Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Formation and Decay of Hydroperoxo-Ferric Heme Complex in Horseradish Peroxidase Studied by Cryoradiolysis

2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês

10.1074/jbc.m207949200

ISSN

1083-351X

Autores

Ilia G. Denisov, Thomas M. Makris, Stephen G. Sligar,

Tópico(s)

Metal complexes synthesis and properties

Resumo

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo π-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150–180 K. After further annealing at 195–205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide. Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo π-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150–180 K. After further annealing at 195–205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide. horseradish peroxidase ferric protoporphyrin IX complex with hydroperoxo anion electron paramagnetic resonance Horseradish peroxidase (HRP)1 is a highly characterized heme enzyme that has historically served as a paradigm for evaluating the rules of formation of redox active heme-oxygen intermediates, including the ferryl-oxo heme complexes commonly known as "Compound I" and "Compound II." A detailed mechanism for Compound I formation in the reaction of H2O2with peroxidases was formulated by Poulos and Kraut on the basis of structural analysis of cytochrome c peroxidase active center (1Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 8199-8205Abstract Full Text PDF PubMed Google Scholar) and has been successfully applied to other enzymes (2English A.M. Tsaprailis G. Adv. Inorg. Chem. 1995; 43: 79-125Crossref Scopus (114) Google Scholar, 3Dunford H.B. Heme Peroxidases. Wiley, New York.1999Google Scholar, 4Nicholls P. Fita I. Loewen P.C. Adv. Inorg. Chem. 2001; 51: 51-106Crossref Google Scholar, 5Veitch N.C. Smith A.T. Adv. Inorg. Chem. 2001; 51: 107-162Crossref Google Scholar), as shown in Reaction FR1. The essential features of this mechanism also provide a framework for the analysis of new systems, which involve formation of redox-active heme-oxygen intermediates (6Watanabe Y. Groves J.T. Sigman D.S. 3rd. Ed. The Enzymes. 20. Academic Press, San Diego1992: 405-452Google Scholar, 7Dawson J.H. Chem. Rev. 1996; 96: 2841-2888Crossref PubMed Scopus (2161) Google Scholar, 8Groves J.T. Wang C.C.-Y. Curr. Opin. Chem. Biol. 2000; 4: 687-695Crossref PubMed Scopus (184) Google Scholar). A crucial point in the formation of the ferryl-oxo species is the second protonation of the distal oxygen atom of the bound hydroperoxo species, followed by the heterolytic scission of O–O bond. The importance of these elementary steps has been confirmed by quantum chemical methods (9Loew G.H. Dupuis M. J. Am. Chem. Soc. 1996; 118: 10584-10587Crossref Scopus (51) Google Scholar, 10Woon D.E. Loew G.H. J. Phys. Chem. A. 1998; 102: 10380-10384Crossref Scopus (19) Google Scholar, 11Wirstam M. Blomberg M.R.A. Siegbahn P.E.M. J. Am. Chem. Soc. 1999; 121: 10178-10185Crossref Scopus (128) Google Scholar). The vast amount of kinetic data obtained in mutagenesis studies of the distal pocket (5Veitch N.C. Smith A.T. Adv. Inorg. Chem. 2001; 51: 107-162Crossref Google Scholar, 12Rodrigues-Lopez J.N. Smith A.T. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 4023-4030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 13Rodrigues-Lopez J.N. Smith A.T. Thorneley R.N.F. J. Biol. Chem. 1997; 272: 389-395Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Tanaka M. Ishimori K. Mukai M. Kitagawa T. Morishima I. Biochemistry. 1997; 36: 9889-9898Crossref PubMed Scopus (76) Google Scholar, 15Tanaka M. Ishimori K. Morishima I. Biochemistry. 1998; 37: 2629-2638Crossref PubMed Scopus (25) Google Scholar) provide an additional support for the evaluation of the second protonation of the distal oxygen as the most important and, in some cases, the rate-limiting step in catalysis. Despite many efforts, experimental characterization of this step, as well as the detailed properties of the hydroperoxo-ferric heme (Fe3+P-OOH−) complex in peroxidases, is still far from complete. One reason is the low stability of such complexes (16Tajima K. Inorg. Chim. Acta. 1989; 163: 115-122Crossref Scopus (34) Google Scholar), because their transient character leads to a lack of accumulation during the reaction course. Several studies of these intermediates in wild-type and mutant HRP (17Baek H.K. van Wart H.E. Biochemistry. 1989; 28: 5714-5719Crossref PubMed Scopus (137) Google Scholar, 18Baek H.K. van Wart H.E. J. Am. Chem. Soc. 1992; 114: 718-725Crossref Scopus (111) Google Scholar, 19Rodrigues-Lopez J.N. Lowe D.J. Hernandez-Ruiz J. Hiner A.N.P. Garcia-Canovas F. Thorneley R.N.F. J. Am. Chem. Soc. 2001; 123: 11838-11847Crossref PubMed Scopus (286) Google Scholar) and microperoxidase (20Primus J.