929-11933Crossref PubMed Scopus (94) Google Scholar, 5Hosten C.M. Sullivan A.M. Palaniappan V. Fitzgerald M.M. Terner J. J. Biol. Chem. 1994; 269: 13966-13978Abstract Full Text PDF PubMed Google Scholar). The intermediates in the complex P450 reaction cycle have been gradually divulged through the use of a wide range of spectroscopic techniques (6Lipscomb J.D. Biochemistry. 1980; 19: 3590-3599Crossref PubMed Scopus (161) Google Scholar, 7Yu C.A. Gunsalus I.C. Katagiri M. Suhara K. Takemori S. J. Biol. Chem. 1974; 249: 94-101Abstract Full Text PDF PubMed Google Scholar, 8Bangcharoenpaurpong O. Rizos A.K. Champion P.M. Jollie D. Sligar S.G. J. Biol. Chem. 1986; 261: 8089-8092Abstract Full Text PDF PubMed Google Scholar) and the use of methods that allow access to the fast steps occurring after O2 binding (2Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1404-1415Crossref Scopus (408) Google Scholar, 9Denisov I.G. Makris T.M. Sligar S.G. J. Biol. Chem. 2001; 276: 11648-11652Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Recently the peroxo and hydroperoxo intermediates have been observed through the use of cryo-enzymology techniques combined with radiolysis, generating these intermediates through gradual temperature annealing (2Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1404-1415Crossref Scopus (408) Google Scholar, 10Davydov R. Macdonald I.D.G. Makris T.M. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10654-10655Crossref Scopus (138) Google Scholar). Although there has been no identification of a Compound I intermediate by these techniques, it has been inferred by indirect measurements (2Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1404-1415Crossref Scopus (408) Google Scholar, 11Aikens J. Sligar S.G. J. Am. Chem. Soc. 1994; 116: 1143-1144Crossref Scopus (90) Google Scholar, 12Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1222) Google Scholar). The Compound I state as well as the peroxo and hydroperoxo states of the enzyme have been proposed to be active in oxygenation events (13Raner G.M. Hatchell A.J. Morton P.E. Ballou D.P. Coon M.J. J. Inorg. Biochem. 2000; 81: 153-160Crossref PubMed Scopus (18) Google Scholar, 14Vaz A.D.N. Kessell K.J. Coon M.J. Biochemistry. 1994; 33: 13651-13661Crossref PubMed Scopus (44) Google Scholar). To probe the nature of Compound I, meta-chloroperoxybenzoic acid (m-CPBA) has been used as an oxidizing agent to produce this high valent iron-oxo through heterolytic cleavage of the organic peroxide in CPO and HRP. The Compound I species of CPO and HRP have sufficient half-lives for resonance Raman and EPR studies and have been well characterized (3Palaniappan V. Terner J. J. Biol. Chem. 1989; 264: 16046-16053Abstract Full Text PDF PubMed Google Scholar, 5Hosten C.M. Sullivan A.M. Palaniappan V. Fitzgerald M.M. Terner J. J. Biol. Chem. 1994; 269: 13966-13978Abstract Full Text PDF PubMed Google Scholar, 15Egawa T. Miki H. Ogura T. Makino R. Ishimura Y. Kitagawa T. FEBS Lett. 1992; 305: 206-208Crossref PubMed Scopus (31) Google Scholar, 16Egawa T. Proshlyakov D.A. Miki H. Makino R. Ogura T. Kitagawa T. Ishimura Y. J. Biol. Inorg. Chem. 2001; 6: 46-54Crossref PubMed Scopus (74) Google Scholar, 17Kincaid J.R. Zheng Y. Al-Mustafa J. Czarnecki K. J. Biol. Chem. 1996; 271: 28805-28811Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The same peroxy acid and its derivatives have been used with P450s in attempts to generate intermediates. Evidence for the existence of Compound I in P450s was demonstrated through substrate oxygenation or hydroxylation in the reaction of various organic peroxides with either liver microsomal P450 or CYP101, respectively (18Guengerich F.P. Vaz A.D.N. Raner G.N. Pernecky S.J. Coon M.J. Mol. Pharmacol. 