Heme Oxygenase-2 Is a Hemoprotein and Binds Heme through Heme Regulatory Motifs That Are Not Involved in Heme Catalysis
1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês
10.1074/jbc.272.19.12568
ISSN1083-351X
AutoresWilliam K. McCoubrey, Tony Jun Huang, Mahin D. Maines,
Tópico(s)Cannabis and Cannabinoid Research
ResumoThe heme oxygenase (HO) system degrades heme to biliverdin and CO and releases chelated iron. In the primary sequence of the constitutive form, HO-2, there are three potential heme binding sites: two heme regulatory motifs (HRMs) with the absolutely conserved Cys-Pro pair, and a conserved 24-residue heme catalytic pocket with a histidine residue, His151 in rat HO-2. The visible and pyridine hemochromogen spectra suggest that the Escherichia coli expressed purified HO-2 is a hemoprotein. The absorption spectrum, heme fluorescence quenching, and heme titration analysis of the wild-type protein versus those of purified double cysteine mutant (Cys264/Cys281 → Ala/Ala) suggest a role of the HRMs in heme binding. While the His151 → Ala mutation inactivates HO-2, Cys264 → Ala and Cys281 → Ala mutations individually or together (HO-2 mut) do not decrease HO activity. Also, Pro265 → Ala or Pro282 → Ala mutation does not alter HO-2 activity. Northern blot analysis of ptk cells indicates that HO-2 mRNA is not regulated by heme. The findings, together with other salient features of HO-2 and the ability of heme-protein complexes to generate oxygen radicals, are consistent with HO-2, like five other HRM-containing proteins, having a regulatory function in the cell. The heme oxygenase (HO) system degrades heme to biliverdin and CO and releases chelated iron. In the primary sequence of the constitutive form, HO-2, there are three potential heme binding sites: two heme regulatory motifs (HRMs) with the absolutely conserved Cys-Pro pair, and a conserved 24-residue heme catalytic pocket with a histidine residue, His151 in rat HO-2. The visible and pyridine hemochromogen spectra suggest that the Escherichia coli expressed purified HO-2 is a hemoprotein. The absorption spectrum, heme fluorescence quenching, and heme titration analysis of the wild-type protein versus those of purified double cysteine mutant (Cys264/Cys281 → Ala/Ala) suggest a role of the HRMs in heme binding. While the His151 → Ala mutation inactivates HO-2, Cys264 → Ala and Cys281 → Ala mutations individually or together (HO-2 mut) do not decrease HO activity. Also, Pro265 → Ala or Pro282 → Ala mutation does not alter HO-2 activity. Northern blot analysis of ptk cells indicates that HO-2 mRNA is not regulated by heme. The findings, together with other salient features of HO-2 and the ability of heme-protein complexes to generate oxygen radicals, are consistent with HO-2, like five other HRM-containing proteins, having a regulatory function in the cell. A great deal remains to be learned about the biochemical and physiological functions of heme oxygenase (HO) 1The abbreviations used are: HO, heme oxygenase; HRM, heme regulatory motif. isozymes, HO-1 and HO-2 (1Maines M.D. Trakshel G.M. Kutty R.K. J. Biol. Chem. 1986; 261: 411-419Abstract Full Text PDF PubMed Google Scholar, 2Trakshel G.M. Kutty R.K. Maines M.D. J. Biol. Chem. 1986; 261: 11131-11137Abstract Full Text PDF PubMed Google Scholar), which have been traditionally viewed only in terms of heme catabolism. And some confusion persists about why there are two forms of an enzyme that, by all appearances, have the same catalytic activity and substrate specificity. More puzzling is the high level expression of the second isozyme, HO-2, in tissues and cells that have no role in hemoglobin heme turnover (3Sun Y. Rotenberg M.O. Maines M.D. J. Biol. Chem. 1990; 265: 8212-8217Abstract Full Text PDF PubMed Google Scholar, 4Ewing J.F. Maines M.D. Mol. Cell. Neurosci. 1992; 3: 559-570Crossref PubMed Scopus (189) Google Scholar, 5Ewing J.F. Maines M.D. Endocrinology. 