Isoporphyrin Intermediate in Heme Oxygenase Catalysis
2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês
10.1074/jbc.m709685200
ISSN1083-351X
AutoresJohn P. Evans, Fernando Niemevz, Graciela Buldain, Paul Ortiz de Montellano,
Tópico(s)Neonatal Health and Biochemistry
ResumoHuman heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the α-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin π-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of α-meso-phenylheme-IX, α-meso-(p-methylphenyl)-mesoheme-III, and α-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the α-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced α-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation. Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the α-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin π-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of α-meso-phenylheme-IX, α-meso-(p-methylphenyl)-mesoheme-III, and α-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the α-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced α-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation. Heme metabolism by heme oxygenase (HO) 2The abbreviations used are: HO, heme oxygenase; hHO-1, truncated human HO-1; CPR, NADPH-cytochrome P450 reductase; MH, mesoheme-III; CH3PMH, α-meso-(p-methylphenyl)-mesoheme-III; CF3PMH, α-meso-(p-trifluoromethylphenyl)-mesoheme-III; PH, α-meso-phenylheme-IX; HPLC, high performance liquid chromatography; LC-ESI-MS, liquid chromatography electrospray ionization mass spectrometry. is an important process in animals that alleviates the potentially deleterious effects of free heme. The reaction releases iron, which is largely recycled, and produces CO and biliverdin. Recent studies have shown that the CO has important physiological functions, in part as a cell signaling molecule (1Kim H.P. Ryter S.W. Choi A.M. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 411-449Crossref PubMed Scopus (372) Google Scholar), whereas the biliverdin is immediately converted to bilirubin, a powerful antioxidant species (2Stocker R. Yamamoto Y. McDonagh A.F. Glazer A.N. Ames B.N. Science. 1987; 235: 1043-1046Crossref PubMed Scopus (3003) Google Scholar, 3Baranano D.E. Rao M. Ferris C.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16093-16098Crossref PubMed Scopus (916) Google Scholar). A growing body of evidence suggests that these heme oxygenase products have significant protective roles in a number of disease processes, including atherogenesis and carcinogenesis (4Stocker R. Perrella M.A. Circulation. 2006; 114: 2178-2189Crossref PubMed Scopus (203) Google Scholar, 5Berberat P.O. Dambrauskas Z. Gulbinas A. Giese T. Giese N. Kunzli B. Autschbach F. Meuer S. Buchler M.W. Friess H. Clin. Cancer Res. 2005; 11: 3790-3798Crossref PubMed Scopus (236) Google Scholar). In addition, heme degradation by pathogenic bacterial heme oxygenases, which is essential for their survival because it serves as a primary source of iron, makes these enzymes of potential interest for antibacterial strategies (6Furci L.M. Lopes P. Eakanunkul S. Zhong S. MacKerell Jr., A.D. Wilks A. J. Med. Chem. 2007; 50: 3804-3813Crossref PubMed Scopus (38) Google Scholar). Human heme oxygenase-1 (hHO-1) utilizes O2 and NADPH in the three-step process shown in Fig. 1 (7Unno M. Matsui T. Ikeda-Saito M. Nat. Prod. Rep. 2007; 24: 553-570Crossref PubMed Scopus (131) Google Scholar, 8Ortiz de Montellano P.R. Curr. Opin. Chem. Biol. 2000; 4: 221-227Crossref PubMed Scopus (213) Google Scholar, 9Colas C. Ortiz de Montellano P.R. Chem. Rev. 2003; 103: 2305-2332Crossref PubMed Scopus (124) Google Scholar). The heme acts as both a substrate and a cofactor that sequentially activates a total of three O2 molecules in oxidation of the macrocycle, a process that requires seven electrons provided by