1997; 51: 147-151Crossref PubMed Scopus (49) Google Scholar, 19Sligar S.G. Shastry B.S. Gunsalus I.C. Ullrich V. Roots I. Hildebrandt A. Estabrook R.W. Conney A.H. Microsomes and Drug Oxidations. Pergamon Press, Oxford1976: 204-205Google Scholar, 21Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar). Contrary to the evidence for Compound I formation in cytochrome P450 using m-CPBA, Blake and Coon (20Blake R.C.I. Coon M.J. J. Biol. Chem. 1989; 264: 3694-3701Abstract Full Text PDF PubMed Google Scholar) reported that the reactions of several peroxy acids with CYP2B4 generated other active species, which are neither Compound I nor its one-electron-reduced adduct (Compound II). However, in previous studies the generation of the Compound I intermediate in cytochrome P450 (CYP101) required a large excess of peroxyacid, leading to the rapid formation, then conversion of Compound I to other species. This inherent reactivity of CYP101 from Pseudomonas putida has made the identification of Compound I tenuous, and, despite numerous efforts, kinetic analysis could not be completed in the 10-ms time regime of the intermediate's existence (21Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar, 22Wagner G.C. Palcic M.M. Dunford H.B. FEBS Lett. 1983; 156: 244-248Crossref PubMed Scopus (25) Google Scholar). To provide a characterization of the spectral properties of Compound I in a thiolate-liganded heme protein, Harris et al.calculated the spectrum of methylmercaptooxyferryl protoporphyrin IX using density functional theory (DFT) (23Harris D. Loew G. Waskell L. J. Inorg. Biochem. 2001; 83: 309-318Crossref PubMed Scopus (50) Google Scholar). The calculated spectrum had a split Soret blue shifted from the ferric Soret maximum by 60 nm with the Q bands appearing at 690 nm. Recently, Mössbauer and EPR spectroscopic studies of freeze-quenched samples from the reaction of m-CPBA with CYP101 demonstrated the existence of an organic radical and a ferryl species (24Schünemann V. Jung C. Trautwein A.X. Mandon D. Weiss R. FEBS Lett. 2000; 479: 149-154Crossref PubMed Scopus (65) Google Scholar). The techniques employed were not fast enough to capture the Compound I intermediate on the path to the observed ferryl-radical species. Again, the high concentration of m-CPBA used in these studies leads to degradation of protein, which further complicates data analysis. CYP119 is a thermostable P450 isolated from Sulfolobus solfataricus with a melting temperature of 91 °C and remarkable stability to pressure denaturation (25Tschirret-Guth R.A. Koo L.S. Hui Bon Hoa G. Ortiz de Montellano P.R. J. Am. Chem. Soc. 2001; 123: 3412-3417Crossref PubMed Scopus (26) Google Scholar, 26McLean M.A. Maves S.A. Weiss K.E. Krepich S. Sligar S.G. Biochem. Biophys. Res. Commun. 1998; 252: 166-172Crossref PubMed Scopus (97) Google Scholar). Thermostable enzymes are thought to have more rigid active site structures at room temperature than their mesophilic counterparts, which could lead to a slowing of individual steps in the catalytic cycle (27Michels P.C. Clark Douglas S. Appl. Environ. Microbiol. 1997; 63: 3985-3991Crossref PubMed Google Scholar). This alteration in relative reaction rates may enable the resolution of intermediates that occur too rapidly to be characterized in their mesophilic counterparts. CYP119 has proven to be an effective model system for the study of the intermediate states of the reaction cycle of this P450. For instance, we recently employed cryoenzymology to define the hydroperoxo state resulting from the one-electron reduction of the ferrous dioxygen state (28Denisov I.G. Hung S.C. Weiss K.E. McLean M.A. Shiro Y. Park S.Y. Champion P.M. Sligar S.G. J. Inorg. Biochem. 2001; 87: 215-226Crossref PubMed Scopus (45) Google Scholar). In this communication, we report the reaction kinetics of ferric CYP119 with m-CPBA using stopped-flow spectroscopy and document the formation of a spectral intermediate with the characteristic spectrum of Compound I. The relevant time scales of formation and breakdown of Compound I in this thermophilic P450 are such that a kinetic characterization of these processes under various conditions can be obtained for the first time. m-CPBA was purchased from Aldrich-Sigma and purified using the method described by Davies et al. (29Davies D.M. Jones P. Mantle D. Biochem. J. 1976; 157: 247-253Crossref PubMed Scopus (65) Google Scholar). Briefly, m-CPBA was re-crystallized in a 1:3 ether/petroleum ether solution and characterized by NMR. Solutions of m-CPBA for kinetic studies were prepared by addition of an acetone stock. Them-CPBA concentrations were determined by iodide oxidation to triiodide with the extinction coefficient of 25.5 mm−1 cm−1 at 353 nm (30Ramette R.W. Sandford R.W. J. Am. Chem. Soc. 1965; 87: 5001-5005Crossref Scopus (139) Google Scholar). Cytochrome P450 CYP119 expression and purification fromEscherichia coli are as published previously (26McLean M.A. Maves S.A. Weiss K.E. Krepich S. Sligar S.G. Biochem. Biophys. Res. Commun. 