1995; 136: 2294-2302Crossref PubMed Scopus (69) Google Scholar, 6Prabhakar N.R. Dinerman J.L. Agani F.H. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1994-1997Crossref PubMed Scopus (187) Google Scholar). The aim of this study was to learn more about HO-2. HO-1 and HO-2 catalyze the conversion of heme (Fe-protoporphyrin IX) to biliverdin and CO and release Fe in a reaction that utilizes 3 mol each of oxygen and NADPH (7Tenhunen R. Marver H.S. Schmid R. J. Biol. Chem. 1969; 244: 6388-6394Abstract Full Text PDF PubMed Google Scholar). All products of HO activity are suspected to be physiologically active (4Ewing J.F. Maines M.D. Mol. Cell. Neurosci. 1992; 3: 559-570Crossref PubMed Scopus (189) Google Scholar, 5Ewing J.F. Maines M.D. Endocrinology. 1995; 136: 2294-2302Crossref PubMed Scopus (69) Google Scholar, 6Prabhakar N.R. Dinerman J.L. Agani F.H. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1994-1997Crossref PubMed Scopus (187) Google Scholar). The constitutive form, HO-2, and the inducible form, HO-1, are different gene products (8Cruse I. Maines M.D. J. Biol. Chem. 1988; 263: 3348-3353Abstract Full Text PDF PubMed Google Scholar, 9Müller R.M. Taguchi H. Shibahara S. J. Biol. Chem. 1987; 262: 6795-6802Abstract Full Text PDF PubMed Google Scholar, 10McCoubrey Jr., W.K. Maines M.D. Gene ( Amst. ). 1994; 139: 155-161Crossref PubMed Scopus (110) Google Scholar). Except for catalyzing heme and sharing a stretch of amino acids known as the "HO signature" (GenBankTM), they have little resemblance to one another. The "HO signature" is part of a 24-residue domain, which forms the heme catalytic pocket and that, except for one residue, is conserved (11Rotenberg M.O. Maines M.D. Arch. Biochem. Biophys. 1991; 290: 336-344Crossref PubMed Scopus (62) Google Scholar) among all HO-1s and HO-2s characterized to date. The HO signature motif has a conserved histidine residue, His-151 in HO-2, that is essential for its activity (12McCoubrey W.K. Maines M.D. Arch. Biochem. Biophys. 1993; 302: 402-408Crossref PubMed Scopus (41) Google Scholar). In HO-1 the conserved histidine stabilizes a distal water ligand, and based on experimental findings (13Wilks A. Ortiz de Montellano P.R. Sun J. Loehr T.M. Biochemistry. 1996; 35: 930-936Crossref PubMed Scopus (49) Google Scholar) it is placed in a position close to the ligand binding site and plays a role in oxygen binding/activation in the distal heme pocket. At the primary amino acid level, the similarity between rat HO-1 and HO-2 is a mere 43% (14Shibahara S. Müller R. Taguchi H. Yoshida T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7865-7869Crossref PubMed Scopus (437) Google Scholar, 15Rotenberg M.O. Maines M.D. J. Biol. Chem. 1990; 265: 7501-7506Abstract Full Text PDF PubMed Google Scholar). 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Sundaresan V. Rothstein D.M. Brown S.E. Ausubel F.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2524-2528Crossref PubMed Scopus (40) Google Scholar, 23Lee-Huang S. Lin J.J. Kung H.F. Huang P.L. Lee L. Gene ( Amst. ). 1993; 137: 203-210Crossref PubMed Scopus (15) Google Scholar) and is found in the erythroprotein gene (21Benyon J. Cannon M. Buchanan-Wollaston V. Cannon F. Cell. 1983; 34: 665-671Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 22Ow D.W. Sundaresan V. Rothstein D.M. Brown S.E. Ausubel F.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2524-2528Crossref PubMed Scopus (40) Google Scholar). Aside from the overall differences in amino acid composition of HO-1 and HO-2, a major difference is the presence of two cysteines in all HO-2s and the absence of this residue in all HO-1s (11Rotenberg M.O. Maines M.D. Arch. Biochem. Biophys. 1991; 290: 336-344Crossref PubMed Scopus (62) Google Scholar, 14Shibahara S. Müller R. Taguchi H. Yoshida T. Proc. Natl. Acad. Sci. U. S. 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Biochemistry. 