NADPH-cytochrome P450 reductase (CPR). In the first step, the two-electron reduction of O2 at the heme iron generates a ferrous hydroperoxide intermediate (Fe(III)-OOH) that inserts the terminal oxygen of the peroxide moiety into the α-meso carbon of the same heme, generating α-meso-hydroxyheme (10Wilks A. Torpey J. Ortiz de Montellano P.R. J. Biol. Chem. 1994; 269: 29553-29556Abstract Full Text PDF PubMed Google Scholar, 11Kumar D. de Visser S.P. Shaik S. J. Am. Chem. Soc. 2005; 127: 8204-8213Crossref PubMed Scopus (77) Google Scholar). The second step utilizes one molecule of O2 to form verdoheme in a reaction that results in elimination of the α-meso carbon as CO. Finally, in the third step the verdoheme is transformed into biliverdin in a reaction that requires both O2 and five reducing equivalents from CPR. An analogous heme degradation reaction can occur spontaneously in a coordinating solvent such as pyridine in the presence of an electron donor such as ascorbate. This process of “coupled oxidation” is non-regiospecific so that random oxidation occurs at all four meso carbons to give a mixture of the corresponding IXα, IXβ, IXγ, and IXδ biliverdin isomers (12Bonnett R. McDonagh A.F. J. Chem. Soc. Perkin Trans. I. 1973; 9: 881-888Crossref PubMed Google Scholar, 13Sano S. Sano T. Morishima I. Shiro Y. Maeda Y. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 531-535Crossref PubMed Scopus (102) Google Scholar). In contrast, the hHO-1-catalyzed reaction regiospecifically oxidizes the α-meso carbon. Multiple factors contribute to this hHO-1 regiospecificity. Of primary importance are the steric constraints imposed by the distal helix in which two conserved glycine residues at its center, Gly-139 and Gly-143, provide the flexibility required to sandwich the heme between two helices (14Takahashi S. Ishikawa K. Takeuchi N. Ikedasaito M. Yoshida T. Rousseau D.L. J. Am. Chem. Soc. 1995; 117: 6002-6006Crossref Scopus (103) Google Scholar, 15Schuller D.J. Wilks A. Ortiz de Montellano P.R. Poulos T.L. Nat. Struct. Biol. 1999; 6: 860-867Crossref PubMed Scopus (314) Google Scholar) while at the same time suppressing conversion of the ferric hydroperoxide intermediate to a ferryl (Fe(IV) = O) species (16Liu Y. Lightning L.K. Huang H.W. Moenne-Loccoz P. Schuller D.J. Poulos T.L. Loehr T.M. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 34501-34507Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Furthermore, the structure of nitric oxide-bound hHO-1 indicates that in addition to this steric constraint, a hydrogen bonding network involving Asp140-water-NO H-bonds cause a tilt of the NO toward the α-meso carbon (17Lad L. Wang J. Li H. Friedman J. Bhaskar B. Ortiz de Montellano P.R. Poulos T.L. J. Mol. Biol. 2003; 330: 527-538Crossref PubMed Scopus (74) Google Scholar). Finally, the orientation of the heme relative to an axis through the iron perpendicular to the heme plane is determined by electrostatic interactions between charged residues on the protein and the heme propionate groups (18Zhou H. Migita C.T. Sato M. Sun D.Y. Zhang X.H. Ikeda-Saito M. Fujii H. Yoshida T. J. Am. Chem. Soc. 2000; 122: 8311-8312Crossref Scopus (36) Google Scholar, 19Wang J. Evans J.P. Ogura H. La Mar G.N. Ortiz de Montellano P.R. Biochemistry. 2006; 45: 61-73Crossref PubMed Scopus (26) Google Scholar). Heme oxygenase can utilize a number of alternate Fe(III)porphyrins as substrates with the only clear prerequisite being the presence of the two vicinal propionate groups (20Frydman R.B. Tomaro M.L. Buldain G. Awruch J. Diaz L. Frydman B. Biochemistry. 1981; 20: 5177-5182Crossref PubMed Scopus (59) Google Scholar). Previous studies have investigated the oxidation of hemes in which the α-meso carbon bears a methyl, formyl, or phenyl substituent (21Torpey J. Lee D.A. Smith K.M. Ortiz de Montellano P.R. J. Am. Chem. Soc. 1996; 118: 9172-9173Crossref Scopus (27) Google Scholar, 22Torpey J. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 22008-22014Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Wang J. Niemevz F. Lad L. Huang L. Alvarez D.E. Buldain G. Poulos T.L. Ortiz de Montellano P.R. J. Biol. Chem. 2004; 279: 42593-42604Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Surprisingly, phenyl-substituted hemes readily bind to the hHO-1 active site due to the presence of a hydrophobic cavity, formed by residues Met-34, Phe-37, and Phe-214, that is postulated to function as a CO trapping site (24Sugishima M. Sakamoto H. Noguchi M. Fukuyama K. J. Mol. Biol. 2004; 341: 7-13Crossref PubMed Scopus (18) Google Scholar). The hHO-1 oxidation of α-meso-methyl and -phenyl-substituted hemes produces α-biliverdin, whereas the oxidation of α-meso-formyl heme does not. The product containing the α-meso carbon in the oxidation of α-meso-methylheme was not identified but was inferred to be acetic acid. More recently, benzoic acid has been identified as a product of the hHO-1-catalyzed oxidation of α-meso-phenylheme. An additional intermediate is required to account for benzoic acid formation (23Wang J. Niemevz F. Lad L. Huang L. Alvarez D.E. Buldain G. Poulos T.L. Ortiz de Montellano P.R. J. Biol. Chem. 2004; 279: 42593-42604Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). To further characterize the oxidation mechanism of hHO-1, we report here additional studies of the hHO-1-catalyzed oxidation of α-meso-phenylheme as well as the symmetrical mesoheme III (MH), α-meso-(p-methylphenyl)-mesoheme-III (CH3PMH), and α-meso-(p-trifluoromethylphenyl)-mesoheme-III (CF3PMH). We anticipated that the addition of substituents with varying electron donating/withdrawing properties would modulate the reactivity in a way that would facilitate the detection of intermediates. A unique absorbance appeared transiently during the catalytic reaction with NADPH-CPR in the near IR region that is characteristic of an isoporphyrin. The formation of a long-lived isoporphyrin was confirmed by mass spectrometry in the reaction supported by H2O2. This is the first actual evidence for formation of this unique tetrahedral intermediate during HO catalytic turnover. Chemicals—MH, CH3PMH, CF3PMH, and α-meso-phenylheme-IX (PH) were synthesized as described previously (25Niemevz F. Vazquez M.S. Buldain G.Y. Synthesis. 2008; 6: 875-882Google Scholar, 26Robinsohn A.E. Maier M.S. Buldain G.Y. Heterocycles. 2000; 53: 2127-2142Crossref Google Scholar). The dimethyl esterified biliverdin isomers were prepared from the corresponding heme by coupled oxidation in the presence of pyridine, O2, and ascorbic acid according to published procedures (27Niemevz F. Alvarez D.E. Buldain G.Y. Heterocycles. 2002; 57: 697-704Crossref Google Scholar, 28Niemevz F. Buldain G.Y. J. Porphyrins Phthalocyanines. 2004; 8: 989-995Crossref Scopus (7) Google Scholar). NADPH and H2O2 were obtained from Sigma-Aldrich. H218O2 (90%) and 18O2 (99%) were from Icon Isotopes (Summit, NJ). H218O (95%) was from Cambridge Isotope Laboratories Inc. (Andover, MA). The concentration of the stock solution of H2O2 was determined from ϵ240 = 43.6 m-1cm-1 (29Hildebrandt A.G. Roots I. Tjoe M. Heinemeyer G. Methods Enzymol. 1978; 52: 342-350Crossref PubMed Scopus (194) Google Scholar). Enzymes—Truncated hHO-1 lacking the 23 C-terminal residues was expressed and purified according to published procedures (23Wang J. Niemevz F. Lad L. Huang L. Alvarez D.E. Buldain G. Poulos T.L. Ortiz de Montellano P.R. J. Biol. Chem. 2004; 279: 42593-42604Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 30Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 31Wilks A. Black S.M. Miller W.L. Ortiz de Montellano P.R. Biochemistry. 