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• W2060174475 abstract "The oxidation of heme to biliverdin IXα by heme oxygenase involves regiospecific α-meso-hydroxylation followed by extrusion of the α-meso-carbon as CO. In an earlier study, enzymatic oxidation of the fourmeso-methylmesoheme isomers suggested that the reaction regiospecificity is sensitive to the electronic properties of themeso-methyl group (Torpey, J. W., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 26067–26073), although we could not exclude the possibility that the altered reaction regiochemistry was due to perturbation of the porphyrin structure by the meso-substituent. To examine this possibility, we have synthesized the fourmeso-formylmesoporphyrin isomers and have examined their oxidation by heme oxygenase. The meso-formyl andmeso-methyl substituents differ in that the former is electron withdrawing and the latter is electron donating. In contrast to α-meso-methylmesoheme, which is exclusively oxidized at the methyl-substituted position, α-meso-formylmesoheme is exclusively oxidized at a non-formyl-substitutedmeso-carbon. The finding that the methyl and formyl groups channel the reaction regiospecificity in opposite directions establishes that the regiochemistry of the heme oxygenase reaction is primarily under electronic rather than steric control. It also confirms that the oxidation involves electrophilic addition of the oxygen to the porphyrin ring. The oxidation of heme to biliverdin IXα by heme oxygenase involves regiospecific α-meso-hydroxylation followed by extrusion of the α-meso-carbon as CO. In an earlier study, enzymatic oxidation of the fourmeso-methylmesoheme isomers suggested that the reaction regiospecificity is sensitive to the electronic properties of themeso-methyl group (Torpey, J. W., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 26067–26073), although we could not exclude the possibility that the altered reaction regiochemistry was due to perturbation of the porphyrin structure by the meso-substituent. To examine this possibility, we have synthesized the fourmeso-formylmesoporphyrin isomers and have examined their oxidation by heme oxygenase. The meso-formyl andmeso-methyl substituents differ in that the former is electron withdrawing and the latter is electron donating. In contrast to α-meso-methylmesoheme, which is exclusively oxidized at the methyl-substituted position, α-meso-formylmesoheme is exclusively oxidized at a non-formyl-substitutedmeso-carbon. The finding that the methyl and formyl groups channel the reaction regiospecificity in opposite directions establishes that the regiochemistry of the heme oxygenase reaction is primarily under electronic rather than steric control. It also confirms that the oxidation involves electrophilic addition of the oxygen to the porphyrin ring. The oxidation of heme 1The abbreviations used are: heme, iron protoporphyrin IX regardless of the iron oxidation and ligation states; HO-1, heme oxygenase isozyme-1; hHO-1, truncated human HO-1; HPLC, high pressure liquid chromatography; P-450 reductase, cytochrome P-450 reductase; mesoheme, iron mesoporphyrin IX; CHO, Chinese hamster ovary.1The abbreviations used are: heme, iron protoporphyrin IX regardless of the iron oxidation and ligation states; HO-1, heme oxygenase isozyme-1; hHO-1, truncated human HO-1; HPLC, high pressure liquid chromatography; P-450 reductase, cytochrome P-450 reductase; mesoheme, iron mesoporphyrin IX; CHO, Chinese hamster ovary.by heme oxygenase yields biliverdin IXα and CO (see Fig. 1), both of which have important physiological activities. Biliverdin is reduced to bilirubin, a powerful antioxidant that is highly lipophilic and, under conditions of impaired excretion, can reach concentrations at which it becomes neurotoxic (1Maines M.D. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Boca Raton, FL1992: 203-266Google Scholar). Carbon monoxide, the second product of the heme oxygenase reaction, is a putative physiological messenger akin to nitric oxide (2Verma A. Hirsch D.J. Glatt C.E. Ronnett G.V. Snyder S.H. Science. 1993; 259: 381-384Crossref PubMed Scopus (1362) Google Scholar, 3Stevens C.F. Wang Y. Nature. 1993; 364: 147-149Crossref PubMed Scopus (243) Google Scholar, 4Marks G.S. Cell. Mol. Biol. 1994; 40: 863-870PubMed Google Scholar). Human HO-1, a 32-kDa protein, is anchored to the endoplasmic reticulum via a C-terminal lipophilic domain (5Yoshida T. Biro P. Cohen T. Müller R.M. Shibahara S. Eur. J. Biochem. 