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• W1987188025 abstract "Heme oxygenase (HO) catalyzes the O2- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568–12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys265 of HRM1 and Cys282 of HRM2, binds heme tightly. Reduction of the disulfide bond increases the Kd for ferric heme from 0.03 to 0.3 μm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the Kd for heme, they do not alter the kcat for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca2+-activated potassium channel. Heme oxygenase (HO) catalyzes the O2- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568–12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys265 of HRM1 and Cys282 of HRM2, binds heme tightly. Reduction of the disulfide bond increases the Kd for ferric heme from 0.03 to 0.3 μm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the Kd for heme, they do not alter the kcat for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca2+-activated potassium channel. Heme oxygenase (HO 2The abbreviations used are: HO, heme oxygenase; HRMs, heme regulatory motifs; GST, glutathione S-transferase; PBS, phosphate-buffered saline; DTNB, 5,5′-dithiobis(nitrobenzoic acid); DTT, dithiothreitol; DSC, differential scanning calorimetry; NBD-Cl, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. ; EC 1.14.99.3) catalyzes the O2- and NADPH-dependent conversion of heme to biliverdin, CO, and iron in a reaction that is coupled to cytochrome P450 reductase (1Maines M.D. Antioxid. Redox Signal. 2004; 6: 797-801Crossref PubMed Scopus (78) Google Scholar, 2Maines M.D. Physiology (Bethesda). 2005; 20: 382-389Crossref PubMed Scopus (111) Google Scholar): heme + 7e– + 3O2 → biliverdin + CO + Fe(II) + 3H2O. Subsequently, biliverdin reductase catalyzes the two-electron reduction of biliverdin to bilirubin. HO is present in organisms from bacteria to eukaryotes and, as the only known enzyme that can degrade heme, plays a critical role in heme and iron homeostasis. Maintaining tight control of heme levels is important because heme is toxic at concentrations >1 μm (3Sassa S. Antioxid. Redox Signal. 2006; 38: 138-155Google Scholar); however, it is the required prosthetic group of many electron transfer proteins and redox enzymes and regulates genes involved in oxygen utilization in lower eukaryotes and prokaryotes (4Qi Z. Hamza I. O'Brian M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13056-13061Crossref PubMed Scopus (145) Google Scholar, 5Ogawa K. Sun J. Taketani S. Nakajima O. Nishitani C. Sassa S. Hayashi N. Yamamoto M. Shibahara S. Fujita H. Igarashi K. EMBO J. 2001; 20: 2835-2843Crossref PubMed Scopus (423) Google Scholar, 6Dioum E.M. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.A. McKnight S.L. Science. 2002; 298: 2385-2387Crossref PubMed Scopus (381) Google Scholar). Regulation of the concentrations of the products of the HO reaction is also important. Iron deficiency is pathological because iron is the essential catalytic center for many heme and non-heme metalloenzymes, yet iron excess is also pathological (7Baker H.M. Anderson B.F. Baker E.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3579-3583Crossref PubMed Scopus (215) Google Scholar). CO is toxic at high levels (>500 ppm), yet HO-derived CO, which is produced locally within tissues, serves a signaling function in various physiological processes, including circadian modulation of heme biosynthesis, regulation of T cell function, and modulation of caveolin-1 status in growth control (8Kim H.P. Ryter S.W. Choi A.M. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 411-449Crossref PubMed Scopus (368) Google Scholar) as well as activation of guanylate cyclase (9Baranano D.E. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10996-11002Crossref PubMed Scopus (267) Google Scholar) and mediation of O2 sensing and the hypoxic response (10Williams S.E. Wootton P. Mason H.S. Bould J. Iles D.E. Riccardi D. Peers C. Kemp P.J. Science. 