1998; 252: 166-172Crossref PubMed Scopus (97) Google Scholar, 31Maves S.A. Sligar S.G. Protein Sci. 2001; 10: 161-168Crossref PubMed Scopus (42) Google Scholar). Protein concentration was determined based on the Soret maximum at 415 nm (ε = 104 mm−1 cm−1). All steady-state UV-visible spectra were recorded on a Hitachi U3300 spectrophotometer. Single wavelength stopped-flow measurements were performed on a KinTek (Austin, TX) model SF-2001 stopped-flow system at 4 °C. For multiple-wavelength absorption studies, a stopped-flow apparatus (model SX-18MV) and the associated computer system fromApplied Photophysics (Surrey, UK) were used. Multiple-wavelength absorption studies were carried out using a photodiode array detector and X-SCAN software (Applied Photophysics, Ltd.). The dead-time of this instrument is about two milliseconds. In a typical experiment one syringe contained CYP119 (6 μm heme) buffered in 100 mm phosphate (at various pH), and the other contained various concentrations of m-CPBA. The reaction temperature was controlled by a water bath. Spectral deconvolution and kinetic analysis were performed by global analysis and numerical integration methods using PRO-K software (Applied Photophysics, Ltd.). Reaction kinetics of m-CPBA with ferric CYP119 were first obtained by following the decay of the ferric signal at 415 nm and a concurrent increase of absorption at 370 nm. Variousm-CPBA/CYP119 concentration ratios were investigated, as shown in Fig. 1. At m-CPBA concentrations less than half that of the ferric protein, the absorption kinetics clearly show the formation of an intermediate by a second order process. The spectral intermediate then decays in a first order manner, with the spectrum returning to that of a ferric enzyme. At 21 μm CYP119 and an m-CPBA concentration of 1.25, 2.5, and 5 μm, the maximal observed absorbance change corresponds to 1, 2.1, and 3.4% of the total enzyme concentration, respectively. At higher m-CPBA concentrations, more complicated kinetic behavior was observed, with a broadband decrease in absorbance indicating protein decomposition (see "Discussion"). Fig. 2 shows intermediate formation by the increase of absorption at 370 nm in the reaction of ferric CYP119 with a substoichiometric concentration of m-CPBA. The data (○) are easily fit by a mechanism involving second order formation and first order decay (solid line, Reaction 1). Moreover, to assess the order of reactions (inset), the initial velocity of the intermediate formation was plotted against m-CPBA concentration, clearly showing the linear increase in velocity with increasing substrate concentration, indicative of a second order reaction. On the other hand, the decay rate constants obtained by the mechanism described above are within the range of 27–33 s−1 and independent of m-CPBA concentration for the reactions of 21 μm ferric CYP119 with 1.25, 2.5, and 5 μm m-CPBA. It is evident that the decomposition of this intermediate goes through a first order process under substoichiometric amount of m-CPBA. Note that at highm-CPBA concentrations there appears to be a departure from first order decomposition process, indicating a combination of protein destruction and side oxidation processes. To better characterize the first intermediate formed in this reaction, multi-wavelength kinetic measurements were carried out at low m-CPBA concentrations. Rapid-scan stopped-flow spectrophotometry, together with singular value decomposition, was used to provide spectral identification of the observed intermediate. In this case, full spectra as a function of time were analyzed using the following model. CYP119 (ferric)+mCPBA →k1 Intermediate I →k2 CYP119 (ferric) REACTION 1The spectra of the ferric protein and Intermediate I as well as the kinetic trace of each species involved were resolved (Fig.3). As indicated, rapid mixing ofm-CPBA with CYP119 produced a spectral intermediate (solid line, Fig. 3 A) with absorbance maxima at 370, 610, and 690 nm. This spectrum is indicative of a Compound I state of the enzyme, as shown by the DFT calculations from Harris et al. (23Harris D. Loew G. Waskell L. J. Inorg. Biochem. 