1995; 34: 4421-4427Crossref PubMed Scopus (111) Google Scholar). This truncated protein is referred to as hHO-1 throughout the paper. The expression and purification of human CPR (32Dierks E.A. Davis S.C. Ortiz de Montellano P.R. Biochemistry. 1998; 37: 1839-1847Crossref PubMed Scopus (59) Google Scholar) and rat biliverdin reductase (33Lightning L.K. Huang H. Moenne-Loccoz P. Loehr T.M. Schuller D.J. Poulos T.L. Ortiz de Montellano P.R. J. Biol. Chem. 2001; 276: 10612-10619Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) were performed as previously reported. Superoxide dismutase and catalase were from Sigma-Aldrich. Reconstitution of hHO-1 with Mesoheme Derivatives—MH or one of the mono meso-arylmesohemes was first fully dissolved in a minimum volume of 0.1 N NaOH (sonication was used to aid in dissolution), diluted in 0.1 m potassium phosphate buffer (pH 7.4) and then added to the purified apo-hHO-1 to give a final 1.2:1 heme:protein ratio. The sample was allowed to stand for at least 2 h at 4 °C before it was applied to a Bio-Gel HTP hydroxyapatite column (12 ml) preequilibrated with 10 mm potassium phosphate buffer (pH 7.4). The column was then washed with the same buffer (5 volumes), and the protein was eluted in 0.1 m potassium phosphate buffer (pH 7.4). The fractions containing the heme-hHO-1 complex were pooled and concentrated. Single Turnover Assays—All spectra were performed on a Varian Cary 50 UV-visible spectrophotometer in 0.1 m potassium phosphate buffer (pH 7.4) at room temperature. Because all the reactions in this study were carried out in 0.1 m potassium phosphate buffer (pH 7.4), this buffer is referred to as the standard buffer. The reaction mixtures (500 μl) contained 5 μm heme-hHO-1 complex, 40 nm CPR, 50 μm NADPH, and 10 μg/ml catalase in standard buffer. Spectra were recorded at 15-s intervals over the range of 300–1000 nm for 20 min. For the H2O2 reactions, 20 μm H2O2 was added in place of NADPH, CPR, and catalase. The reaction was also performed under an argon atmosphere in the presence of 20 μm H2O2 using an anaerobic cuvette with a side arm, where H2O2 was contained in the side-arm until gas exchange was complete, as described previously (34Liu Y. Moenne-Loccoz P. Loehr T.M. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). To monitor the stability of the isoporphyrin-hHO-1 complex, 10 μg/ml catalase was added to the reaction of 5 μm arylheme-hHO-1 and 40 μm H2O2 after 20 min, and the decay was monitored over 6 h. CO Formation Assay—Horse myoglobin (Sigma) was used as a CO trap according to the protocol previously described (23Wang J. Niemevz F. Lad L. Huang L. Alvarez D.E. Buldain G. Poulos T.L. Ortiz de Montellano P.R. J. Biol. Chem. 2004; 279: 42593-42604Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The reactions (1 ml) consisted of 10 μm arylmesoheme-hHO-1 complex, 60 nm CPR, and 100 μm NADPH in standard buffer and were allowed to proceed for 20 min in a sealed container before the addition of myoglobin via syringe to a final concentration of 4 μm. Biliverdin Isomer Analysis—A solution (300 μl) of 100 μm heme-hHO-1 complex, 2.7 μm CPR, 5 mm NADPH, 10 μg/ml catalase, and 17 units/ml superoxide dismutase in standard buffer was incubated for 1.5 h at room temperature. The reaction was quenched with two drops of hydrochloric acid (37%) plus a few drops of acetic acid and was then extracted with 700 μl of CH2Cl2. The work-up, esterification, and HPLC analysis were carried out as described previously (19Wang J. Evans J.P. Ogura H. La Mar G.N. Ortiz de Montellano P.R. Biochemistry. 