2004; 306: 2093-2097Crossref PubMed Scopus (406) Google Scholar). There are two forms of HO (HO-1 and HO-2), which share similar physical and kinetic properties, but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. HO-1, considered to be the inducible HO, is also known as the heat-shock protein HSP32 and is found in most tissues, with particularly high levels in the spleen and liver. Expression of HO-1 is regulated by heat shock and various oxidative stress conditions such as ischemia, hypoxia, hyperoxia, and alteration of glutathione levels (11Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2218) Google Scholar, 12Maines M.D. Heme Oxygenase: In Clinical Applications and Functions. 1992; (, CRC Press LLC, Boca Raton, FL)Google Scholar). On the other hand, HO-2 appears to be constitutively expressed, but exhibits a narrow tissue distribution, with high levels in the brain and testes (11Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2218) Google Scholar). HO-2 has been implicated in oxygen sensing by the carotid body (10Williams S.E. Wootton P. Mason H.S. Bould J. Iles D.E. Riccardi D. Peers C. Kemp P.J. Science. 2004; 306: 2093-2097Crossref PubMed Scopus (406) Google Scholar, 13Hoshi T. Lahiri S. Science. 2004; 306: 2050-2051Crossref PubMed Scopus (31) Google Scholar), mediating oxidative stress in neurons (14Regan R.F. Chen J. Benvenisti-Zarom L. BMC Neurosci. 2004; 5: 34Crossref PubMed Scopus (57) Google Scholar), and regulating cerebral blood flow and vascular tone in certain tissues (15Galbraith R. Proc. Soc. Exp. Biol. Med. 1999; 222: 299-305Crossref PubMed Scopus (84) Google Scholar). Human HO-1 and HO-2 exhibit similar catalytic activities and share a high level of homology (55% identity and 76% similarity), including related stretches of 20 hydrophobic residues at their C termini that anchor them to the microsomal membrane (11Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2218) Google Scholar). However, HO-1 and HO-2 diverge significantly around residue 127 and between residues 240 and 295 (HO-2 numbering throughout unless stated otherwise). Although HO-1 typically lacks Cys residues, there are three Cys-Pro sequences in regions that have been proposed to contain heme regulatory (or responsive) motifs (HRMs) centered at Cys127, Cys265, and Cys282. It has been proposed that the HRM per se can bind heme (4Qi Z. Hamza I. O'Brian M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13056-13061Crossref PubMed Scopus (145) Google Scholar, 16Huang T.J. McCoubrey Jr., W.K. Maines M.D. Antioxid. Redox Signal. 2001; 3: 685-696Crossref PubMed Scopus (43) Google Scholar). Interactions between heme and the HRM have also been proposed to control the activity or stability of several regulatory proteins, including the iron-responsive regulator Irr (4Qi Z. Hamza I. O'Brian M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13056-13061Crossref PubMed Scopus (145) Google Scholar, 17Yang J. Ishimori K. O'Brian M.R. J. Biol. Chem. 2005; 280: 7671-7676Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 18Yang J. Panek H.R. O'Brian M.R. Mol. Microbiol. 2006; 60: 209-218Crossref PubMed Scopus (50) Google Scholar), eukaryotic initiation factor-2α kinase (19Uma S. Matts R.L. Guo Y. White S. Chen J.J. Eur. J. Biochem. 2000; 267: 498-506Crossref PubMed Scopus (29) Google Scholar), the mammalian transcriptional repressor Bach1 (5Ogawa K. Sun J. Taketani S. Nakajima O. Nishitani C. Sassa S. Hayashi N. Yamamoto M. Shibahara S. Fujita H. Igarashi K. EMBO J. 2001; 20: 2835-2843Crossref PubMed Scopus (423) Google Scholar, 20Chefalo P.J. Oh J. Rafie-Kolpin M. Kan B. Chen J.J. Eur. J. Biochem. 1998; 258: 820-830Crossref PubMed Scopus (62) Google Scholar), 5-aminolevulinate synthase (21Munakata H. Sun J.Y. Yoshida K. Nakatani T. Honda E. Hayakawa S. Furuyama K. Hayashi N. J. Biochem. (Tokyo). 