1995; 34: 4421-4427Crossref PubMed Scopus (111) Google Scholar). This residue is the axial ligand for the heme prosthetic moiety in various hemoproteins, including all cytochrome P450s and nitric-oxide synthase isozymes (29Haniu M. Yasunobu K.T. Gunsalus I.C. Biochem. Biophys. Res. Commun. 1983; 116: 30-38Crossref PubMed Scopus (3) Google Scholar, 30Kalb V.K. Cooper J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7221-7225Crossref PubMed Scopus (87) Google Scholar, 31Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Abstract Full Text PDF PubMed Google Scholar, 32McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (366) Google Scholar, 33White K.A. Marletta M.A. Biochemistry. 1992; 31: 6627-6631Crossref PubMed Scopus (588) Google Scholar). In HO-2 this residue is flanked downstream by a proline residue followed by phenylalanine (Cys-Pro-Phe); two copies of such arrangements are present in the predicted sequence of the protein. The Cys-Pro dipeptide, often flanked downstream by a hydrophobic residue, phenylalanine, is the absolutely conserved core of the recently identified motif called the heme regulatory element (HRM) (34Zhang K. Guarente L. EMBO J. 1995; 14: 313-320Crossref PubMed Scopus (256) Google Scholar). There is a tendency for a positively charged residue (arginine or lysine) to flank the core upstream. The HO-2 core and the surrounding residues are as follows: Val261-Arg-Lys-Cys-Pro-Phe-Tyr-Ala-Ala-Gln and Gly278-Ser-Asp-Cys-Pro-Phe-Arg-Thr-Ala-Met. In the second (Cys281-Pro) dipeptide, the upstream residues (Gly-Ser-Asp) are polar; polar residues (glycine and serine) also flank two copies of heme lyase HRM (34Zhang K. Guarente L. EMBO J. 1995; 14: 313-320Crossref PubMed Scopus (256) Google Scholar). HO-2 is among only six proteins identified to have this core motif and the characteristic flanking residues (34Zhang K. Guarente L. EMBO J. 1995; 14: 313-320Crossref PubMed Scopus (256) Google Scholar). The others are Saccharomyces cerevisiae heme lyase (35Dumont M.E. Ernst J.F. Hampsey D.M. Sherman F. EMBO J. 1987; 6: 235-247Crossref PubMed Scopus (177) Google Scholar), human erythroid δ aminolevulinate synthase (36Lanthrop J.T. Tinko M.P. Science. 1993; 259: 522-525Crossref PubMed Scopus (251) Google Scholar),Escherichia coli catalase (37Triggs-Raine B.L. Doble B.W. Mulvey M.R. Sorby P.A. Loewen P.C. J. Bacteriol. 1988; 170: 4415-4419Crossref PubMed Google Scholar), rabbit heme-regulated initiator factor α kinase (38Chen J.J. Throop M.S. Gehrke L. Kuo I. Pal J.K. Brodsky M. London U.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7729-7733Crossref PubMed Scopus (174) Google Scholar), and S. cerevisiae HAP1, a transcriptional activator that responds to oxygen/heme (34Zhang K. Guarente L. EMBO J. 1995; 14: 313-320Crossref PubMed Scopus (256) Google Scholar, 39Creusot F. Verediere J. Gaisne M. Slonimski P. J. Mol. Biol. 1988; 204: 263-276Crossref PubMed Scopus (110) Google Scholar, 40Pfeiffer K. Kim K.S. Kogan S. Guarente L. Cell. 1989; 56: 291-301Abstract Full Text PDF PubMed Scopus (252) Google Scholar). The HRMs bind heme and confer regulation by heme to proteins; in fact, only one copy of HRM is adequate for such activity (34Zhang K. Guarente L. EMBO J. 1995; 14: 313-320Crossref PubMed Scopus (256) Google Scholar). Given the fact that in HO-2, two copies of the core HRM are present, we undertook the present study to examine whether, in intact HO-2 protein, the HRMs are involved in heme binding and to investigate their function in heme catalysis. In the course of this investigation we have found that HO-2 is a hemoprotein and provide good evidence to suggest that HRMs are involved in binding of heme but not in heme catalysis. Furthermore, we confirm that the His151 in the 24-residue heme pocket is essential for HO-2 activity. Oligonucleotides for sequencing and mutagenesis were obtained from Midland