2006; 45: 61-73Crossref PubMed Scopus (26) Google Scholar). Product Analysis by HPLC and LC-ESI-MS—Reactions (500 μl) containing 10 μm heme-hHO-1 and either 80 μm H2O2 or 10 μg/ml catalase, 160 nm CPR, and 100 μm NADPH in standard buffer were quenched after 20 min with 100 μl of 1 m HCl. Additional reactions were performed by first incubating with 80 μm H2O2 for 20 min followed by the addition of 10 μg/ml of catalase, 160 nm CPR, and 100 μm NADPH for an additional 20 min before the acid quench. The quenched reactions were immediately extracted with 700 μl of CH2Cl2, and the organic phase was evaporated under a stream of air. The resulting residue was dissolved in 100% acetonitrile and was then analyzed on either an Agilent 1200 Series HPLC attached to a G1315B diode array detector using a YMC ODS-AQ column (S-5, 120 Å, 4.5 × 250 mm) or by LC-ESI-MS performed on a Waters Micromass ZQ coupled to a Waters Alliance HPLC system using a Symmetry ODS column (Waters, 3.5 μm, 2.1 × 150 mm). Solvents were water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The running conditions were identical for each case differing only in flow rate, 1.0 ml/min for the 4.5-mm inner diameter column or 0.2 ml/min for the 2.1-mm inner diameter column, and were as follows: 30% B for 5 min, 30–80% B in 35 min, 80–95% B in 5 min, 95% B for 5 min, 95–30% B in 5 min, and finally 30% B for 5 min. The settings for the mass spectrometer were as follows: mode, ES+; capillary voltage, 3.5 kV; cone voltage, 25 V; desolvation temperature, 300 °C; source temperature, 120 °C. Origin of the Oxygen Atoms in the Products—Oxidation of arylmesohemes by hHO-1 was carried out in the presence of either 18O2, H218O, or H218O2. Reactions in the presence of 18O2 and H218O were performed as described previously (23Wang J. Niemevz F. Lad L. Huang L. Alvarez D.E. Buldain G. Poulos T.L. Ortiz de Montellano P.R. J. Biol. Chem. 2004; 279: 42593-42604Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). All reactions in the presence of H218O2 were performed as described above but with H218O2 in place of H2O2. UV-visible Spectra of meso-Phenylheme-reconstituted hHO-1—The symmetrical MH, CH3PMH, and CF3PMH were combined with hHO-1 followed by hydroxyapatite chromatography to remove excess and non-specifically bound heme. The UV spectra of the ferric, ferrous, and ferrous-CO forms at pH 7.4 (supplemental Fig. S1) reveal that all three heme analogues bind to the active site in a similar mode as heme and, most importantly, that the heme-iron can undergo redox cycling. In the spectrum of MH, replacement of the vinyl groups of heme by ethyl groups places greater electron density on the iron and causes a blue shift of the Soret band from the normal 404 to 394 nm. Upon reduction to the ferrous state, the Soret maximum shifts to 421 nm and, upon further exposure to CO, to 410 nm, consistent with formation of the ferrous-CO complex. In comparison to the MH-hHO-1 complex, the CH3PMH and CF3PMH complexes exhibit a red-shifted Soret maximum at 400 nm that shifts to 426–428 nm in the ferrous state and to ∼416 nm in the ferrous-CO complex. This red-shift is consistent with the presence of an electron-withdrawing substituent (i.e. aryl group) on the heme α-meso carbon that removes electron density from the iron. Formation of CO during the NADPH/CPR-supported Reaction—The normal degradation of heme by hHO-1, supported by electrons from NADPH via CPR, produces biliverdin IXα and CO. An assay based on the binding of CO to ferrous deoxymyoglobin was used to investigate its formation in the NADPH/CPR-supported reactions of MH, CH3PMH, and CF3PMH. In the presence of CO, the Soret maximum of Fe(II) Mb at λmax = 434 nm shifts to λmax = 422 nm (Fe(II)-CO Mb) with α/β bands at 540 and 570 nm, respectively. In the reaction of MH with hHO-1, degradation of the heme clearly generates CO, as shown by the resulting peak at 422 nm. The reactions of CH3PMH and CF3PMH do not show the clear shift in the Soret maximum of Fe(II) MB, instead giving peaks at 431 and 428 nm, respectively, without α/β bands. The absence of the expected peak shifts indicates that no CO was formed. Biliverdin Isomer Analysis by HPLC—To facilitate the isolation and identification of the biliverdin isomers produced from the normal HO reaction, the products were dimethylesterified before HPLC analysis. Chemical oxidation in the absence of hHO-1 using ascorbic acid under coupled oxidation conditions gives a mixture of the isomers from reaction at the non-substituted meso positions of each heme analogue (Fig. 2) (28Niemevz F. Buldain G.Y. J. Porphyrins Phthalocyanines. 