2004; 136: 233-238Crossref PubMed Scopus (88) Google Scholar), and the yeast transcription factor Hap1 (which mediates the effects of oxygen on transcription) (22Hon T. Hach A. Lee H.C. Cheng T. Zhang L. Biochem. Biophys. Res. Commun. 2000; 273: 584-591Crossref PubMed Scopus (50) Google Scholar, 23Lee H.C. Hon T. Lan C. Zhang L. Mo. Cell. Biol. 2003; 23: 5857-5866Crossref PubMed Scopus (32) Google Scholar). In 5-aminolevulinate synthase, there are three HRMs that reverse the heme-mediated inhibition of mitochondrial import of the synthase (21Munakata H. Sun J.Y. Yoshida K. Nakatani T. Honda E. Hayakawa S. Furuyama K. Hayashi N. J. Biochem. (Tokyo). 2004; 136: 233-238Crossref PubMed Scopus (88) Google Scholar), whereas in the human iron-responsive regulator IRR, heme binding to the HRMs appears to regulate protein degradation (18Yang J. Panek H.R. O'Brian M.R. Mol. Microbiol. 2006; 60: 209-218Crossref PubMed Scopus (50) Google Scholar). In the case of HO-2, it was concluded that one heme binds to each of the two HRMs at the C terminus as well as to the active site, where it undergoes conversion to biliverdin (24McCoubrey Jr., W.K. Huang T.J. Maines M.D. J. Biol. Chem. 1997; 272: 12568-12574Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In the work described here, we have addressed whether or not heme binds to the HRMs in HO-2 and examined the role of the HRMs by comparing the properties of wild-type HO-2 and variants generated by substituting the Cys residues (singly or in combination) with Ala. Our results demonstrate that the HRMs have no effect on the steady-state catalytic activity and stability of HO-2. However, the C-terminal HRMs appear to act as a thiol/disulfide redox switch that regulates the affinity of HO-2 for heme and the spin state of the heme iron. Our results indicate that reduction of the disulfide bond between Cys265 and Cys282 lowers the affinity of HO-2 for heme by 10-fold, thus increasing the dissociation constant from 0.03 μm (near the concentration of free heme found in cells) to 0.35 μm. This increase in the heme Kd would be expected to increase the free heme pool and thus lower CO, iron, and biliverdin levels in the cell. Cloning, Overexpression, and Purification of Human HO-2—The human full-length HO-2 cDNA in a pGEX-4T-2 vector (Amersham Biosciences), which was graciously contributed by Dr. Mahin D. Maines (University of Rochester, School of Medicine), was transformed into Escherichia coli strain BL21. In this construct, the human HO-2 fragment is fused to glutathione S-transferase (GST) and thus can be overexpressed and purified using a glutathione affinity column. BL21 cells carrying the pGEX-4T-2/HO-2 plasmid were cultured in 1 liter of LB medium containing 100 μg/ml ampicillin at 37 °C. When the absorbance at 600 nm reached 0.8–1.0, 60 mg of 5-aminolevulinic acid hydrochloride (Sigma) was added along with 300 μlof1 m isopropyl β-d-thiogalactopyranoside (Gold BioTechnology, St. Louis, MO) to induce the expression of HO-2. The cells were incubated for another 15 h at 25 °C and harvested by centrifugation at 7000 rpm for 10 min at 4 °C in a Beckman J2-HS centrifuge. The cell pellet was resuspended in 5 volumes of 1× phosphate-buffered saline (PBS; 140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, and 1.8 mm KH2PO4, pH 7.4), containing Triton X-100 (0.04% (v/v); Sigma), protease inhibitor (1 tablet/50 ml of extraction solution; Roche Applied Science), lysozyme (0.5 mg/ml; Sigma), and DNase I (5 units/ml; Sigma) at 4 °C. Cells were then lysed by sonication, and the suspension was centrifuged at 17,000 rpm for 1 h at 4°C in the Beckman J2-HS centrifuge. The supernatant was loaded onto a 10-ml glutathione-Sepharose 4B column (Amersham Biosciences), which was washed with 1× PBS containing 0.04% (v/v) Triton X-100. The HO-2/GST fusion protein was eluted with buffer containing 50 mm Tris-HCl, 0.04% (v/v) Triton X-100, and 10 mm reduced glutathione, pH 7.4; and the combined fractions containing the HO-2/GST fusion protein were extensively dialyzed