-L. Grunenwald S. Hagedoorn P.-L. Albrecht-Gay A.-M. Mandon D. Veeger C. J. Am. Chem. Soc. 2002; 124: 1214-1221Crossref PubMed Scopus (49) Google Scholar, 21Veeger C. J. Inorg. Biochem. 2002; 91: 35-45Crossref PubMed Scopus (42) Google Scholar, 22Yeh H.-C. Wang J.-S., Su, Y.O. Lin W.-Y. J. Biol. Inorg. Chem. 2001; 6: 770-777Crossref PubMed Scopus (11) Google Scholar) by means of stopped-flow methods have produced absorption spectra that could be tentatively assigned to Fe3+P-OOH−(hydroperoxo anion) or Fe3+P-H2O2(neutral hydrogen peroxide) complex via comparison with theoretical estimates (23Harris D.L. Loew G.H. J. Am. Chem. Soc. 1996; 118: 10588-10594Crossref Scopus (65) Google Scholar). Even so, we are aware of only one successful isolation of an (Fe3+P-OOH−) intermediate in an H2O2-driven heme protein system, which was obtained using a distal pocket mutant of Mb (24Brittain T. Baker A.R. Butler C.S. Little R.H. Lowe D.J. Greenwood C. Watmough N.J. Biochem. J. 1997; 326: 109-115Crossref PubMed Scopus (55) Google Scholar). The difficulties in the kinetic resolution of the (Fe3+P-OOH−) intermediate on this pathway may be caused by the fact that both protons necessary for the Compound I formation are brought into the active site of the enzyme with the H2O2molecule itself. Apparently, the subsequent 1-2 proton shift is fast and not rate-limiting under most conditions (5Veitch N.C. Smith A.T. Adv. Inorg. Chem. 2001; 51: 107-162Crossref Google Scholar, 12Rodrigues-Lopez J.N. Smith A.T. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 4023-4030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 13Rodrigues-Lopez J.N. Smith A.T. Thorneley R.N.F. J. Biol. Chem. 1997; 272: 389-395Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Tanaka M. Ishimori K. Mukai M. Kitagawa T. Morishima I. Biochemistry. 1997; 36: 9889-9898Crossref PubMed Scopus (76) Google Scholar, 15Tanaka M. Ishimori K. Morishima I. Biochemistry. 1998; 37: 2629-2638Crossref PubMed Scopus (25) Google Scholar, 19Rodrigues-Lopez J.N. Lowe D.J. Hernandez-Ruiz J. Hiner A.N.P. Garcia-Canovas F. Thorneley R.N.F. J. Am. Chem. Soc. 2001; 123: 11838-11847Crossref PubMed Scopus (286) Google Scholar). An alternative way of generating (Fe3+P-OOH−) intermediates with high yield is the cryoradiolytic reduction of the corresponding oxy-ferrous complexes using ionizing radiation (25Symons M.C.R. Petersen R.L. Proc. R. Soc. Lond. B Biol. Sci. 1978; 201: 285-300Crossref PubMed Scopus (82) Google Scholar, 26Symons M.C.R. Petersen R.L. Biochim. Biophys. Acta. 1978; 535: 241-247Crossref PubMed Scopus (46) Google Scholar). This method mimics the oxidase/oxygenase pathway of oxygen activation, which uses molecular oxygen instead of H2O2, and includes the reduction of the bound oxygen and delivery of two external protons to form Compound I (6Watanabe Y. Groves J.T. Sigman D.S. 3rd. Ed. The Enzymes. 20. Academic Press, San Diego1992: 405-452Google Scholar, 27Groves J.T. Han Y. Ortiz de Montellano P.R. Cytochrome P450. Structure, Mechanism, and Biochemistry. Plenum Publishing Corp., New York1995: 3-48Crossref Google Scholar). Because of the one-electron reduction of oxy-ferrous precursors at 77 K, peroxo/hydroperoxo-ferric heme enzyme complexes can be stabilized at low temperatures (26Symons M.C.R. Petersen R.L. Biochim. Biophys. Acta. 1978; 535: 241-247Crossref PubMed Scopus (46) Google Scholar, 28Davydov R.M. Biofizika. 1980; 25: 203-207PubMed Google Scholar, 29Kappl R. Hohn-Berlage M. Huttermann J. Bartlett N. Symons M.C.R. Biochim. Biophys. Acta. 1985; 827: 327-343Crossref Scopus (73) Google Scholar, 30Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar). The (Fe3+P-OOH−) intermediates of cytochromes P450 (31Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (68) Google Scholar, 32Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (411) Google Scholar, 33Denisov 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, 34Denisov 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), heme oxygenase (35Davydov R. Kofman V. Fujii H. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 2002; 124: 1798-1808Crossref PubMed Scopus (149) Google Scholar), Mb (26Symons M.C.R. Petersen R.L. Biochim. Biophys. Acta. 1978; 535: 241-247Crossref PubMed Scopus (46) Google Scholar, 29Kappl R. Hohn-Berlage M. Huttermann J. Bartlett N. Symons M.C.R. Biochim. Biophys. Acta. 1985; 827: 327-343Crossref Scopus (73) Google Scholar, 30Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar), and Hb (25Symons M.C.R. Petersen R.L. Proc. R. Soc. Lond. B Biol. Sci. 1978; 201: 285-300Crossref PubMed Scopus (82) Google Scholar, 28Davydov R.M. Biofizika. 