Certified Reagent Co. (Midland, TX) or Life Technologies, Inc. Two oligonucleotides 10 residues long encompassing the HRMs of HO-2 (Val261-Arg-Lys-Cys-Pro-Phe-Tyr-Ala-Ala-Gln) and (Gly278-Ser-Asn-Cys-Pro-Phe-Arg-Thr-Ala-Met) were synthesized and purified by high pressure liquid chromatography (95%) by Primm Laboratories (Cambridge, MA). These peptides herein are referred to as the Cys264 and Cys281 peptides, respectively. Nitrocellulose for Western blot analysis and Nytran for Northern hybridization were from Schleicher and Schuell (Keene, NH). Sequenase, version 2.0, random primer labeling system, restriction enzymes, and other DNA modification enzymes were purchased from U.S. Biochemical Corp. [α-32P]dCTP and [α-35S] dATP were obtained from U.S. Biochemical Corp. or DuPont NEN. Reagents for protein determination were obtained from Bio-Rad. All other reagents were of the highest quality commercially available. Adult male Harlan Sprague Dawley rats and New Zealand rabbits were obtained from Harlan Industries (Madison, WI). Rat biliverdin reductase was purified essentially by the method described previously (41Huang T.-J. Trakshel G.M. Maines M.D. J. Biol. Chem. 1989; 264: 7844-7849Abstract Full Text PDF PubMed Google Scholar). NADPH-cytochrome P450 reductase was purified as described by Yasokuchi and Masters (42Yasukochi Y. Masters B.S.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar). E. coli Inv α F′ (F′ end A1rec A1 hsd R17 (rk−, mk+) sup E44 thi-1gyrA96 relA1 φ80 lac ZΔM15Δ (lac ZYA-arg F) U169 λ−) carrying HO-2 plasmids were grown to saturation overnight at 37 °C in 2 × YT medium containing 50 μg/ml ampicillin. Cultures were diluted 1:100 in the same medium and grown to an A 600 of ∼1.0. One milliliter was removed from the culture and prepared for SDS-polyacrylamide gel electrophoresis as previously detailed (19McCoubrey Jr., W.K. Ewing J.F. Maines M.D. Arch. Biochem. Biophys. 1992; 295: 13-20Crossref PubMed Scopus (174) Google Scholar). To assay HO-2 expression, 30-μl aliquots of bacterial lysates were examined on a 12.5% polyacrylamide gel. The gel was electroblotted onto nitrocellulose and probed with HO-2 polyclonal antibody as described previously (8Cruse I. Maines M.D. J. Biol. Chem. 1988; 263: 3348-3353Abstract Full Text PDF PubMed Google Scholar). Cell lysates were prepared essentially by the method of Scopes (43Scopes R. Cantor C.R. Protein Purification. Springer-Verlag, Heidelberg, Germany1982: 21-37Crossref Google Scholar) in a buffer containing 20 mmTris-HCl, pH 7.5, 1 mm EDTA, 20% (v/v) glycerol, and 0.4% (v/v) Triton X-100. Total extracted protein in bacterial lysates was quantitated by the method of Bradford (44Bradford M.M. Arch. Biochem. 1976; 72: 248-254Crossref Scopus (222621) Google Scholar) using bovine serum albumin as the standard. HO activity was measured as previously detailed (15Rotenberg M.O. Maines M.D. J. Biol. Chem. 1990; 265: 7501-7506Abstract Full Text PDF PubMed Google Scholar) in the presence of added purified rat liver biliverdin reductase and NADPH-cytochrome P450 reductase. One unit of activity was defined as producing 1 nmol of bilirubin/h. Plasmid DNA from the rat HO-2 expression clone, pRHOP (12McCoubrey W.K. Maines M.D. Arch. Biochem. Biophys. 1993; 302: 402-408Crossref PubMed Scopus (41) Google Scholar, 15Rotenberg M.O. Maines M.D. J. Biol. Chem. 1990; 265: 7501-7506Abstract Full Text PDF PubMed Google Scholar) was utilized as the substrate to carry out site-directed mutagenesis of cysteine residues using the mutagenic primers indicated below as detailed previously (12McCoubrey W.K. Maines M.D. Arch. Biochem. Biophys. 