with 1× PBS containing 0.04% (v/v) Triton X-100 to remove glutathione. The GST domain then was cleaved by limited proteolysis using thrombin, and HO-2 was separated from GST by chromatography on a glutathione-Sepharose 4B column. All of these steps were carried out at 4 °C. Site-directed Mutagenesis of HO-2—As described below, the truncated HO-2 variants are more stable and soluble than the full-length protein. A number of variants were generated to determine the functional roles of the HRMs in HO-2. Two kinds of truncated HO-2 were generated: one lacking the C-terminal membrane-binding region (HO-2Δ289–316) the other lacking the two C-terminal HRMs and the membrane-binding region (HO-2Δ265–316). The C265A, C282A, C127A, C265A/C282A, and C127A/C282A variants were generated from HO-2Δ289–316, and C127A/Δ265–316 was from HO-2Δ265–316. All mutations were generated using the QuikChange site-directed mutagenesis protocol (Stratagene, La Jolla, CA). The pGEX-4T-2/HO-2 plasmid was the template for PCRs using primers from Integrated DNA Technologies (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)Google Scholar). All of these variants were purified by the same procedure described above for the wild-type enzyme. HO-2 Assay—The HO-2 enzymatic assay was slightly modified from that described previously (26Ishikawa K. Matera K.M. Zhou H. Fujii H. Sato M. Yoshimura T. Ikeda-Saito M. Yoshida T. J. Biol. Chem. 1998; 273: 4317-4322Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The 1-ml reaction mixture contained 0.1 m potassium phosphate buffer, pH 7.4, 20 μm freshly prepared hemin (froma2mm stock containing 0.1 m NaOH and 5% Me2SO), 0.1 mg/ml bovine serum albumin, 30 μg of NADPH-cytochrome P450 reductase, 60 μg of purified human biliverdin reductase, and 30–100 μg of HO-2. The mixture was incubated at 37 °C, and the reaction was started by the addition of 10 μl of 100 mm NADPH and monitored by following the increase in absorbance at 468 nm using a Beckman DU7400 spectrophotometer. The specific activities of the various wild-type and variant HO-2 proteins were calculated using the difference extinction coefficient between heme and bilirubin at 468 nm (43.5 mm–1 cm–1). P450 reductase was donated by Dr. Bettie Sue Masters (University of Texas Health Sciences Center, Arlington, TX). Biliverdin reductase was purified from E. coli cells containing pGEX-4T-2/BVR. The BVR gene was purchased from American Type Culture Collection (Manassas, VA) and then subcloned into the pGEX-4T-2 plasmid. Quantification of Heme Binding by the Pyridine Hemochrome Assay—HO-2 ·heme complexes were prepared by incubating purified HO-2 variants with a 5-fold molar excess of Fe3+ heme from a stock that was freshly prepared in 5% Me2SO to prevent aggregation. After incubation at 4 °C for 10 min, excess free heme was removed by chromatography on a Sephadex G-15 gel filtration column (Sigma). The amount of heme bound to HO-2 was calculated using a difference extinction coefficient at 556 nm of 28.32 mm–1 cm–1 (27Berry E.A. Trumpower B.L. Anal. Biochem. 1987; 161: 1-15Crossref PubMed Scopus (749) Google Scholar). Determination of Free Thiol Groups by the 5,5′-Dithiobis(nitrobenzoic Acid) (DTNB) Assay—Free thiol quantification of HO-2 was conducted by the DTNB assay basically as described (28Ellman G.L. Arch. Biochem. Biophys. 1958; 74: 443-450Crossref PubMed Scopus (933) Google Scholar, 29Bulaj G. Kortemme T. Goldenberg D.P. Biochemistry. 1998; 37: 8965-8972Crossref PubMed Scopus (325) Google Scholar). After incubation of the 1-ml reaction mixture containing HO-2, 100 mm Tris-HCl, pH 8.0, and 100 μm DTNB at room temperature for 15 min, the absorbance at 412 nm was recorded, and the free thiol concentrations were calculated by reference to a standard curve generated using dithiothreitol (DTT). The DTNB titration was performed in the presence and absence of 8 m urea. When the thiol groups in DTT-reduced HO-2 were measured, the protein was extensively dialyzed in an anaerobic chamber (Vacuum Atmospheres Co., Hawthorne, CA) before the assay. Alkylation of Cysteine Residues and Mass Spectrometric Analysis—One- and two-step alkylation reactions were performed as described previously (30Elliott J.I. Brewer J.M. Arch. Biochem. Biophys. 