1988; 171: 457-461Crossref PubMed Scopus (267) Google Scholar). Removal of the 23 C-terminal amino acids by mutation of the cDNA coding for the protein, followed by heterologous expression in Escherichia coli, yields a truncated human HO-1 (hHO-1) that is both soluble and fully active (6Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar). An active and soluble rat heme oxygenase without the 26 C-terminal amino acids and with mutations in the last two amino acids (S262R,S263L) has been independently reported (7Ishikawa K. Sato M. Ito M. Yoshida T. Biochem. Biophys. Res. Commun. 1992; 182: 981-986Crossref PubMed Scopus (46) Google Scholar). The first step of the reaction catalyzed by heme oxygenase is the NADPH-, cytochrome P-450-reductase-, and O2-dependent oxidation of heme to α-meso-hydroxyheme (see Fig. 1) (8Liu Y. Moënne-Loccoz P. Loehr T.M. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Matera K.M. Takahashi S. Fujii H. Zhou H. Ishikawa K. Yoshimura T. Rousseau D.L. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1996; 271: 6618-6624Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 10Saito S. Itano H.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1393-1397Crossref PubMed Scopus (66) Google Scholar, 11Yoshida T. Noguchi M. J. Biochem. ( Tokyo ). 1984; 96: 563-570Crossref PubMed Scopus (36) Google Scholar, 12Schacter B.A. Nelson E.B. Marver H.S. Masters B.S.S. J. Biol. Chem. 1972; 247: 3601-3607Abstract Full Text PDF PubMed Google Scholar). In the second step, α-meso-hydroxyheme undergoes an O2-dependent but NADPH-independent (8Liu Y. Moënne-Loccoz P. Loehr T.M. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 6909-6917Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) reaction that results in extrusion of the hydroxylated α-meso-carbon as CO with the concomitant formation of verdoheme (Fig. 1). Finally, in the third step, heme oxygenase catalyzes the formation of biliverdin from verdoheme in a reaction that also requires NADPH, cytochrome P-450 reductase, and O2 (10Saito S. Itano H.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1393-1397Crossref PubMed Scopus (66) Google Scholar, 11Yoshida T. Noguchi M. J. Biochem. ( Tokyo ). 1984; 96: 563-570Crossref PubMed Scopus (36) Google Scholar). The heme oxygenase reaction is highly regiospecific and exclusively produces biliverdin IXα, the isomer resulting from oxidation of the α-meso-carbon (13Tenhunen R. Marver H.S. Schmid R. J. Biol. Chem. 1969; 244: 6388-6394Abstract Full Text PDF PubMed Google Scholar). The central role of α-meso-hydroxylation in the oxidation of heme by heme oxygenase led us earlier to synthesize the four possible meso-methylmesoheme isomers and to examine their enzyme-catalyzed oxidation (14Torpey J. Ortiz de Montellano P.R. J. Org. Chem. 1995; 60: 2195-2199Crossref Scopus (14) Google Scholar, 15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Surprisingly, α-meso-methylmesoheme is still oxidized to mesobiliverdin IXα. The reaction does not result in the formation of CO, however, in accord with the fact that normal α-meso-hydroxylation is impossible due to the presence of the methyl substituent. Equally surprising was the finding that γ-meso-methylmesoheme, despite the presence of an unsubstituted α-meso position, is oxidized exclusively at the γ-meso position to give γ-mesobiliverdin (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). δ-meso-Methylmesoheme was similarly oxidized at the δ-meso position to give δ-mesobiliverdin, but oxidation at the unsubstituted meso positions to give a mixture of methyl-substituted biliverdin isomers was also observed. The finding that the electron donating methyl group enhances reaction at the substituted carbon suggests that the oxidation reaction involves electrophilic addition of the activated oxygen to the mesocarbon. An electrophilic oxidation mechanism is also suggested by the earlier finding that the oxidation of heme by heme oxygenase in the presence of ethylhydroperoxide rather than NADPH and P-450 reductase produces α-meso-ethoxyheme (16Wilks A. Torpey J. Ortiz de Montellano P.R. J. Biol. Chem. 1994; 269: 29553-29556Abstract Full Text PDF PubMed Google Scholar). Furthermore, the results argue that the regiochemistry of the heme oxygenase reaction is controlled not by steric orientation of the oxidizing species as previously suggested (17Brown S.B. Chabot A.A. Enderby E.A. North A.C.T. Nature. 