1980; 25: 203-207PubMed Google Scholar) have been isolated and characterized using EPR and optical absorption spectroscopy. Several x-ray structures of intermediates produced by radiolytic reduction of the crystals of corresponding oxy-ferrous precursors, including Compound III in HRP (36Berglund G.I. Carlsson G.H. Smith A.T. Szoke H. Henriksen A. Hajdu J. Nature. 2002; 417: 463-468Crossref PubMed Scopus (765) Google Scholar), are also available (36Berglund G.I. Carlsson G.H. Smith A.T. Szoke H. Henriksen A. Hajdu J. Nature. 2002; 417: 463-468Crossref PubMed Scopus (765) Google Scholar, 37Schlichting 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 (1231) Google Scholar, 38Sjogren T. Hajdu J. J. Biol. Chem. 2001; 276: 13072-13076Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), although none of these studies was able to detect the (Fe3+P-OOH−) complex. Earlier investigations using cryoradiolysis (30Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar), or pulse radiolysis at ambient conditions (39Kobayashi K. Hayashi K. J. Biol. Chem. 1981; 256: 12350-12354Abstract Full Text PDF PubMed Google Scholar) for reduction of oxy-ferrous HRP, have not provided unambiguous assignment of observed intermediate states. Detailed studies of the reductive pathway of oxygen activation in HRP, where the associated Compound I state is relatively stable, could provide critical input into the mechanism of oxygen-oxygen bond scission and the requirements for specific distal pocket residues in generating higher valent metal-oxo complexes thought to be operating in the heme-thiolate oxygenases such as cytochrome P450. To investigate this path, we used the cryogenic radiolytic reduction of oxy-ferrous HRP to prepare the peroxo/hydroperoxo ferric intermediate and monitor its protonation and possible Compound I formation using EPR and optical absorption spectroscopy. As a reference point, we also document the cryoradiolytic reduction of Compound I, prepared using the classic reaction of the ferric enzyme with hydrogen peroxide. Ferrous HRP was prepared by anaerobic reduction of the ferric HRP (Type XII; Sigma) with dithionite and subsequent anaerobic chromatography on a Sephadex G-25 column (Pharmacia) to remove the excess dithionite and decomposition products. The enzyme solution was mixed with buffered deoxygenated glycerol (gas chromatography grade; Sigma) to give a final concentration of 25–50 μmHRP in 70% (v/v) glycerol and 0.1 m phosphate-citrate buffer, pH 6.2, oxygenated at 270 K by bubbling air and stirring for 30 s, and cooled to 77 K, as described previously (33Denisov 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, 34Denisov 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). All samples for optical spectroscopy were prepared in UV-enhanced methacrylate semimicro spectroscopic cells (Fisher) and used with 4.3-mm path. The ferric HRP samples used for the control were prepared in the same fashion, cooled to 77 K and irradiated together with other samples. Compounds I and II were prepared according to published procedures (3, and references therein). Compound I was obtained by manual mixing 70% glycerol/buffer solutions of the ferric HRP and 1.2 molar excess of H2O2 at low temperature (260–270 K). After mixing for 20–30 s, the sample was placed into a thermostat and quickly cooled to 200 K. The sample was then slowly cooled to 77 K using UV-visible spectroscopy to monitor the purity of the preparation. Compound II was prepared by mixing of 75% glycerol/buffer solutions of the ferric HRP, containing 4-fold molar excess of the substrate, 3,5-dimethylphenol, with a 4-fold molar excess of H2O2 in the similar conditions. The spectra of Compounds I and II showed weak temperature dependence and were similar to those reported in the literature (40Blumberg W.E. Peisach J. Wittenberg B.A. Wittenberg J.B. J. Biol. Chem. 1968; 243: 1854-1862Abstract Full Text PDF PubMed Google Scholar, 41Gasyna Z. Browett W.R. Stillman M.J. Biochemistry. 