2004; 8: 989-995Crossref Scopus (7) Google Scholar). However, the hHO-1-catalyzed oxidation of each heme analogue in the presence of NADPH/CPR produces only the α-mesobiliverdin isomer. Even using sodium ascorbate as the electron donor, hHO-1 enforces the specificity for the α-meso position, producing only the α-mesobiliverdin derivative (not shown). Single Turnover Reaction Supported by NADPH-CPR—We examined the reactivity of each of the modified heme-hHO-1 complexes with NADPH/CPR (Fig. 3). Upon the addition of NADPH (50 μm, 10 eq relative to heme-hHO-1 complex), CPR (0.04 μm), and catalase, the heme-hHO-1 complex changed immediately to the ferrous-oxy form, as indicated by a shift of the Soret maximum from 405 to 408 nm and the appearance of 530- and 566-nm peaks. Under these conditions this intermediate decays over the course of 20 min with a corresponding increase in the visible region at 680 nm due to the formation of verdoheme and at 640 nm due to CO-bound ferrous verdoheme. Ultimately biliverdin is formed with a broad peak centered at 688 nm (Fig. 3A). The PH-hHO-1 complex, upon the addition of NADPH/CPR, also converted immediately to the ferrous-oxy form as indicated by the Soret shift from 404 to 408 nm (Fig. 3B). However, there are two major differences from the heme-hHO-1 reaction. First, there are transient increases in absorbance at 440 and 900 nm, suggestive of an additional intermediate, and second, there is no detectable CO-ferrous verdoheme absorbance at 640 nm. Oxidation of the MH-hHO-1 complex is noticeably slower than the oxidation of heme but proceeds through the obligatory verdoheme intermediate to mesobiliverdin with an absorption maximum at 650 nm (Fig. 3C). The intensity of the verdoheme intermediate and mesobiliverdin product are significantly lower than those of the equivalent species in the oxidation of heme itself. The CH3PMH-hHO-1 (Fig. 3D) and CF3PMH-hHO-1 (not shown) complexes underwent a similar Soret shift, going from 400 to 409 nm on conversion to the ferrous oxy form, and a transient increase in absorbance at 440 and 900 nm. In all the arylheme reactions there was a transient increase in absorbance at both 440 and 900 nm that was not observed in the oxidation of heme or MH. These absorbances are characteristic of both isoporphyrins (35Ator M.A. David S.K. Ortiz de Montellano P.R. J. Biol. Chem. 1989; 264: 9250-9257Abstract Full Text PDF PubMed Google Scholar) and benzoylbiliverdins (36Ongayi O. Fronczek F.R. Vicente M.G.H. Chem. Commun. 2003; : 2298-2299Crossref PubMed Google Scholar), but it was first necessary to determine whether this species was a side product or an intermediate in the pathway of biliverdin formation. Single Turnover Reaction Supported by H2O2—Fe(III)-verdoheme can be generated by reaction of the heme-hHO-1 complex with H2O2 (30Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar, 34Liu Y. Moenne-Loccoz P. Loehr T.M. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The single turnover reaction of MH with 4 eq of H2O2 (20 μm) resulted in the accumulation of verdoheme, as indicated by a decrease in the Soret band and a concomitant increase at 650 nm (Fig. 4A). In contrast, when the α-meso position is blocked with a phenyl group, as in CH3PMH (Fig. 4B), CF3PMH, and PH (not shown), the reaction exclusively generates what looks like an isoporphyrin intermediate (Fig. 4B), as shown by the decrease in the Soret band with concomitant increases in absorbance at 440 and 930 nm. The spectral features of the proposed isoporphyrin-hHO-1 complexes formed in the reactions of the CH3PMH and CF3PMH hHO-1 complexes with H2O2 are very similar (Fig. 5A). Both intermediates exhibit peaks at ∼360 and 435 nm in the UV region and a strong peak with a maximum at ∼935 nm in the near IR region with a side band at 840 nm. This species can be removed from the protein by mild quenching with acid and extraction with dichloromethane. The optical absorption spectra of the extracted isoporphyrins of CH3PMH and CF3PMH dissolved in CH2Cl2 (Fig. 5B) are very similar to those of ferric and zinc isoporphyrins (37Gold A. Ivey W. Toney G.E. Sangaiah R. Inorg. Chem. 1984; 23: 2932-2935Crossref Scopus (52) Google Scholar, 38Dolphin D. Felton R.H. Borg D.C. Fajer J. J. Am. Chem. Soc. 1970; 92: 743-745Crossref Scopus (151) Google Scholar) but not to Ni(II) and Cu(II) benzoylbiliverdins (36Ongayi O. Fronczek F.R. Vicente M.G.H. Chem. Commun. 2003; : 2298-2299Crossref PubMed Google Scholar), which have very broad and featureless peaks in the near-IR. Although both CH3PMH and CF3PMH look very similar in CH2Cl2, there is a significant difference between their maxima in the near-IR. Both show a shift of the 435 peak to 428 nm, but CH3PMH has a peak at 949 nm and a side band at 855, whereas CF3PMH has a peak at 927 nm with a side band at 842 nm. To confirm that the intermediate observed spectroscopically was an isoporphyrin rather than the more oxidized benzoylbiliverdin, we performed the peroxide reaction under anaerobic conditions. A reaction analogous to that seen under aerobic conditions, with the same rate of Soret disappearance and intermediate formation, was observed. This evidence is consistent with formation of an isoporphyrin rather than a more oxidized benzoylbiliverdin species, although it is possible that peroxide could facilitate further oxidation of the isoporphyrin. Next we asked whether the isoporphyrin complex formed with H2O2 was a true intermediate in the conversion of the heme to a biliverdin analogue or a dead end side product. The verdoheme intermediate formed from the reaction of MH-hHO-1 with peroxide, when exposed to additional reducing equivalents in the form of NADPH/CPR, rapidly converts to mesobiliverdin (Fig. 6A). The addition of NADPH/CPR to the CH3PMH-hHO-1 isoporphyrin complex resulted in a shift of the 935-nm peak to 900 nm, a broadening with loss of the sideband, and conversion to mesobiliverdin over 10 min (Fig. 6B). A very similar but more rapid decay was seen with the PH and CF3PMH-hHO-1 isoporphyrin complexes (supplemental Fig. S2). The increase in absorbance at 650 nm is indicative of the formation of biliverdin as the primary reaction product. Product Analysis by HPLC and LC-ESI-MS—We analyzed the products formed in the reactions of the CH3PMH-hHO-1 (Fig. 7), CF3PMH-hHO-1, and PH-hHO-1 (not shown) complexes in the presence of either NADPH/CPR or H2O2 by quenching and extracting the reaction mixtures. The reaction of CH3PMH-hHO-1 with NADPH/CPR in the presence of catalase generates primarily α-mesobiliverdin (Fig. 7A, top). The reaction with H2O2 shows two products eluting on HPLC at 20.1 and 25.5 min. The major product eluting first has an absorbance spectrum (Fig. 7B, middle) similar to that of the observed spectroscopic product (Fig. 5). ESI-MS analysis obtained in the ES+ mode identified this major product as the isoporphyrin that is detected as the formate conjugate with m/z 772 and as its fragment at m/z 608 due to the loss of the α-meso carbon (Fig. 7C, middle, and Fig. 8). The second HPLC peak has a unique absorbance spectrum with peaks at 310 an
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