1981; 289: 93-95Crossref PubMed Scopus (30) Google Scholar) but rather by changes in the frontier orbital electron density at the meso positions of the heme group. However, the possibility that the effect of the meso-methyl group was due to deformation of the heme structure rather than to an electronic effect could not be excluded. To further test the hypothesis that the regiochemistry of the heme oxygenase reaction is governed by electronic rather than steric effects, we have synthesized the four possible isomers ofmeso-formylmesoheme and examined their oxidation by heme oxygenase. The formyl group is an electron-withdrawing substituent and should exert an effect opposite to that of a methyl group if the reaction regiochemistry is electronically controlled. On the other hand, the effects of the methyl and formyl substituents on the reaction regiochemistry should be parallel if they stem from a deformation of the heme structure or a steric interaction of themeso-substituent with the protein. The finding that the formyl and methyl substituents channel the reaction regiochemistry in opposite directions provides persuasive evidence that the regiochemistry of the heme oxygenase reaction is electronically rather than sterically controlled. Human heme oxygenase truncated of its 23 C-terminal amino acids (hHO-1) was expressed in E. coli and purified as reported (6Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar). Cytochrome P-450 reductase was kindly provided by Prof. B. S. S. Masters (University of Texas Health Sciences Center, San Antonio, TX). Mesoheme was prepared from mesoporphyrin IX dimethyl ester (Porphyrin Products, Logan, UT), and the individual meso-formylmesohemes were prepared from the corresponding synthetic meso-formyl mesoporphyrin IX dimethyl esters as described previously (14Torpey J. Ortiz de Montellano P.R. J. Org. Chem. 1995; 60: 2195-2199Crossref Scopus (14) Google Scholar). Thin layer chromatography was done on silica gel GF (250 micron) plates (Analtech, Newark, DE). HPLC was performed on a Varian 9010 solvent delivery system equipped with a Hewlett Packard 1040A detector and a reverse-phase Whatman analytical (4.6 × 250 mm) Partisil 10-mm ODS-3 column. The mobile phase was either 100% Solvent A (acetone/0.1% aqueous formic acid, 50:50) monitored at 374 nm and referenced at 474 nm or 100% Solvent B (acetonitrile/water/AcOH, 55:40:10) monitored at 404 nm and referenced at 550 nm. Absorption spectra were recorded on a Hewlett Packard 8452A diode array spectrophotometer. Mass spectra were obtained by (+)LSIMS on a Kratos Concept instrument using a 1:1 (1% trifluoroacetic acid) glycerol-thioglycerol matrix. 1H NMR spectra were measured in deuterated chloroform (porphyrin concentration 3–4 mg/ml) on a General Electric QE 300-MHz instrument. 13C NMR spectra were acquired on the same 300-MHz instrument and are completely decoupled. The meso-formylmesoheme isomers were individually reconstituted into hHO-1 in a 2:1 heme to enzyme ratio, and the resulting mixtures were purified over a Bio-Rad HTP column to give the 1:1 complexes in 100 mm potassium phosphate buffer (pH 7.4) (6Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar). The regioisomeric meso-hydroxymethyl mesoporphyrin IX dimethyl esters were synthesized as previously reported (14Torpey J. Ortiz de Montellano P.R. J. Org. Chem. 1995; 60: 2195-2199Crossref Scopus (14) Google Scholar). The meso-hydroxymethyl substituent was oxidized to the meso-formyl by dissolving themeso-(hydroxymethyl)-mesoporphyrin IX isomers (α = 16 mg, 25.6 μmol; β = 14 mg, 22.4 μmol; γ = 5 mg, 8.0 μmol; δ = 42 mg, 67.3 μmol) in CH2Cl2 (20 ml) and pyridine (5 ml) containing three equivalents of pyridinium chlorochromate. After standing overnight at room temperature in the dark, the reaction was partitioned between 1.0 n HCl (300 ml) and CH2Cl2 (50 ml). The organic phase was then washed with water (400 ml) and concentrated under vacuum. Thin layer chromatography of the reaction mixture with diethyl ether yielded themeso-formylmesoporphyrin dimethyl ester product (Rf = 0.88). The meso-formyl porphyrins were purified on silica gel columns with hexanes-diethyl ether (30:70) as the eluting solvent. The four isomers were oxidized in the same manner, yielding the α (6.0 mg, 9.6 μmol, 38%), β (9.3 mg, 15.0 μmol, 67%), γ (4.0 mg, 6.4 μmol, 80%), and δ (11 mg, 17.7 μmol, 26%) meso-formylmesoporphyrins. The spectroscopic and analytical data for the isomers are as follows: α isomer, λmax (CH3Cl) 404, 504, 538, 576 nm;1H NMR (CDCl3) δ 1.69 (t, 2H,J = 7.3 Hz), 1.76 (t, 2H, J = 7.5 Hz), 3.23 (t, 4H, J = 6.5 Hz), 3.34 (s, 3H), 3.52 (s, 3H), 3.56 (s, 3H), 3.58 (s, 3H), 3.63 (s, 3H), 3.64 (s, 3H), 3.79 (m, 2H) 3.98 (m, 2H), 4.31 (m, 4H), 9.94 (s, 1H), 10.01 (s, 2H), 12.72 ppm (s, 1H); 13C NMR (CDCl3) δ 11.6, 15.2, 16.0, 16.7, 17.5, 18.5, 19.8, 21.7, 29.7, 36.8, 51.8, 98.4, 113.6, 115.2, 137.9, 138.6, 142.2, 143.1, 143.4, 144.0, 144.1, 145.1, 145.5, 150.2, 173.5, 197.6 ppm; HRMS m/z 622.3163, calculated for C37H42N4O6 622.3155; β isomer, λmax (CH3Cl) 404, 506, 538, 574 nm; 1H NMR (CDCl3) δ 1.79 (brd m, 2H),1.81 (t, 2H, J = 7.5 Hz), 3.21 (m, 4H), 3.37 (s, 3H), 3.51 (s, 3H), 3.54 (s, 3H), 3.55 (s, 3H), 3.62 (s, 3H), 3.66 (s, 3H), 3.82 (m, 2H), 4.00 (m, 2H), 4.33 (m, 4H), 9.93 (s, 1H), 