1993; 302: 402-408Crossref PubMed Scopus (41) Google Scholar), and the products were transformed into E. coli XL-1 Blue (recA1, lac−(F′, proAB, lacIqZΔM15, Tn10(tetR))) cells. The HO-2 mutagenesis primers were 5′-AGCATAAAAGGGGGCTTTACGTACATC-3′, complimentary to nucleotides 778–804 (15Rotenberg M.O. Maines M.D. J. Biol. Chem. 1990; 265: 7501-7506Abstract Full Text PDF PubMed Google Scholar) with a GC mismatch (boldface type) for CA to convert Cys264 into alanine; 5′-CCGGAAGGGGGCGTTGCTGCCTCC-3′, complimentary to nucleotides 829–852 also with a GC for CA mismatch to convert Cys281into alanine; and 5′-CGAGTGTAAGCCGCGGCCACCAG-3′, complementary to nucleotides 442–464 with mismatches to convert His151 to alanine. Transformants were screened initially for the loss of the unique NdeI site and the gain of a single NcoI site and were subsequently sequenced by the method of Chen and Seeburg (45Chen E.Y. Seeburg P.H. DNA. 1985; 4: 165-1170Crossref PubMed Scopus (1858) Google Scholar) to identify mutants. DNA from mutant clones and the parental plasmid were separately transformed into InvαF′ cells (Invitrogen, San Diego, CA) which were assayed for immunoreactive protein and heme oxygenase activity as described above. The double mutant Cys264/Cys281 → Ala/Ala, referred to as "HO-2 mut," was generated from the Cys264 → Ala mutant using the Cys281 → Ala mutagenesis primer. The same method was also used to generate Pro265 and Pro282 → Ala mutants using oligonucleotide primers complimentary to nucleotides 778–810 (5′-CTGAGCAGCATAAAATGCGCATTTACGTACATC-3′) and nucleotides 832–850 (5′-GGCTGTCCGGAATGCGCAGTTGCTGGC-3′), respectively (mismatched nucleotides are in boldface type). Both mutagenic primers introduce an FspI site, and transformants were initially screened for the presence of the restriction site prior to sequencing. The wild-type and mutant HO-2 constructs were subjected to an additional round of polymerase chain reaction-mediated mutagenesis. DNA from the carboxyl-terminal substitution mutant, pRHOP (12McCoubrey W.K. Maines M.D. Arch. Biochem. Biophys. 1993; 302: 402-408Crossref PubMed Scopus (41) Google Scholar), and the double Cys → Ala mutant above were utilized as template for the polymerase chain reaction utilizing the primers 5′-CGCAATTAACCCTCACTAAAG-3, which represents the sequence upstream of the polylinker of the vector, pBS+, and 5′-GTCGA CTA ATGATGATGATGATGATG CTTATCGTCATCGTCCTGCAGGCTAGGCTTCCTG-3′, which represents the reverse complement of HO-2 nucleotides 870–888 plus a histidine "tag" of six codons (boldface type) and an enterokinase cleavage site (underlined) to facilitate removal of the tag from the fusion protein. The primer also introduces a stop codon (double underline) and a SalI restriction site (italics) following the histidine tag (5′ to it in the primer). Polymerase chain reaction products were cloned into the vector pCRII (Invitrogen Corporation, San Diego, CA). Transformants were screened for the presence of a 934-base pair SalI fragment representing the tagged HO-2 coding region. The inserts were sequenced to confirm their identity and subcloned into the SalI site of pBS+ (Stratagene, La Jolla, CA), and the resultant plasmids were transformed into Inv α F′. The orientation of the insert was determined by restriction analysis and confirmed by sequencing. A similar construct was generated for HO-1. In this case first strand cDNA was generated from 1 μg of testis poly(A) RNA using the cDNA cycle kit (Invitrogen) priming with oligo(dT) and was used as a template for polymerase chain reaction using the primers 5′-GGGAAGCTTGGAGCGCCCACAGCTCG 3′, representing nucleotides 2–18 of HO-1 (14Shibahara S. Müller R. Taguchi H. Yoshida T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7865-7869Crossref PubMed Scopus (437) Google Scholar) and a HindIII restriction site (italics) and 5′-AAGCTT ATGATGATGATGATGATG CTTATCGTCATCGTCCATGGCATAAATTCCCACTG-3′, which contains the reverse complements of nucleotides 848→867, an enterokinase cleavage site (underlined), histidine tag (boldface type), stop codon (double underline), and HindI III site (italics). The fragment was cloned into the HindIII site of the pBS+. Fusion proteins were