1988; 27: 2502-2509Crossref Scopus (8) Google Scholar). UV-visible spectra were measured on a Cary 3 spectrophotometer (Varian) in an unsilvered Dewar (33Denisov 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) using liquid nitrogen as a cooling agent. Electron spin resonance spectra were obtained at the University of Illinois EPR Resource Center on a Varian E-122 X-Band (9.08 GHz) spectrometer with a protein concentration of 0.4 mm. Typical conditions include a microwave power of 0.5 milliwatts with modulation amplitude of 12.5 G at 100 kHz. A liquid helium flow system (Air Products, Allentown, PA) was used for measurements at 4–20 K. Samples for EPR were prepared in a similar manner as those for UV-visible experiments, with a final glycerol concentration of 20% (v/v). The radiolytic reduction of the enzyme and reference samples was achieved by exposing to γ-radiation from a 60Co source (dose rate, 11 kilograys/h for 4 or 8 h) (Notre Dame Radiation Laboratory, Notre Dame University, South Bend, IN). The samples were fully immersed in liquid nitrogen during irradiation. Background absorption from the radiolysis products was subtracted as described previously (33Denisov 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) using irradiated buffer/glycerol reference samples. These samples were irradiated at 77 K together with the protein samples, and 40 absorption spectra were taken at different temperatures from 80 to 240 K. The background spectra for any arbitrary temperature in this range were then calculated using linear interpolation. The spectra of oxy-HRP before and after irradiation at 77 K are shown in Fig. 1. The radiolytic reduction of the oxy-ferrous heme complex in HRP results in formation of a primary intermediate, peroxo/hydroperoxo-ferric intermediate, as has been observed with other heme proteins (26Symons M.C.R. Petersen R.L. Biochim. Biophys. Acta. 1978; 535: 241-247Crossref PubMed Scopus (46) Google Scholar, 29Kappl R. Hohn-Berlage M. Huttermann J. Bartlett N. Symons M.C.R. Biochim. Biophys. Acta. 1985; 827: 327-343Crossref Scopus (73) Google Scholar, 30Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar, 31Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (68) Google Scholar, 32Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (411) Google Scholar, 33Denisov 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, 34Denisov 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, 35Davydov R. Kofman V. Fujii H. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 2002; 124: 1798-1808Crossref PubMed Scopus (149) Google Scholar, 42Davydov R.M. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10656-10657Crossref Scopus (147) Google Scholar, 43Davydov 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). The yield of this intermediate (35–50%) was estimated from the dose-dependent absorption spectra, using the spectra of oxy-HRP as the reference. The spectrum of the pure (Fe3+P-OOH−) HRP complex, calculated from multiple experiments with different yields, is also shown in Fig. 1. The absorption spectrum of this intermediate has a red-shifted Soret band (maximum at 419 nm, compared with 416 nm for oxy-ferrous HRP) and blue-shifted Q-bands (556 and 526 nm). This spectrum is in very good agreement with the spectrum of the (Fe3+P-OOH−) complex of heme oxygenase, 2I. G. Denisov, M. Ikeda-Saito, T. Yoshida, and S. G. Sligar, submitted for publication. and with the kinetically resolved spectrum of the same intermediate obtained in stopped-flow studies of the reaction of Mb mutant H64Q with H2O2 (24Brittain T. Baker A.R. Butler C.S. Little R.H. Lowe D.J. Greenwood C. Watmough N.J. Biochem. J. 1997; 326: 109-115Crossref PubMed Scopus (55) Google Scholar). This intermediate of the reaction of H2O2 with heme enzymes is very unstable at ambient conditions and previously was characterized only via decomposition of transient absorption spectra in stopped-flow studies or via freeze-quench techniques (12Rodrigues-Lopez J.N. Smith A.T. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 4023-4030Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 13Rodrigues-Lopez J.N. Smith A.T. Thorneley R.N.F. J. Biol. Chem. 1997; 272: 389-395Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Tanaka M. Ishimori K. Mukai M. Kitagawa T. Morishima I. Biochemistry. 1997; 36: 9889-9898Crossref PubMed Scopus (76) Google Scholar, 15Tanaka M. Ishimori K. Morishima I. Biochemistry. 1998; 37: 2629-2638Crossref PubMed Scopus (25) Google Scholar, 16Tajima K. Inorg. Chim. Acta. 1989; 163: 115-122Crossref Scopus (34) Google Scholar, 17Baek H.K. van Wart H.E. Biochemistry. 1989; 28: 5714-5719Crossref PubMed Scopus (137) Google Scholar, 18Baek H.K. van Wart H.E. J. Am. Chem. Soc. 1992; 114: 718-725Crossref Scopus (111) Google Scholar, 19Rodrigues-Lopez J.N. Lowe D.J. Hernandez-Ruiz J. Hiner A.N.P. Garcia-Canovas F. Thorneley R.N.F. J. Am. Chem. Soc. 2001; 123: 11838-11847Crossref PubMed Scopus (286) Google Scholar, 20Primus J.-L. Grunenwald S. Hagedoorn P.-L. Albrecht-Gay A.-M. Mandon D. Veeger C. J. Am. Chem. Soc. 2002; 124: 1214-1221Crossref PubMed Scopus (49) Google Scholar, 21Veeger C. J. Inorg. Biochem. 