10.01 (s, 1H), 10.07 (s, 1H), 12.73 ppm (s, 1H); 13C NMR (CDCl3) δ 11.6, 14.1, 17.6, 19.6, 21.7, 22.0, 22.7, 27.4, 31.9, 36.8, 51.7, 97.2, 948.0, 98.5, 113.6, 114.4, 128.7, 135.1, 135.7, 136.2, 138.5, 140.7, 142.0, 142.9, 143.4, 144.7, 146.0, 146.2, 173.5, 174.0, 197.8 ppm; HRMS m/z 622.3149, calculated for C37H42N4O6 622.3155; γ isomer, λmax (CH3Cl) 406, 504, 538, 574 nm; 1H NMR (CDCl3) δ 1.81 (t, 6H,J = 9.0 Hz), 2.33 (t, 2H, J = 7.5 Hz), 3.11 (brd t, 2H), 3.53 (, 3H), 3.54 (s, 3H), 3.55 (s, 3H), 3.56 (s, 3H), 3.63 (m, 2H), 3.72 (s, 3H), 3.73 (s, 3H), 3.99 (t, 2H,J = 9.0 Hz), 4.12 (t, 2H, J = 9.0 Hz), 4.29 (m, 2H), 9.92 (s, 1H), 10.02 (s, 2H), 12.73 ppm (s, 1H);13C NMR (CDCl3) δ 11.4, 11.9, 17.5, 19.7, 24.4, 29.7, 35.5, 51.8, 98.5, 98.6, 98.8, 136.9, 139.7, 142.9, 143.1, 143.2, 143.8, 145.2, 173.4, 173.5, 196.0 ppm; HRMSm/z 622.3142, calculated for C37H42N4O6 622.3155; δ isomer, λmax 434, 574, 628 (CH3Cl) nm;1H NMR (CDCl3) δ 1.77 (t, 2H,J = 7.5 Hz), 1.81 (t, 2H, J = 7.5 Hz), 3.18 (t, 2H, J = 7.5 Hz), 3.24 (t, 2H,J = 7.5 Hz), 3.37 (s, 3H), 3.38 (s, 3H), 3.55 (s, 3H), 3.57 (s, 3H), 3.63 (s, 3H), 3.65 (s, 3H), 3.98 (q, 4H,J = 7.5 Hz), 4.34 (m, 4H), 9.94 (s, 1H), 10.01 (s, 1H), 10.07 (s, 1H), 12.73 ppm (s, 1H); 13C NMR (CDCl3) δ 11.4, 11.5, 16.5, 16.7, 17.5, 19.7, 21.7, 29.7, 36.8, 51.7, 98.1, 98.3, 98.6, 129.2, 133.9, 135.7, 136.6, 138.8, 141.3, 142.4, 142.8, 143.2, 144.1, 144.7, 144.9, 145.3, 173.4, 174.0, 198.1 ppm; HRMS m/z 622.3151, calculated for C37H42N4O6622.3155. P-450 reductase (12 μg, 0.15 nmol) and NADPH (100 nmol) were added to a 1-ml cuvette containing, in a final 1-ml volume, a solution of one of the meso-formylmesoheme-hHO-1 complexes (α-CHO, 0.29 mg, 9.8 nmol; β-CHO, 0.37 mg, 12.2 nmol; γ-CHO, 0.23 mg, 7.8 nmol; and δ-CHO, 0.41 mg, 13.8 nmol) in 100 mmpotassium phosphate buffer (pH 7.4). The solution was presaturated with CO by bubbling with the gas before addition of the P-450 reductase and NADPH. The progress of the reaction was followed spectrophotometrically by monitoring both the appearance of the Fe(II)mesoverdoheme-CO complex at λmax 616 nm and the loss of the Soret band of the starting complex. H2O2 (one equivalent) was added to a cuvette containing, in a final 1-ml volume, one of the meso-formylmesoheme-hHO-1 complexes (pH 7.4) (α-CHO, 0.27 mg, 9.1 nmol; β-CHO, 0.46 mg, 15.2 nmol; γ-CHO, 0.23 mg, 7.8 nmol; δ-CHO, 0.33 mg, 11.11 nmol) in 100 mmpotassium phosphate buffer. The progress of the reaction was monitored spectrophotometrically by the appearance of the Fe(III)mesoverdoheme absorption at 600–700 nm and the decrease in the intensity of the Soret absorbance of the starting complex. P-450 reductase (900 μg, 13.4 nmol) and NADPH (83.3 mg, 120 μmol) were added to a solution of the α-meso-formylmesoheme-hHO-1 complex (81.0 mg, 2.7 μmol) in 100 mm potassium phosphate buffer (pH 7.4). The final incubation volume was 40 ml. The reaction was allowed to stand at 25 °C for 60 min and was then extracted and analyzed as described below. The products formed in the reactions of the other threemeso-formyl isomers were similarly obtained in smaller scale to determine their absorption spectra and HPLC properties. To the α-meso-formylmesoheme-hHO-1 reaction mixture was added concentrated HCl (1 drop) and acetic acid (1 ml) before the solution was extracted with CH2Cl2 (50 ml), and the organic phase was washed with brine (50 ml). The concentrate was dissolved in 100% Solvent A and analyzed by HPLC at a flow rate of 1.0 ml·min−1. The UV-visible absorption spectrum was acquired, and the mesobiliverdin was dissolved in 50 ml of 5% H2SO4 in MeOH (v/v) and left at room temperature for 8 h. The mesobiliverdin dimethyl ester was extracted into the organic phase after adding CHCl3 (50 ml) and water (50 ml), and the organic phase was washed with water (100 ml) and concentrated under vacuum. The electron impact mass spectrum of the α-meso-formylmesobiliverdin dimethyl ester was then determined. To a 1-ml solution of the α-meso-formylmesoheme-hHO-1 complex (1.6 mg, 54.0 nmol) was added P-450 reductase (0.24 mg, 3.1 nmol) and NADPH (0.42 mg, 500 nmol). The tube was immediately sealed with a rubber septum. After 15 min at 25 °C, the solution had turned green. By injection with a syringe through a septum, 525 μl of ferrous deoxymyoglobin (0.45 mg, 27 nmol) was added to the α-meso-formylmesoheme-hHO-1 reaction mixture. The solution was shaken, the septum was removed, and the UV-visible spectrum was recorded. The ferrous deoxymyoglobin used to detect CO was prepared by adding sodium dithionite (5 mg) to 2 ml of a solution of horse skeletal muscle myoglobin (1.7 mg, 100 nmol) in 100 mm potassium phosphate buffer (pH 7.4). CO assays were similarly performed by sealing the tubes after