purified from overnight bacterial cultures utilizing ProBondTM (Invitrogen) columns in accordance with the manufacturer's instructions. Pooled peak fractions from the ProBondTM column elution were buffer-exchanged into 50 mm Tris-HCl, pH 8.0, containing 10% glycerol, 1 mm CaCl2, and 0.1% Tween 20 and concentrated to approximately 1 mg/ml. When necessary, the histidine tag was removed using enterokinase (EKMaxTM, Invitrogen) utilizing 1 unit/mg protein and digesting for 16 h at 14 °C. Removal of enterokinase was accomplished using soybean trypsin inhibitor-agarose (Sigma) as described by the manufacturer. Eluted proteins were then buffer-exchanged into a buffer appropriate to the final application (20 mm Tris-HCl, pH 7.5, containing 1 mm EDTA and 20% glycerol for storage at −80 °C) using Centricon 10 microconcentrators. Preparations were judged to be >90% homogeneous as assessed by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue. The SDS gel profile of final preparations is shown in Fig. 1. (The lower molecular weight band in HO-2 mut is often observed in SDS gels of HOs and is due to the cleavage of the proteins.) The hemoprotein nature of HO-2 was established by examining spectral properties of the purified protein preparations over the range of 350–650 nm at 2 nm/s. The reference was 0.1 mTris-HCl, pH 7.5, with 0.01% Tween 20, while the test cuvette contained purified protein in the same buffer at the concentrations indicated in the figure legends. Heme was quantitated by a pyridine hemochromogen assay (46Paul K-G. Theorell H. Åkeson Å. Acta Chem. Scand. 1953; 7: 1284-1289Crossref Google Scholar). The change in OD between 557 and 575 nm was used to determine the heme concentration using an extinction coefficient of 32.4 mm−1 cm−1. For heme binding studies, heme was prepared fresh by dissolving in a 1:1 (v/v) mixture of 1 m NH4OH/methanol, and volume was adjusted by the addition of 0.1 m Tris-HCl (pH 7.5) containing 0.01% Tween 20. Heme binding was determined by absolute absorption spectroscopy using buffer solution as the reference. Reconstitution of HO-2 with heme was carried out by incubating purified HO-2 with a 5-fold molar excess concentration of heme. After incubation at 4 °C for 1 h, excess heme was removed by chromatography through a G25 column. 0.5-ml fractions were collected, and heme and protein concentrations were measured in each fraction. The heme binding of the purified wild-type HO-2 and HO-2 mut proteins was also examined by UV fluorescence quenching (47Brill A. Williams R.J. Biochem. J. 1961; 78: 246-251Crossref PubMed Google Scholar, 48Schechter A.N. Epstein C.J. J. Mol. Biol. 1968; 35: 567-589Crossref PubMed Scopus (75) Google Scholar), and for comparison that of rat HO-1 was also examined. The fluorescence of a solution of protein (0.5 μm) in 0.1 mTris-HCl, pH 7.5, was measured using 280 nm as the excitation wavelength and scanning emissions from 300 to 450 nm. The emission spectrum had a maximum at ∼330 nm. Subsequently, incremental additions of heme were made to the cuvette, and the fluorescence was measured following each addition. A full-length (1300-base pair) HO-2 cDNA isolated from a rat testis DNA library (15Rotenberg M.O. Maines M.D. J. Biol. Chem. 1990; 265: 7501-7506Abstract Full Text PDF PubMed Google Scholar) was used as HO-2 hybridization probe. Mouse α-actin and HO-2 cDNA probes were labeled with [32P]dCTP according to the manufacturer's instructions, using the random primer DNA labeling system, and further purified by spin column chromatography. ptk cells were maintained at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium containing 10% bovine calf serum supplemented with 4.1 mml-glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin sulfate. For RNA analysis, cells were grown to 60–70% confluence. The medium was replaced with serum-free medium supplemented with GMS-X (Life Technologies, Inc.) for 