2001; 83: 309-318Crossref PubMed Scopus (50) Google Scholar). The formation of Compound I at a ratio of m-CPBA to protein of 1:2.5 (pH 7.0) had a second order rate (k 1) of 3.20 (± 0.3) × 105m−1s−1, and a first order decomposition rate (k 2) of 29.4 ± 3.4 s−1. Whenm-CPBA concentrations were higher than the concentration of CYP119, protein decay occurred in multiple complicated kinetic processes. To probe the role of proton concentration on Compound I formation, the reaction between CYP119 and m-CPBA was studied as a function of pH by means of multi-wavelength measurements. The rates of formation and decay for compound I in CYP119 at different pH are shown in TableI. Additionally, the rates of formation and decay for Compound I were increased with an increase in reaction temperature. The Arrhenius plot (data not shown) for the Compound I formation reaction (k 1) equates to anE a = 14.1 kcal/mole and the decay rate (k 2) E a = 18.1 kcal/mol.Table IThe observed second order formation (k1 ) and first order decomposition (k2 ) rate constants of CYP119 Compound I at various pH valuespHk 1 (M −1 s −1)k 2(s −1)6.24.28 (±1.5) × 10522.0 ± 3.16.63.69 (±0.4) × 10527.5 ± 5.27.03.20 (±0.3) × 10529.4 ± 3.47.42.49 (±0.3) × 10532.1 ± 3.67.81.86 (±0.3) × 10537.6 ± 3.1 Open table in a new tab The thermostable cytochrome P450 CYP119 has proven to be an excellent system for investigating the nature of intermediates with higher oxidation states of this important class of heme proteins. Previous attempts to form and isolate a Compound I intermediate in CYP101 and other P450s were hampered by the use of a large excess of peroxyacids, leading to rapid progression from Compound I to other intermediates and concomitant protein destruction (21Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar, 24Schünemann V. Jung C. Trautwein A.X. Mandon D. Weiss R. FEBS Lett. 2000; 479: 149-154Crossref PubMed Scopus (65) Google Scholar). The formation of Compound I in CYP119 was slowed both by the use of lower concentrations of m-CPBA and by the temperature at which the experiments were carried out (4 °C), which was well below the physiological temperature for this thermophilic enzyme (80 °C). This allowed the detection of an intermediate clearly defined as Compound I well within the time scale accessible by stopped-flow spectroscopy. Protein degradation, which can often complicate kinetic analysis, was largely avoided. These properties of Compound I formation in CYP119 have made the spectral and kinetic characterization of this previously elusive chemical species in P450s possible. CYP119 is capable of forming an intermediate with the spectral characteristics of a Compound I, ferryl-oxo-(π) porphyrin cation radical, when rapidly mixed with the peroxy acid m-CPBA. The spectral similarities of the CYP119 Compound I to the optical spectra of Compound I in other heme proteins are summarized in TableII. The Soret band of this Compound I was asymmetric and single-peaked at 370 nm (Fig. 3 A), while asymmetric Soret bands of both CYP101 and CPO are at 367 nm (21Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar, 32Hager L.P. Doubek D.L. Silverstein R.M. Hargis J.H. Martin J.C. J. Am. Chem. Soc. 1972; 94: 4364-4366Crossref PubMed Scopus (92) Google Scholar). This asymmetric feature has been deconvoluted using Gaussian-type transition bands and assigned as the splitting Soret band of thed-type hyper-porphyrin in the recent spectroscopic studies of CPO Compound I (16Egawa T. Proshlyakov D.A. Miki H. Makino R. Ogura T. Kitagawa T. Ishimura Y. J. Biol. Inorg. Chem. 2001; 6: 46-54Crossref PubMed Scopus (74) Google Scholar). Moreover, the visible spectrum of CYP119 Compound I has almost identical absorption intensity at 610 and 690 nm, whereas the intensity at 610 nm of CPO Compound I is lower than its absorption at 688 nm. Overall, these findings indicate the similarity between the Compound I entity of these two thiolate-ligated heme proteins and the minor differences in the absorption spectra can be attributed to their electronic structure differences. Similar spectral characteristics of P450 Compound I have also been generated from DFT calculations by Harris et al. (23Harris D. Loew G. Waskell L. J. Inorg. Biochem. 