1978; 190: 351-357Crossref PubMed Scopus (90) Google Scholar). Samples (2 mg/ml) of reduced (with 10 mm DTT) and oxidized (lacking DTT) HO-2 were dissolved in 0.5 ml of 8 m urea, 50 mm Tris-HCl, pH 8.2, and1mm Na-EDTA. The first alkylation step was performed by the addition of 50 mm iodoacetamide (final concentration) and incubation for 30 min at room temperature. The excess iodoacetamide was removed by precipitating the protein by adding a solution containing cold acetone and 1 N HCl/H2O (98:2:10), followed by centrifugation at 5000 rpm for 10 min at 4 °C. After washing the pellet three times with the same solution, the protein was reduced by reacting with 10 mm DTT for 30 min at 37 °C, and the second alkylation step was performed by the addition of 2 μlof9.5 m 4-vinylpyridine and incubation for 20 min at room temperature. The 4-vinylpyridine addition was repeated three times. The unbound 4-vinylpyridine was removed using the acidic acetone solution as described above. After the one- and two-step alkylation reactions, samples were digested with trypsin overnight at room temperature, and the tryptic peptides were isolated and analyzed by nano-liquid chromatography/tandem mass spectrometry using a QSTAR XL mass spectrometer (Applied Biosystems). Peptide isolation was accomplished with a C18 PepMap100 column (75 μm × 15 cm, 3 μm, 100 Å; LC Packings, Sunnyvale, CA) using a linear gradient from 0.3% formic acid in H2O to 0.3% formic acid in acetonitrile at a flow rate of 170 nl/min. The mass spectroscopic results were analyzed using the MASCOT search engine. Spectroscopic and Analytical Methods—Protein concentration was calculated based on the rose bengal method (30Elliott J.I. Brewer J.M. Arch. Biochem. Biophys. 1978; 190: 351-357Crossref PubMed Scopus (90) Google Scholar) using a standard curve generated using known amounts of HO-2, the concentration of which was determined by dry weight. The heme binding affinity of each of the HO-2 variants was determined by adding heme to the reference and sample (containing HO-2) cuvettes and measuring the difference spectrum from 350 to 750 nm in an Olis updated Cary 14 double-beam spectrophotometer. To determine the binding affinity of Fe3+ heme, freshly prepared hemin (from a stock solution of 500 μm hemin in 5% Me2SO) was added in 1–2-μl aliquots to the sample cuvette containing 8 μm HO-2 in 1 ml of 100 mm potassium phosphate buffer, pH 7.4, and to the reference cuvette containing the same buffer. The stock hemin (Fe3+ heme) solution was prepared by dissolving hemin in 0.1 m NaOH and 5% Me2SO and filtration with a 0.2-μm syringe filter (Amicon, Beverly, MA). The heme concentration was calculated using an extinction coefficient at 385 nm of 58.4 mm–1 cm–1 (31Kirschner-Zilber I. Rabizadeh E. Shaklai N. Biochim. Biophys. Acta. 1982; 690: 20-30Crossref PubMed Scopus (70) Google Scholar). To determine the binding affinity of reduced HO-2 for hemin, the as-isolated protein was incubated with a 50-fold molar excess of DTT in the anaerobic chamber for 30 min. DTT was then removed by dialyzing the protein in anaerobic buffer (50 mm Tris-HCl and 50 mm KCl, pH 7.4). Titration was performed as just described for the oxidized protein, except that it was performed under anaerobic conditions in serum-stoppered cuvettes. To determine the binding affinity of HO-2 for ferrous heme, freshly prepared sodium dithionite (2 mm final concentration) was added to a stock solution of 200 μm Fe2+ heme in 20% Me2SO in the anaerobic chamber. Titrations with the reduced protein and Fe2+ heme were performed as described above in serum-stoppered cuvettes to maintain anaerobic conditions. The heme