adding cytochrome P-450 reductase (6.0 μg, 78 pmol) and NADPH (167 μg, 200 nmol) to 1-ml solutions (final volume) of the othermeso-formylmesoheme-hHO-1 complexes (β-CHO, 0.27 mg, 9.1 nmol; γ-CHO, 0.21 mg, 7.0 nmol; δ-CHO, 0.26 mg, 8.6 nmol). After 5 min at 25 °C, the reactions had turned green, and freshly prepared ferrous deoxymyoglobin (88 μg, 5 nmol) was injected through a septum. The solutions were mixed, the septa were removed, and the UV-visible spectra were recorded. In separate experiments, quantitative CO assays were repeated with 0.5 ml of a solution of the α-meso-formylmesoheme-hHO-1 complex (α-CHO = 1.1 mg, 35 nmol) to which were added cytochrome P-450 reductase (0.24 mg, 3.1 nmol) and NADPH (0.42 mg, 500 nmol). The sealed reaction tubes were allowed to stand for 15 min at 25 °C. Freshly prepared ferrous deoxymyoglobin (0.73 mg, 32 nmol) was then injected via syringe, the solutions were mixed, the septa were removed, and the UV-visible spectra were recorded. The four meso-formylmesoporphyrin IX dimethyl ester regioisomers were synthesized by pyridinium chlorochromate oxidation of the corresponding regioisomericmeso-hydroxymethylmesoporphyrin IX dimethyl esters (Fig. 2). Themeso-hydroxymethylmesoporphyrin IX dimethyl esters were obtained by Vilsmeier formylation of the dimethyl ester of copper mesoporphyrin followed by reduction of the formyl to the hydroxymethyl group, as previously reported (14Torpey J. Ortiz de Montellano P.R. J. Org. Chem. 1995; 60: 2195-2199Crossref Scopus (14) Google Scholar). Reduction of the formyl to the hydroxymethyl group was required to separate the four regioisomeric products because the alcohols are readily separated by chromatography but the formyl precursors are not. The position of the substitution in each of the meso-hydroxymethyl isomers was established by NMR in the earlier studies (14Torpey J. Ortiz de Montellano P.R. J. Org. Chem. 1995; 60: 2195-2199Crossref Scopus (14) Google Scholar). Reoxidation of the separated hydroxymethyl isomers provides the individual, regiochemically defined, meso-formyl isomers. The synthesis of the requisitemeso-formylmesoheme regioisomers is completed by ester hydrolysis and insertion of the iron atom (14Torpey J. Ortiz de Montellano P.R. J. Org. Chem. 1995; 60: 2195-2199Crossref Scopus (14) Google Scholar). Incubation of the α-, β-, γ-, and δ-meso-formylmesoheme-hHO-1 complexes with NADPH and P-450 reductase under an atmosphere of O2 and CO causes a decrease in the intensity of the Soret band and the accumulation of an intermediate with an absorption maximum at λmax = 616 nm (Fig. 3). The absorption spectrum of the intermediate resembles that of the Fe(II)mesoverdoheme-CO complex formed under similar conditions in incubations of Fe(III)mesoheme with hHO-1 (18Yoshida T. Noguchi M. Kikuchi G. Sano S. J. Biochem. ( Tokyo ). 1981; 90: 125-131Crossref PubMed Scopus (52) Google Scholar, 19Lagarias J.C. Biochim. Biophys. Acta. 1982; 717: 12-19Crossref PubMed Scopus (45) Google Scholar). The CO arrests the reaction at the spectroscopically convenient mesoverdoheme stage by chelating to the iron and thus preventing conversion of the mesoverdoheme to the final mesobiliverdin product. The Soret band changes, involving a broadening and decrease in the absorbance maximum with a small shift to lower wavelengths (from 400 to 396 nm), are similar in the reactions of the α- (Fig. 3 A), β- (Fig. 3 B), and δ-meso-formyl (Fig. 3 D) isomers. However, for the γ-formyl isomer, the decrease in the Soret band intensity is associated with a marked red shift from 396 to 424 nm (Fig. 3 C). The reason for the different spectroscopic change observed with the γ-isomer is unclear but may be due to an interaction of the γ-formyl group with the flanking propionic acid side chains. Earlier studies have shown that H2O2 can substitute for O2 and NADPH-P-450 reductase in the hHO-1-catalyzed oxidation of heme to verdoheme but not in the subsequent conversion of verdoheme to biliverdin (6Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar). The H2O2-dependent reaction can therefore be monitored without adding CO because the reaction is automatically arrested at the Fe(III)mesoverdoheme stage. As shown in Fig. 4, one equivalent of H2O2 similarly supports conversion of themeso-formylmesohemes to mesoverdohemes. For all fourmeso-formyl regioisomers, the reaction with H2O2 brings about a decay in the intensity of the Soret maximum at 400 nm without changing its position. This change is associated with the appearance of a broad absorbance with a maximum at 642 nm. These changes are similar to those reported for the H2O2-dependent oxidation of heme by hHO-1 (6Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 22357-22362Abstract