2001; 83: 309-318Crossref PubMed Scopus (50) Google Scholar). The calculated spectra of the methylmercaptooxyferryl protoporphyrin IX model structure are blue-shifted by 50–60 nm in both the ferric resting state and Compound I; however, the other features and the peak shapes are quite similar to the results presented here. The major peak in the split Soret band of the calculated Compound I spectrum is blue-shifted from the ferric Soret by 60 nm, similar to the 45 nm shift in the CYP119 Compound I. Additionally, the Q-bands of the calculated Compound I are shifted to 690 nm, with the equivalent bands in the same location for CYP119 Compound I.Table IIComparison of the absorption maxima (in nm) of Compounds I and II for various heme proteinsCompound ICompound IIRef.CYP119370, 610, 690This workCYP101367, 694(21Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar)Chloroperoxidase367, 688438, 542, 571(32Hager L.P. Doubek D.L. Silverstein R.M. Hargis J.H. Martin J.C. J. Am. Chem. Soc. 1972; 94: 4364-4366Crossref PubMed Scopus (92) Google Scholar)Catalase405, 660429, 536, 568(4Benecky M.J. Frew J.E. Scowen N. Jones P. Hoffman B.M. Biochemistry. 1993; 32: 11929-11933Crossref PubMed Scopus (94) Google Scholar)Horseradish peroxidase400, 577, 622, 651420, 527, 554(29Davies D.M. Jones P. Mantle D. Biochem. J. 1976; 157: 247-253Crossref PubMed Scopus (65) Google Scholar) Open table in a new tab The reaction of CYP101 with m-CPBA was shown by Egawaet al. (21Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar) to form an intermediate with spectral characteristics similar to those of CYP119 Compound I. However, this intermediate was formed in the dead-time of the stopped-flow instrument used and decomposed rapidly into a series of unidentified intermediates. The high concentration of peracid used also caused breakdown of the protein. The experimental conditions necessary to generate the putative Compound I in CYP101 included the use of a 30-fold excess of m-CPBA, increasing the formation rate to the point that the intermediate was fleetingly formed and rapidly decayed. The Compound I, formed under the conditions presented here, in CYP119 has a half-life more than an order of magnitude longer than the apparent CYP101 half-life with high concentrations ofm-CPBA. This longer half-life has allowed the first kinetic characterization of the formation and decomposition processes for Compound I in a P450. To compare the reaction kinetics of CYP119 Compound I with other heme proteins, rate constants for Compound I formation and decay in various systems are summarized in TableIII. While the Compound I states of the other three proteins are stable enough to detect at room temperature, characterization of a P450 Compound I species necessitated the utilization of a thermophilic protein and low temperature (4 °C).Table IIIThe formation (k1 ) and decomposition (k2 ) rate constants of Compounds I of several heme proteinsk 1k 2M−1s−1CYP1193-apH 7.0, 4 °C, m-chloroperoxybenzoic acid, this work.3.20 × 10529.4 s−1CPO3-bpH 4.7, 25 °C, peroxyacetic acid (38).3.8 × 1060.5 s−13-eObtained by first order approximation, the decay of CPO Compound I goes to either the ferric form or CPO Compound II.Catalase3-cpH 7, 25 °C, peroxyacetic acid, ox liver catalase (33).1.44 × 1041.6 × 10−4s−1HRP3-dpH 7, 25 °C, m-chloroperoxybenzoic acid (39).3.5 × 1071.1 × 106 M −1 s−13-fThe HRP Compound I reacts with a second m-CPBA molecule so that the decay is a second order reaction.3-a pH 7.0, 4 °C, m-chloroperoxybenzoic acid, this work.3-b pH 4.7, 25 °C, peroxyacetic acid (38Araiso T. Rutter R. Palcic M.M. Hager L.P. Dunford H.B. Can. J. Biochem. 1981; 59: 233-236Crossref PubMed Scopus (35) Google Scholar).3-c pH 7, 25 °C, peroxyacetic acid, ox liver catalase (33Jones P. Middlemiss D.N. Biochem. J. 1972; 130: 411-415Crossref PubMed Scopus (42) Google Scholar).3-d pH 7, 25 °C, m-chloroperoxybenzoic acid (39Rodriguez-Lopez J.N. Hernández-Ruiz J. Garcia-Cánovas F. Thorneley R.N.F. Acosta N. Arnao M.B. J. Biol. Chem. 1997; 272: 5469-5476Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).3-e Obtained by first order approximation, the decay of CPO Compound I goes to either the ferric form or CPO Compound II.3-f The HRP Compound I reacts with a second m-CPBA molecule so that the decay is a second order reaction. Open table in a new tab The kinetic competence in the conversion of the observed intermediate to an oxygenated product would be a valuable piece of information. Unfortunately, the physiological substrate for this enzyme is not known. However, the addition of laurate to the reaction mixture in a double mixing configuration after the addition of m-CPBA leads to complete quenching of the observed spectral intermediate within the dead-time of the instrument. The reaction of a Compound I