2002; 91: 35-45Crossref PubMed Scopus (42) Google Scholar, 24Brittain T. Baker A.R. Butler C.S. Little R.H. Lowe D.J. Greenwood C. Watmough N.J. Biochem. J. 1997; 326: 109-115Crossref PubMed Scopus (55) Google Scholar). The same (Fe3+P-OOH−) complex is firmly identified as an intermediate of the reductive activation of the bound dioxygen in heme proteins, as demonstrated by cryoradiolytic reduction studies (26Symons M.C.R. Petersen R.L. Biochim. Biophys. Acta. 1978; 535: 241-247Crossref PubMed Scopus (46) Google Scholar,29Kappl R. Hohn-Berlage M. Huttermann J. Bartlett N. Symons M.C.R. Biochim. Biophys. Acta. 1985; 827: 327-343Crossref Scopus (73) Google Scholar, 30Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar, 32Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (411) Google Scholar, 33Denisov 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, 34Denisov 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, 35Davydov R. Kofman V. Fujii H. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 2002; 124: 1798-1808Crossref PubMed Scopus (149) Google Scholar) and quantum chemical calculations (10Woon D.E. Loew G.H. J. Phys. Chem. A. 1998; 102: 10380-10384Crossref Scopus (19) Google Scholar, 11Wirstam M. Blomberg M.R.A. Siegbahn P.E.M. J. Am. Chem. Soc. 1999; 121: 10178-10185Crossref Scopus (128) Google Scholar, 23Harris D.L. Loew G.H. J. Am. Chem. Soc. 1996; 118: 10588-10594Crossref Scopus (65) Google Scholar). Unfortunately, it was impossible to resolve the spectra of (Fe3+P-OOH−) complex at wavelengths shorter than 360 nm because of the high absorption of the products of radiolysis of glycerol and the protein matrix. Thus, we cannot confirm the true hyperporphyrin shape of the Soret band of (Fe3+P-OOH−) complex, as calculated by Harris and Loew (23Harris D.L. Loew G.H. J. Am. Chem. Soc. 1996; 118: 10588-10594Crossref Scopus (65) Google Scholar) and often used as a criterion to distinguish between the neutral peroxide-ferric (Fe3+P-HOOH) and hydroperoxo-anion-ferric (Fe3+P-OOH−) complexes based on their absorption spectra in the Soret region (19Rodrigues-Lopez J.N. Lowe D.J. Hernandez-Ruiz J. Hiner A.N.P. Garcia-Canovas F. Thorneley R.N.F. J. Am. Chem. Soc. 2001; 123: 11838-11847Crossref PubMed Scopus (286) Google Scholar, 44Hiner A.N.P. Raven E.L. Thorneley R.N.F. Garcia-Canovas F. Rodrigues-Lopez J.N. J. Inorg. Biochem. 2002; 91: 27-34Crossref PubMed Scopus (132) Google Scholar). Fig. 2 shows the annealing of the (Fe3+P-OOH−) complex monitored by EPR and UV-visible spectra. In the EPR spectra, the presence of two species in the primary intermediate is clearly resolved. Based on the analogous studies of other heme proteins (25Symons M.C.R. Petersen R.L. Proc. R. Soc. Lond. B Biol. Sci. 1978; 201: 285-300Crossref PubMed Scopus (82) Google Scholar, 29Kappl R. Hohn-Berlage M. Huttermann J. Bartlett N. Symons M.C.R. Biochim. Biophys. Acta. 1985; 827: 327-343Crossref Scopus (73) Google Scholar, 42Davydov R.M. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10656-10657Crossref Scopus (147) Google Scholar, 43Davydov 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), these are assigned as the unprotonated peroxo-ferric heme complex (Fe3+P-OO2−, g3 = 2.27) and the protonated, hydroperoxo-ferric heme complex (Fe3+P-OOH−, g3 = 2.32). Gradual annealing at 140–160 K results in disappearance of the feature at g3 = 2.27 and increase at g3 = 2.32 as the first protonation of peroxo-ferric complex and formation of hydroperoxo intermediate is completed. These results are in agreement with EPR spectra obtained using cryoradiolytic reduction studies of oxy-HRP in 50% ethylene glycol (30Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar). The analysis of absorption spectra of the similar samples measured at 90–160 K does not reveal any significant changes that can be assigned to the protonation of peroxo-ferric intermediate. Thus, at least under the conditions of our experiments, the optical spectra in this region are not sensitive enough to distinguish clearly between the unprotonated and protonated peroxo/hydroperoxo-ferric heme complex of HRP. Recently we obtained EPR and UV-visible spectra of peroxo/hydroperoxo intermediates of the distal mutants of cytochrome P450 CYP101, in which the first protonation of a peroxo-ferric heme complex could be detected in optical absorption spectra as a 3-nm blue shift of Soret and a similar blue shift of the Qα band from 561 to 558 nm. 3T. M. Makris, I. G. Denisov, S.