Full Text PDF PubMed Google Scholar). The regiospecificity of the oxidation of α-meso-formylmesoheme by hHO-1 was examined with the NADPH-P-450 reductase system. For these studies, the incubations were carried out in the absence of exogenous CO, and the final biliverdin product was isolated and characterized by HPLC, absorption spectroscopy, and mass spectrometry. Oxidation of the α-meso-formylmesoheme by NADPH-P-450 reductase followed by HPLC of the product yields a single broad mesobiliverdin-like peak (Fig. 5). A single product peak with a similar retention time is also obtained when each of the othermeso-formylmesoheme isomers is oxidized by heme oxygenase. However, the HPLC system readily separates the four biliverdin regioisomers derived from heme and the methyl-substituted regioisomers from the unsubstituted mesobiliverdins, but it only partially separates the methyl-substituted mesobiliverdin regioisomers from each other (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Because its ability to resolve the meso-formylmesoheme regioisomers has not been independently established, it cannot be inferred from the observation of a single peak in the chromatograms that a single isomer is formed. The HPLC retention times do show, however, that the products are not unsubstituted biliverdins and therefore that the biliverdin products retain the formyl group. The absorption and mass spectra of the product obtained from α-meso-formylmesoheme confirm that it is a mesobiliverdin that retains the α-meso-formyl group. The maxima in the absorption spectrum (λmax 364, 640 nm) are slightly shifted with respect to those of authentic mesobiliverdin IXα (λmax 366, 636) (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the product expected if the α-meso-carbon and the attached formyl group were eliminated in the reaction. The mass spectrum does not give a molecular ion peak but yields fragments that (a) confirm that the product retains the formyl function and (b) tentatively identify the axis along which the molecule fragments. Previous mass spectrometric studies have demonstrated that biliverdins fragment at the meso-carbon directly opposite the site of the original oxidative cleavage when subjected to electron impact ionization (20Bonnett, R., and McDonagh, A. F. (1973) J. Chem. Soc. Perkin Trans. I 881–888.Google Scholar). The two fragments that are formed allow one to distinguish between, for example, biliverdin IXα and biliverdin IXβ but not biliverdin IXα and biliverdin IXγ. When the HPLC-purified mesobiliverdin dimethyl ester obtained from reaction of the α-meso-formylmesoheme-hHO-1 complex with NADPH-P-450 reductase was subjected to electron impact ionization, prominent fragments appeared in the mass spectrum at m/z273 and 370 (Fig. 6). The sum of the two fragments is 643, a value close to that of the mass of the parent mesoformylmesoheme (642), in agreement with the earlier finding that the sum of the fragments gives a value that differs by 1–3 mass units from that of the parent molecule (20Bonnett, R., and McDonagh, A. F. (1973) J. Chem. Soc. Perkin Trans. I 881–888.Google Scholar). Furthermore, the ions are most consistent with fragmentation of the molecule along the β-δ axis to give structures similar to those shown in Fig. 7, although the fragment ion at 370 requires a dehydrogenation of the structure shown in the figure. Dehydrogenation reactions that occur in the ion source have been observed in related molecules (24Lightner D.A. Moscowitz A. Petryka Z.J. Jones S. Weimer M. Davis E. Beach N.A. Watson C.J. Arch. Biochem. Biophys. 1969; 131: 566-576Crossref PubMed Scopus (9) Google Scholar). Fragmentation along the β-δ axis indicates that the parent porphyrin is oxidized by heme oxygenase either at the β- or δ-meso carbon. Thus, in contrast to the α-meso-methyl analogue (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), α-meso-formylmesoheme is oxidized at one or more non-formyl-substituted meso positions rather than at the normally favored α-meso carbon.Figure 7Proposed structures for the principal ions observed in the electron impact mass spectrum of the mesobiliverdin isolated from oxidation of α-meso-formylmesoheme by hHO-1, P-450 reductase, and NADPH.