intermediate with juxtaposed substrate has been suggested to be very fast, and hence we do not expect to directly observe the kinetic conversion to product. Analysis of the reaction mixture following addition of laurate in this double-mix experiment demonstrated the presence of hydroxylaurate as a product (data not shown), suggesting the coupling of intermediate decay to substrate oxygenation. The pH dependence for Compound I formation reflects the pK ofm-CPBA of 7.4. Previous data have shown that the protonated form of m-CPBA is the active species for Compound I formation, leading to increased rates of Compound I formation at lower pH (20Blake R.C.I. Coon M.J. J. Biol. Chem. 1989; 264: 3694-3701Abstract Full Text PDF PubMed Google Scholar). The pH dependence of the m-CPBA reaction with CYP119 is similar to the behavior of catalase reacting with peroxyacetic acid (33Jones P. Middlemiss D.N. Biochem. J. 1972; 130: 411-415Crossref PubMed Scopus (42) Google Scholar). In both cases the results are most simply interpreted as a reaction between the enzyme and non-ionized peroxy acid molecules. This is substantiated by similar pH dependence for the reaction between peroxybenzoic acid and horseradish peroxidase (29Davies D.M. Jones P. Mantle D. Biochem. J. 1976; 157: 247-253Crossref PubMed Scopus (65) Google Scholar). Additionally, the E a for Compound I formation (14.1 kcal/mol) in CYP119 is relatively high compared with the reaction of hydrogen peroxide with peroxidases (2.5–5 kcal/mol) that utilize hydrogen peroxide as a substrate (34Marklund S. Ohlsson P.I. Opara A. Paul K.G. Biochim. Biophys. Acta. 1974; 350: 304-313Crossref PubMed Scopus (81) Google Scholar). This is in agreement with the recent findings for the reaction of prostaglandin endoperoxide synthase with hydrogen peroxide, which also has an elevatedE a (∼24 kcal/mol) of its Compound I formation and does not use hydrogen peroxide as a native substrate (35Bakovic M. Dunford H.B. Biochem. Cell Biol. 1996; 74: 117-124Crossref PubMed Scopus (2) Google Scholar). The disappearance of CYP119 Compound I is a first order process. This makes it unlikely that the degradation of Compound I involves O2 formation by nucleophilic attack of a secondm-CPBA molecule as observed in chloroperoxidase (32Hager L.P. Doubek D.L. Silverstein R.M. Hargis J.H. Martin J.C. J. Am. Chem. Soc. 1972; 94: 4364-4366Crossref PubMed Scopus (92) Google Scholar). Similarly, the possibility of the formation of Compound II from the reaction of Compound I with a second peroxy acid molecule as in HRP can be ruled out. One possibility is that the oxene oxygen is transferred to the protein. Alternatively, it has been suggested that the slow regeneration of the ferric state in catalase, from Compound I, is carried out by a reducing equivalent originating from the protein matrix (33Jones P. Middlemiss D.N. Biochem. J. 1972; 130: 411-415Crossref PubMed Scopus (42) Google Scholar, 36Pfister T.D. Gengenbach A.J. Syn S. Lu Y. Biochemistry. 2001; 40: 14942-14951Crossref PubMed Scopus (43) Google Scholar). The CYP119 Compound I decays to the low-spin ferric form at a rate about 105 times faster than that of catalase Compound I. These active species can be reduced by any adventitious reducing equivalent from the protein matrix (4Benecky M.J. Frew J.E. Scowen N. Jones P. Hoffman B.M. Biochemistry. 1993; 32: 11929-11933Crossref PubMed Scopus (94) Google Scholar). The pH dependence of the Compound I decomposition rate is similar to the decay of ferryl intermediate in cytochrome c oxidase, which was explained by an auto-reduction pathway (37Fabian M. Palmer G. Biochemistry. 2001; 40: 1867-1874Crossref PubMed Scopus (31) Google Scholar). Taken together, the mechanism of degradation of CYP119 Compound I and the regeneration of the ferric enzyme can only be determined with more experimental analysis. The kinetic characterization of Compound I formation in CYP119 has allowed for the optimization of conditions for the maximal production of this putative reaction intermediate in time regimes accessible to other spectroscopic methods. The information thus obtained will aid in the further determination of the nature of Compound I in P450 reactions. We thank Prof. Robert Gennis and Dr. Joel Morgan for the use of their Applied Photophysics rapid scanning stopped-flow spectrophotometer.
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