titration data were plotted and fit to a one-binding site model to determine the dissociation constant (Equations 1 and 2), ΔA=ΔɛċEL1 EL=0.5((Eo+Lo+Kd-(Eo+Lo+Kd)2-4Eo.Lo)1/2)2 where ΔA is the absorbance difference between sample and reference cuvettes; Δɛ is the difference extinction coefficient between bound and free heme; and EL is the concentration of the HO-2 ·heme complex, which is calculated using the quadratic Equation 2, in which EO is the total HO-2 concentration, LO is the total heme concentration, and Kd is the dissociation constant. EPR measurements were performed on a Bruker ESP 300E spectrometer recently upgraded to an EMX, operating at ∼9.39 GHz and equipped with an Oxford ITC4 temperature controller, a Hewlett-Packard Model 5340 automatic frequency counter to monitor microwave frequency, and a Bruker gaussmeter to determine the magnetic flux density. All of the EPR samples were prepared in 50 mm phosphate buffer at pH 7.4. Protein Stability Measured by Differential Scanning Calorimetry (DSC)—Heat-induced unfolding was performed in a DSC microcalorimeter (MicroCal, LLC, Northhampton, MA). Purified protein was dialyzed against 1× PBS, pH 7.4, to remove the Triton X-100, and a 0.2 mg/ml protein solution was degassed for 5 min at room temperature before loading into the cells. The scan was performed at a rate of 1 °C/min from 20 to 80 °C. Thermogram analysis was performed using Origin Version 7.0 software supplied with the instrument. The major difference between HO-1 and HO-2 is the lack of cysteine residues in HO-1 and the presence of three Cys-Pro or heme regulatory motifs (HRMs) in HO-2. To determine the function of the HRMs, we compared the biochemical properties of HO-2 variants in which the cysteine residues in the three HRMs were substituted with alanine, individually and in combination (Fig. 1). We refer to the two C-terminal HRMs as HRM1 and HRM2 and the one including residue 127 as HRM3. The comparisons were made among various HO-2 variants lacking the C-terminal membrane-spanning region (HO-2Δ289–316) because the full-length protein is fairly insoluble and undergoes cleavage, whereas the truncated forms of HO-2, like HO-1 (26Ishikawa K. Matera K.M. Zhou H. Fujii H. Sato M. Yoshimura T. Ikeda-Saito M. Yoshida T. J. Biol. Chem. 1998; 273: 4317-4322Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), are soluble and stable. To generate HO-2Δ289– 316, a TAA stop codon was inserted after position 288. All of the truncated HRM variants could be obtained in yields of 15–20 mg/liter of culture. Because the sequence between residues 240 and 295 in HO-2 contributes the major difference between HO-1 and HO-2, HO-2 was further truncated at position 264 to generate HO-2Δ265–316, which lacks both C-terminal HRMs. In addition, Cys127 was substituted with Ala in HO-2Δ265–316 to generate a variant that lacks all three Cys residues (C127A/Δ265–316). Effect of HRMs on HO-2 Stability—Similar amounts of purified HO-2 were recovered from cells expressing wild-type protein or any of the variants. Each of the purified proteins showed predominantly a single band when analyzed by SDS-PAGE, with a purity between 91 and 96% based on densitometric analysis (Fig. 2A). In addition, DSC experiments were carried out to evaluate the influence of HRMs on HO-2 stability. The DSC profiles for the variants overlay that for the wild-type protein and fit a non-two-state model (Fig. 3) with similar melting temperatures in the range of 52.4–53.7 °C (Table 1). These results indicate that neither the HRMs nor the sequence between the HRMs at the C terminus affects protein stability.FIGURE 3DSC experiments of HO-2 and HRM variants. All DSC data were fit to a non-two-state model. Solid lines, experimental data; dotted lines, fit of the DSC data to a non-two-state model. Trace a, HO-2Δ289–316; trace b, C127A; trace c, C265A; trace d, C282A; trace e, C127A/C282A. The fit parameters are shown in Table 