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The affinity of ferrous deoxymyoglobin for CO has been used to assay the formation of CO in the NADPH-P-450 reductase-dependent oxidation of α-meso-formylmesoheme by hHO-1. The assay is based on the shift of the Soret band of ferrous deoxymyoglobin at 434 nm to that of the CO-complex at 422 nm. Addition of deoxymyoglobin to the incubation mixture after completion of the reaction in a sealed tube resulted in immediate formation of the ferrous myoglobin-CO complex. For technical reasons, a substoichiometric amount (0.90 equivalents) of deoxymyoglobin was added in these studies, and all of the deoxymyoglobin was converted to the ferrous-CO complex. The same results are obtained with unsubstituted mesoheme, the control substrate. In view of our earlier demonstration that oxidation of the substituted position in meso-methylmesohemes does not produce CO (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the extent of CO formation observed must be due to the nearly quantitative oxidation of unsubstituted positions in the α-meso-formylmesoheme. α-meso-Hydroxylation, the first step in the hHO-1-catalyzed oxidation of heme (Fig. 1), does not proceed normally if a substituent is attached to the α-meso carbon. Earlier studies demonstrated that α-meso-methylmesoheme, despite the α-meso-substituent, is enzymatically oxidized at the α-meso-carbon to give mesobiliverdin IXα, but the reaction proceeds without the concomitant formation of CO (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Thus, the α-meso-carbon and the attached methyl group are eliminated by a mechanism that does not converge on the normal α-meso-hydroxymesoheme intermediate. The excised two-carbon fragment is not released as acetaldehyde or acetic acid (21Torpey, J. (1997) Mechanistic Studies of Home Oxygenase.Ph.D. Dissertation, University of California at San Francisco.Google Scholar), but the identity of the fragment remains unknown. The methyl-substituted position is also exclusively (γ) or preferentially (δ) oxidized in enzymatic turnover of the γ- or δ-meso-methylmesoporphyrin isomers, giving rise to the corresponding unsubstituted mesobiliverdins (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). As found for the α-meso-methyl isomer, no CO is detected in the enzymatic oxidation of γ-meso-methylmesoheme (21Torpey, J. (1997) Mechanistic Studies of Home Oxygenase.Ph.D. Dissertation, University of California at San Francisco.Google Scholar). A methyl substituent thus favors oxidation of the methyl-substitutedmeso-carbon, albeit by a mechanism that diverges from that which leads to formation of the normal meso-hydroxy intermediate. The regiochemistries of the hHO-1-catalyzed oxidations of α-meso-formyl- and α-meso-methylmesoheme are diametrically opposed in that the former results exclusively in oxidation of the substituted position and the latter in oxidation of an unsubstituted position. A single biliverdin-like product peak is observed when the α-meso-formylmesoheme reaction product is analyzed by HPLC (Fig. 5). The retention time and the absorption and mass spectra of the product clearly identify it as a formyl-substituted mesobiliverdin. Although a molecular ion is not observed in the mass spectrum (Fig. 6), the two principal peaks observed atm/z 273 and 370 are consistent with fragmentation of a formyl-substituted mesobiliverdin with a molecular mass of 642. Earlier studies showed that biliverdins fragment to give ions such as those observed here (Fig. 7) (20Bonnett, R., and McDonagh, A. F. (1973) J. Chem. Soc. Perkin Trans. I 881–888.Google Scholar). The molecular masses of the fragments are consistent with fragmentation of the parent mesobiliverdin at either the β- or δ-meso position, which implies that the heme oxygenase reaction occurred at one of these two positions rather than at the formyl-substituted α-meso position. Independent evidence for oxidation at the unsubstituted positions is provided by the finding that CO is formed in amounts (>90%) approaching those expected for quantitative oxidation of the unsubstituted rather than the formyl-substituted, mesopositions. The results unambiguously establish that ameso-formyl substituent, in contrast to a methyl substituent, directs the oxidation away from the substituted carbon toward the unsubstituted positions. The opposite effects of the formyl and methyl groups could be due to the differences in their steric or electronic properties. In steric terms, a formyl group is more compact and therefore presents a smaller steric profile than a methyl group. A value for the steric parameter Es is not available for a formyl group, but the value for the slightly more compact cyano group is −0.51. The corresponding value for methyl group is −1.24 (22Hansch C. Leo A. Exploring QSAR. Fundamentals and Applications in Chemistry and Biology. American Chemical Society, Washington, D.C.1995: 1-96Google Scholar). Steric effects therefore cannot be used to rationalize the opposite effects on the reaction regiospecificity of the meso-methyl andmeso-formyl substituents, particularly when the substituent is at the normally favored α-meso carbon. However, the changes in regiospecificity are readily rationalized by the