-C. Hung, V. Kulik, I. Schlichting, K. Chu, R. M. Sweet, and S. G. Sligar, manuscript in preparation. However, the relative spectral differences between oxy-ferrous and peroxo/hydroperoxo-ferric heme complexes in the Soret region are weaker in HRP and in heme oxygenase than in P450 enzymes (33Denisov 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, 34Denisov 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). This difference, which is caused by the different proximal ligands in these enzymes, imidazole/imidazolate in HRP and heme oxygenaseversus thiolate in P450, also results in different sensitivity of the optical absorption spectra to the protonation of the distal peroxo- ligand. Further annealing by increasing the sample temperature from 180 to 210 K shows the decay of (Fe3+P-OOH−) complex with no evidence for the formation of a Compound I intermediate. EPR spectra of the samples annealed at 195, 200, and 210 K, show gradual decrease of the signal from (Fe3+P-OOH−) complex and formation of the ferric HRP with no other observable intermediates. This result is consistent with two scenarios. One would involve the transient formation of Compound I, which is quickly reduced to generate the EPR-silent Compound II and after the second reduction returns to the ferric enzyme. Another possibility is the direct decay of (Fe3+P-OOH−) intermediate through dissociation of the ligand and return to the ferric resting state of HRP. Because EPR-silent species may be involved in the evolution of this system, EPR spectroscopy does not provide complete characterization of the (Fe3+P-OOH−) fate in HRP. Thus, the optical absorption spectra of the similar samples were studied, and the reference experiments using cryoradiolytic reduction of Compound I were performed to assign the annealing pathway of (Fe3+P-OOH−) complex. Fig. 2 B shows the absorption spectra of the radiolytically reduced oxy-ferrous HRP measured at 190–210 K. At temperatures above the glass transition of the solvent matrix, the hydroperoxo-ferric complex gradually disappears, as clearly seen by simultaneous decrease of the sharp maximum at 557 nm and of the Soret band at 420 nm. No increase of absorption at 650 nm characteristic for Compound I porphyrin π-cation radical was detected in annealing experiments. On the contrary, the bands at 407 and 635 nm are observed, corresponding to the spectrum of the resting form of HRP, possibly perturbed by the presence of the dissociated hydroperoxo-anion/hydrogen peroxide at the distal pocket of the enzyme. At higher temperatures, this spectrum, as well as the spectrum of the remaining oxy-HRP, disappears, and the characteristic spectrum of the carbonmonoxy-ferrous complex of HRP emerges with Soret band at 421 nm and Q bands at 540 and 572 nm (not shown). The CO adduct is the result of the nonspecific reduction of the heme enzyme by the products of radiolysis and binding of CO, which is one of the products of glycerol radiolysis (45Woods R.J. Pikaev A.K. Applied Radiation Chemistry: Radiation Processing. Wiley Interscience, New York.1994Google Scholar) and is always observed after thawing of irradiated frozen solutions of the heme enzymes (33Denisov 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,34Denisov 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, 46Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu, A. Mol. Biol. (Mosc.). 1978; 12: 947-957PubMed Google Scholar, 47Magonov S.N. Davydov R.M. Blyumenfeld L.A. Arutyunyan A.M. Sharonov Yu, A. Mol. Biol. (Mosc.). 1978; 12: 1191-1197PubMed Google Scholar, 48Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu, A. Mol. Biol. (Mosc.). 1978; 12: 1182-1190PubMed Google Scholar). In a control experiment, the dissolution of nonirradiated metMb into a glycerol/buffer reference sample, which was irradiated at 77 K and thawed, resulted in formation of fully reduced Mb-CO. To probe the reactivity of Compound I under radiolytic conditions we separately generated this intermediate using H2O2, as described under "Materials and Methods." Fig. 3 shows the spectra of Compound I before and after radiolytic reduction. The spectrum of Compound I obtained at low temperature is in a good agreement with earlier reports (40Blumberg W.E. Peisach J. Wittenberg B.A. Wittenberg J.B. J. Biol. Chem. 1968; 243: 1854-1862Abstract Full Text PDF PubMed Google Scholar, 41Gasyna Z. Browett W.R. Stillman M.J. Biochemistry. 