2.5 h and then subsequently replaced with fresh serum-free medium or the same medium containing 25 μm heme. After 1 or 4 h, total RNA was prepared from control or heme-treated cells. Poly(A) RNA was isolated by oligo(dT)-cellulose chromatography, fractionated on a 1.2% (w/v) agarose gel, and transferred to Nytran. Prehybridization, hybridization of the appropriate 32P-labeled cDNA, and posthybridization treatment of the blots were performed essentially as described earlier (3Sun Y. Rotenberg M.O. Maines M.D. J. Biol. Chem. 1990; 265: 8212-8217Abstract Full Text PDF PubMed Google Scholar). The involvement of HRMs and HO signature domains of HO-2 in heme catalysis and binding was examined using wild-type and mutant HO-2 proteins. The oligonucleotide primers indicated under "Experimental Procedures" were utilized to substitute alanine for Cys264 and Cys281 of the HRM sequences or His151 in the conserved 24-residue domain in bacterial plasmids, and HO-2 was expressed as a LacZ fusion protein in E. coli. Mutations were confirmed by sequence analysis, and expressed proteins were analyzed for HO activity; data are presented in Fig. 2. As shown in panel a, the bacterial strain expressing the His151 mutant does not have detectable activity, confirming the previous report (12McCoubrey W.K. Maines M.D. Arch. Biochem. Biophys. 1993; 302: 402-408Crossref PubMed Scopus (41) Google Scholar). Neither the mutation of Cys264nor that of Cys281, however, caused a notable effect on activity. Also, substitution of alanine for Pro265 or Pro282 had no discernible effect on enzyme activity, and essentially the same results were obtained as with Cys264→ Ala and Cys281 → Ala mutants (data not shown). To address the possibility that only a single copy of the HRM sequence is required for activity, a construct was generated combining both cysteine mutations. As is shown in the figure, the double mutant also did not display a considerable difference in activity from either of the single mutant or the wild-type constructs. To investigate whether the absence of activity in the His151 mutant was due to decreased expression of the mutated protein as well as whether there was an overexpression of Cys → Ala mutants, Western blot analysis of the same E. coli expression cultures used for activity analysis was carried out using equal amounts of bacterial cell lysate. Fig. 2 b shows that the lack of a detectable activity of His151 → Ala clearly was not due to the absence of the expressed protein (lane 3). Indeed, we consistently observe a higher expression of His151 → Ala mutant than of the wild-type protein (lane 2). Another important observation is that an overexpression of Cys mutants, particularly the Cys double mutant (lane 6) was not the reason for the unaffected heme-degrading activity of the expressed protein. The finding that HRMs are not involved in heme degradation encouraged us to question whether HO-2 is a hemoprotein. The inserts from the wild-type and the Cys264/281 → Ala double mutant plasmids were utilized to generate plasmids expressing the same proteins with a histidine tag (His6), which could be removed by enterokinase digestion, at their carboxyl termini. Wild-type HO-2 and HO-2 mut (double cysteine mutant) were expressed in E. coli and purified. The purified proteins, which were >90% homogenous, as assessed by SDS-polyacrylamide gel electrophoresis (see "Experimental Procedures"), were used to assess heme content and for spectral analysis; results are presented in Fig. 3. The purified wild-type protein has an intense Soret band at 406 nm; upon reduction with dithionite, the maximum shifts to 424 nm, and the peak is at 421 nm for the ferrous CO complex. The absorption in the visible region (500–700 nm) of the ferrous heme (inset a) shows a 632-nm absorption band. The 630-nm band is typical of high spin hemoprotein
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