differences in the electronic effects of the two substituents. A methyl is electron donating (Hammett ςp = −0.17), whereas a formyl substituent is electron withdrawing (Hammett ςp = +0.42) (22Hansch C. Leo A. Exploring QSAR. Fundamentals and Applications in Chemistry and Biology. American Chemical Society, Washington, D.C.1995: 1-96Google Scholar). The opposite effects of meso-formyl andmeso-methyl substituents on the heme oxygenase reaction regiochemistry indicate that electron donating substituents promote oxidation of the substituted carbon, whereas electron-withdrawing substituents disfavor it. A corollary of this inference is that the regioselectivity of the heme oxygenase reaction is primarily determined by electronic rather than steric features of the active site. A second corollary is that the reaction involves electrophilic attack at themeso carbon, because electron donation facilitates the reaction, whereas electron withdrawal impedes it. Electrophilic attack is consistent with the finding that the ethylhydroperoxide-dependent oxidation of heme by hHO-1 produces α-meso-ethoxyheme (16Wilks A. Torpey J. Ortiz de Montellano P.R. J. Biol. Chem. 1994; 269: 29553-29556Abstract Full Text PDF PubMed Google Scholar). Furthermore, the1H NMR spectrum of the hHO-1-heme complex suggests that the distribution of electron density in the highest occupied porphyrin molecular orbital is similar to that seen when the heme bears an electron donating or withdrawing meso-substituent (23Hernández G. Wilks A. Paolesse R. Smith K.M. Ortiz de Montellano P.R. La Mar G.N. Biochemistry. 1994; 33: 6631-6641Crossref PubMed Scopus (62) Google Scholar). Although the normal heme substrate does not have such a substituent, the results suggest that the protein favors α-meso-hydroxylation by causing an analogous asymmetry in the heme electronic structure. A comparison of the enzymatic oxidation of the meso-methyl and meso-formyl hemes reveals two additional significant differences. The oxidation of the β-meso-methylmesoheme regioisomer was unusual in that it was particularly slow and provided low to negligible yields of mesobiliverdin-like products (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In contrast, β-meso-formylmesoheme is oxidized just as readily as the other meso-formyl isomers and gives similar amounts of mesoverdoheme-like products (Fig. 5). This difference is presumably due to the fact that oxidation of the β-meso-formyl substrate disfavors the β-meso-carbon, whereas the β-meso-carbon is favored in the case of the β-meso-methyl substrate. The β-formyl result clearly shows that a substituent at the β position does not cause a general perturbation that abrogates the normal catalytic mechanism. This conclusion strengthens our earlier suggestion (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) that a specific steric interaction prevents oxidation of the β-meso position, an interaction that does not come into play in the case of the β-meso-formyl heme because oxidation occurs at other positions. The second anomaly is provided by the finding that the H2O2-dependent heme oxygenase reaction proceeds normally with the α-meso-formyl substrate, whereas no reaction was observed earlier with the α-meso-methyl substrate (15Torpey J.W. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 26067-26073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). This difference is again rationalized by the fact that an α-meso-formyl substituent directs the oxidation to the β- or δ-unsubstituted mesopositions, whereas an α-meso-methyl favors oxidation of the α-meso carbon. Thus, a specific interaction that blocks the H2O2-dependent oxidation of the α-meso-substituted carbon would not interfere in the case of the α-meso-formyl substrate, which channels the oxidation to the other meso positions. The key finding of this study is that meso-methyl andmeso-formyl substituents have opposite effects on the regiospecificity of the heme oxygenase reaction, the former favoring oxidation of the substituted position and the latter oxidation of an unsubstituted position. This finding leads to two conclusions: (a) that the regiochemistry of the reaction is primarily under electronic rather than steric control and (b) that oxidation involves electrophilic addition to the heme. The normal α-meso regiospecificity of the heme oxygenase reaction thus appears to be enforced by interactions of active site residues with the heme that selectively enrich the frontier orbital electron density at the α-meso carbon. We thank Weiping Jia for obtaining the mass spectra of the mesobiliverdin products and Bettie Sue Siler Masters for the cytochrome P-450 reductase." @default.
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