1988; 27: 2502-2509Crossref Scopus (8) Google Scholar). Radiolytic reduction of Compound I at 77 K results in formation of a new species with the absorption spectra similar to those reported for Compound II. Direct formation of Compound II confirms the chemical competence of cryoradiolysis on HRP intermediates and the true one-electron reduction caused by cryogenic radiolytic treatment of heme enzyme intermediates. At temperatures below 190 K, the shape of the spectrum of Compound II does not change significantly, which implies that it is relatively stable and does not undergo further reduction by products of radiolysis until annealed above the glass transition temperature. At 198 K, this intermediate begins to disappear gradually, with concomitant formation of ferric HRP (Fig. 3, spectra at 198–205 K). The latter process is consistent with one-electron reduction of Compound II, as occurs in the peroxidase enzymatic cycle under ambient conditions. Results shown in Fig. 3 suggest that the annealing of (Fe3+P-OOH−) complex in HRP (Fig.2 B) to form ferric HRP does not follow a path through Compound I and Compound II. Comparison of Fig. 2 B and Fig. 3reveals substantial differences in observed spectra and annealing behavior of reduced oxy-HRP and Compound II. Although the spectrum of Compound II has almost the same peak positions (418, 526, and 558 nm, Fig. 3) as the (Fe3+P-OOH−) intermediate (419, 526, and 556 nm), the spectra of these two species are not identical. The Qα band at 556 nm has substantially higher amplitude than the Qβ band at 526 nm in the spectrum of (Fe3+P-OOH−) complex, whereas in the spectrum of Compound II, these two bands are of almost the same intensity. Thus, the appearance of Compound II spectra in the optical annealing studies of (Fe3+P-OOH−) species would have been detected if the pathway through Compounds I and II was the operative one. Instead, in all such experiments, the spectrum of ferric HRP emerged at the expense of (Fe3+P-OOH−) spectrum, and the direct decay of hydroperoxo-ferric complex, or the so-called peroxide shunt, was observed. The lack of Compound I formation from its immediate precursor, (Fe3+P-OOH−) complex, may be considered as an unexpected result, because HRP is known for its facile formation of the stable Compound I species. However, there are important mechanistic differences between the enzymatic cycle of this enzyme, which uses H2O2 as the oxygen donor, and the oxidase/oxygenase path explored in the current studies. The (Fe3+P-OOH−) complex is formed as the precursor of Compound I in both systems, and the second protonation of the β oxygen atom in this complex is the decisive step to form Compound I. The specific delivery of this second proton is crucial for Compound I formation from (Fe3+P-OOH−) complex (10Woon D.E. Loew G.H. J. Phys. Chem. A. 1998; 102: 10380-10384Crossref Scopus (19) Google Scholar, 11Wirstam M. Blomberg M.R.A. Siegbahn P.E.M. J. Am. Chem. Soc. 1999; 121: 10178-10185Crossref Scopus (128) Google Scholar, 23Harris D.L. Loew G.H. J. Am. Chem. Soc. 1996; 118: 10588-10594Crossref Scopus (65) Google Scholar, 52Filizola M. Loew G.H. J. Am. Chem. Soc. 2000; 122: 18-25Crossref Scopus (68) Google Scholar), because the latter is very unstable and quickly disappears even at low temperatures (16Tajima K. Inorg. Chim. Acta. 1989; 163: 115-122Crossref Scopus (34) Google Scholar, 17Baek H.K. van Wart H.E. Biochemistry. 1989; 28: 5714-5719Crossref PubMed Scopus (137) Google Scholar, 18Baek H.K. van Wart H.E. J. Am. Chem. Soc. 1992; 114: 718-725Crossref Scopus (111) Google Scholar). The perturbation of this proton delivery system caused by mutations at the active site was shown to result in nonproductive reduction of oxygen and formation of hydrogen peroxide, or "uncoupling" in the cytochrome P450 catalytic cycle (49Imai M. Shimada H. Watanabe Y. Matsushima-Hibiya Y. Makino R. Koga H. Horiuchi T. Ishimura Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7823-7827Crossref PubMed Scopus (358) Google Scholar, 50Martinis S.A. Atkins W.M. Stayton P.S. Sligar S.G. J. Am. Chem. Soc. 1989; 111: 9252-9253Crossref Scopus (256) Google Scholar). In HRP, the (Fe3+P-OOH−) complex is formed from H2O2 via deprotonation of the neutral hydrogen peroxide at the distal pocket of the enzyme, and the same proton can be used for the second protonation of the β oxygen via the rearrangement catalyzed by the distal His42 and Arg38 side chains (1Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 8199-8205Abstract Full Text PDF PubMed Google Scholar, 51Dunford H.B. Everse J. Everse K.E. Grisham M.B. Peroxidases in Chemistry and Biology. 2. CRC Press, Boca Raton, FL1991: 2-24Google Scholar, 52Filizola M. Loew G.H. J. Am. Chem. Soc. 2000; 122: 18-25Crossref Scopus (68) Google Scholar). If dioxygen is used instead of H2O2, both protonation steps have to be provided by the enzyme, as occurs in the P450 monooxygenases (32Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (411) Google Scholar, 53Vidakovic M. Sligar S.G., Li, H. Poulos T.L. Biochemistry. 