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1DSC experimentsProteinΔHTmkcal/mol°CHO-2Δ289-316120.2 ± 0.153.5 ± 0.03C127A104.8 ± 0.652.4 ± 0.04C265A118.0 ± 0.653.7 ± 0.03C282A123.8 ± 0.953.4 ± 0.02C127A/C282A113.6 ± 0.552.7 ± 0.03 Open table in a new tab Effect of HRMs on HO-2 Activity—The steady-state enzymatic activities of the various forms of HO-2 were determined by following biliverdin formation using the assay described under “Experimental Procedures.” In all experiments, 5% Me2SO was present in the heme stock solution (0.05% in the final assay mixture) to prevent heme aggregation. We established that the addition of Me2SO in the assay mixture at concentrations up to 0.2% had no effect on HO-2 activity. As shown in Fig. 2B, both full-length HO-2 and the HO-2Δ289–316 truncation variant, which lacks the membrane-spanning region, exhibited similar activities of ∼5.2 nmol/min/mg. Therefore, because the C-terminal membrane-binding region does not affect enzymatic activity, Cys substitutions were generated in the more soluble and stable HO-2Δ289–316 truncation variant to explore the function(s) of the HRMs in HO-2 (Fig. 2B). To simplify their descriptions, we have omitted the Δ289–316 designation from the variants; thus, substitution of Cys265 with Ala in HO-2Δ289–316 generated a variant that we labeled simply C265A. All of the Cys variants, including C265A/C282A, C127A, C265A, C282A, and C127A/C282A, exhibited similar heme degradation activities in the range of 5.0–5.5 nmol/min/mg. The HO-2Δ265–316 truncation variant, lacking the entire HRM1-HRM2 region, and C127A/Δ265–316, lacking all three Cys residues, had similar enzymatic activities of 5.5 and 5.3 nmol/min/mg, respectively. These results strongly indicate that HRM1, HRM2, and HRM3 do not affect steady-state HO-2 activity. Evaluation of Heme Binding to the HRMs in HO-2—A previous study indicated that rat HO-2 binds three hemes: one heme at the active site (coordinated by His45) plus one each at the two C-terminal HRMs (24McCoubrey Jr., W.K. Huang T.J. Maines M.D. J. Biol. Chem. 1997; 272: 12568-12574Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). However, the UV-visible spectra of the Fe(III), Fe(II), and Fe(II)-CO states of all the HRM variants are identical to those of the wild-type enzyme (Fig. 4, A–C). The spectra show a Soret peak at 406 nm and four long wavelength range peaks at 503, 532, 571, and 630 nm (Fig. 4A). The Fe(II) states exhibit a Soret peak at 430 nm and a long wavelength peak at 557 nm with a shoulder at 527 nm (Fig. 4B), whereas the Fe(II)-CO adducts show a Soret peak at 422 nm and two long wavelength peaks at 541 and 572 nm (Fig. 4C). These spectra, including the extinction coefficients, are very similar to those of the Fe(III), Fe(II), and Fe(II)-CO states of human HO-1 (32Sun J. Wilks A. Ortiz de Montellano P.R. Loehr T.M. Biochemistry. 1993; 32: 14151-14157Crossref PubMed Scopus (108) Google Scholar) and of myoglobin (33Dou Y. Admiraal S. Ikeda-Saito M. Krzywda S. Wilkinson A. Li T. Olson J. Prince R. Pickering I. George G.N. J. Biol. Chem. 1995; 270: 15993-16001Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), which bind only one heme/monomer and do not contain any HRMs, strongly indicating that HO-2 binds only one heme/monomer. To further assess the possibility of heme binding to the HRMs, a 5-fold excess of heme was incubated with the wild-type enzyme and each of the variants; unbound heme was removed by gel filtration chromatography; and the heme content was measured by the pyridine hemochrome assay (Fig. 5). Oxidized HO-2Δ289–316 and C282A at 1.58 and 1.51 μm, respectively, were found to bind ∼1.5 μm ferric heme. The other HO-2 variants also exhibi" @default.
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• W1987188025 title "Evidence That the Heme Regulatory Motifs in Heme Oxygenase-2 Serve as a Thiol/Disulfide Redox Switch Regulating Heme Binding" @default.
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