1998; 37: 9211-9219Crossref PubMed Scopus (229) Google Scholar,54Mueller E.J. Loida P.J. Sligar S.G. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Publishing Corp., New York1995: 83-124Crossref Google Scholar). Because this path is non-physiological for HRP, the enzyme fails to deliver the second proton required for Compound I formation, and an uncoupling is observed. In conclusion, we prepared the main redox intermediates of the HRP enzymatic cycle and performed the radiolytic reduction of these species. The (Fe3+P-OOH−) intermediate, the precursor of Compound I formation, was isolated via radiolytic reduction of oxy-HRP. Absorption and EPR spectra of this intermediate are reported with much higher resolution and precision than in previous studies. The first protonation of the peroxo-ferric heme complex, the primary product of the reduction of oxy-ferrous HRP, is completed only at the temperatures higher than 150 K, as was observed in D251N mutant of P450 CYP101 (32Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (411) Google Scholar, 43Davydov 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), where the proton delivery mechanism is impaired (50Martinis S.A. Atkins W.M. Stayton P.S. Sligar S.G. J. Am. Chem. Soc. 1989; 111: 9252-9253Crossref Scopus (256) Google Scholar, 53Vidakovic M. Sligar S.G., Li, H. Poulos T.L. Biochemistry. 1998; 37: 9211-9219Crossref PubMed Scopus (229) Google Scholar, 55Gerber N.C. Sligar S.G. J. Biol. Chem. 1994; 269: 4260-4266Abstract Full Text PDF PubMed Google Scholar). In wild-type P450 CYP101, the primary intermediate of radiolytic reduction of oxy-ferrous enzyme at 77 K is the fully protonated hydroperoxo-ferric complex (32Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (411) Google Scholar, 43Davydov 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). Comparison with similar studies on other heme proteins (25Symons M.C.R. Petersen R.L. Proc. R. Soc. Lond. B Biol. Sci. 1978; 201: 285-300Crossref PubMed Scopus (82) Google Scholar, 26Symons M.C.R. Petersen R.L. Biochim. Biophys. Acta. 1978; 535: 241-247Crossref PubMed Scopus (46) Google Scholar, 28Davydov R.M. Biofizika. 1980; 25: 203-207PubMed Google Scholar, 29Kappl R. Hohn-Berlage M. Huttermann J. Bartlett N. Symons M.C.R. Biochim. Biophys. Acta. 1985; 827: 327-343Crossref Scopus (73) Google Scholar, 30Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar, 35Davydov R. Kofman V. Fujii H. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 2002; 124: 1798-1808Crossref PubMed Scopus (149) Google Scholar, 42Davydov R.M. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10656-10657Crossref Scopus (147) Google Scholar) shows that the ability to deliver a proton to the peroxo-ferric heme complex correlates with the function of the protein, as this protonation is most effective in P450s and least effective in Mb, which does not carry out dioxygen bond scission during its normal function. Annealing of the reduced oxy-ferrous complex of HRP results in completion of the first protonation of the peroxide ligand and formation of the ferric hydroperoxo intermediate. Formally, this intermediate is the same as the one formed in the reaction of H2O2 with heme proteins, which is a precursor for the formation of Compound I in HRP and other peroxidases. However, when obtained by reduction of the oxy-ferrous heme complex, the peroxo-ferric intermediate via this path still needs two protons provided by the enzyme to affect O–O bond scission. This difference between peroxidase (using H2O2) and oxygenase/oxidase (using O2) pathways is essential in the subsequent evolution of this intermediate. Annealing at higher temperatures of (Fe3+P-OOH−) intermediate in HRP did not result in Compound I formation. We conclude, that Compound I is not formed on this pathway, because the delivery of the second proton to the distal (β) oxygen atom of the hydroperoxo-anion ligand is not optimized in HRP. These results confirm the importance of protonation mechanisms in heme enzymes, which use dioxygen for the formation of the redox active heme oxygen intermediates. We gratefully appreciate the help provided by Dr. John Bentley on using the 60Co source in the Notre Dame Radiation Laboratory (Notre Dame University, South Bend, IN). Irradiations were conducted at the Notre Dame Radiation Laboratory, which is a facility of the United States Department of Energy, Office of Basic Energy Sciences. Useful discussions with Dr. S.- C. Hung are gratefully acknowledged. We thank Aretta Weber for expert editorial assistance.

Referência(s)