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• W2168427863 abstract "HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 μm-1 s-1 and 0.22 s-1, respectively, yielding an O2 affinity of 21 μm-1, which is ∼20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3–4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O2 binding. Resonance Raman spectra show that the Fe–O2 vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe–O–O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe–O–O angle is 110°, the O–O bond is pointed toward the heme α-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O2 and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O–O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin α-meso-carbon by preventing premature O–O bond cleavage. HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 μm-1 s-1 and 0.22 s-1, respectively, yielding an O2 affinity of 21 μm-1, which is ∼20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3–4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O2 binding. Resonance Raman spectra show that the Fe–O2 vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe–O–O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe–O–O angle is 110°, the O–O bond is pointed toward the heme α-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O2 and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O–O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin α-meso-carbon by preventing premature O–O bond cleavage. Heme oxygenase, HO, 1The abbreviations used are: HO, heme oxygenase; Mb, myoglobin; Hb, hemoglobin; Wat, water molecule. catalyzes regioselective oxidative conversion of iron protoporphyrin IX (heme) to biliverdin IX, iron, and CO (1Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2218) Google Scholar). HO is not a hemeprotein by itself but utilizes heme as both a prosthetic group and a substrate. In mammalian systems, where electrons are supplied by NADPH through NADPH-cytochrome P450 reductase (2Schacter 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), HO is the enzyme responsible for excess heme degradation (1Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2218) Google Scholar) and iron recycling (3Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10919-10924Crossref PubMed Scopus (870) Google Scholar). The product CO has been implicated as a messenger molecule in various physiological functions (4Dioum E.J. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.-A. McKnight S.L. Science. 2002; 298: 2385-2387Crossref PubMed Scopus (381) Google Scholar, 5Suematsu M. Ishimura Y. Hepatology. 2000; 31: 3-6Crossref PubMed Scopus (119) Google Scholar). Although most of the structural and functional studies have been conducted on the soluble, truncated form of isoform-1 of mammalian heme oxygenase, HO-1 (6Ortiz de Montellano P.R. Curr. Opin. Chem. Biol. 2000; 4: 221-227Crossref PubMed Scopus (210) Google Scholar, 7Ikeda-Saito M. Fujii H. Tesler J. Paramagnetic Resonance of Metallobiomolecules. 858. American Chemical Society, Washington, D. C.2003: 97-112Google Scholar, 8Colas C. Ortiz de Montellano P.R. Chem. Rev. 2003; 103: 2305-2332Crossref PubMed Scopus (122) Google Scholar), the enzyme is also present in some pathogenic bacteria where it is essential for heme-based iron acquisition from a host lacking in free extracellular iron (9Wandersman C. Stojilijkovic I. Curr. Opin. Microbiol. 2000; 3: 215-220Crossref PubMed Scopus (254) Google Scholar, 10Genco C. Dixon D.W. Mol. Microbiol. 2000; 39: 1-11Crossref Scopus (188) Google Scholar, 11Schmitt M.P. J. Bacteriol. 1997; 179: 838-845Crossref PubMed Google Scholar). Two HO proteins from pathogenic bacteria have been characterized in some detail, namely HmuO from Corynebacterium diphtheriae and HemO from Neisseria meningitidis (12Chu G.C. Katakura K. Zhang X. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 21319-21325Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 13Chu G.C. Tomita T. Sonnichsen F.D. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 24490-24496Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Hirotsu S. Chu G.C. Unno M. Lee D.-S. Yoshida T. Park S.-Y. Shiro Y. Ikeda-Saito M. J. Biol. Chem. 2004; 279: 11937-11947Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 15Zhu W. Wilks A. Stijilijkovic I. J. Bacteriol. 2000; 182: 6783-6790Crossref PubMed Scopus (168) Google Scholar, 16Schuller D.J. Zhu W. Stijilijkovic I. Wilks A. Poulos T.L. Biochemistry. 2001; 40: 11552-11558Crossref PubMed Scopus (128) Google Scholar). In comparison with the mammalian HO, neither of them is membrane-bound. Instead, they are soluble and have smaller molecular masses, i.e. 24 kDa for HmuO and 26 kDa for HemO. HmuO has 33% sequence identity to the first 221 amino acids of human HO-1. Despite their differences in size, the two bacterial HO proteins have overall protein folds, heme environments, and catalytic mechanisms very similar to those for mammalian HO-1 (12Chu G.C. Katakura K. Zhang X. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 21319-21325Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 13Chu G.C. Tomita T. Sonnichsen F.D. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 24490-24496Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Hirotsu S. Chu G.C. Unno M. Lee D.-S. Yoshida T. Park S.-Y. Shiro Y. Ikeda-Saito M. J. Biol. Chem. 2004; 279: 11937-11947Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 15Zhu W. Wilks A. Stijilijkovic I. J. Bacteriol. 2000; 182: 6783-6790Crossref PubMed Scopus (168) Google Scholar, 16Schuller D.J. Zhu W. Stijilijkovic I. Wilks A. Poulos T.L. Biochemistry. 2001; 40: 11552-11558Crossref PubMed Scopus (128) Google Scholar). In its catalytic cycle (Fig. 1), HO first binds one equivalent of heme to form a ferric heme-HO complex. The first electron donated from the reducing substrate converts the heme iron to the ferrous state. Then O2 binds to reduced 5-coordinate heme to form a meta-stable oxy complex. A one-electron reduction of the oxy form generates a ferric hydroperoxo complex, which self-hydroxylates the α-meso-carbon of the porphyrin ring (6Ortiz de Montellano P.R. Curr. Opin. Chem. Biol. 2000; 4: 221-227Crossref PubMed Scopus (210) Google Scholar, 17Davydov R.M. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10656-10657Crossref Scopus (147) Google Scholar). The latter reaction is different from what occurs in P450 enzymes, in which the O–O bond of the hydroperoxo complex is heterolytically cleaved to generate an actively hydroxylating, ferryl (Fe4+=O) intermediate (18Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Scopus (2125) Google Scholar). Ferric α-meso-hydroxyheme is then converted to biliverdin by multiple oxidoreductive steps involving a verdoheme intermediate (6Ortiz de Montellano P.R. Curr. Opin. Chem. Biol. 2000; 4: 221-227Crossref PubMed Scopus (210) Google Scholar, 12Chu G.C. Katakura K. Zhang X. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 21319-21325Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Mansfield Matera K. 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 (97) Google Scholar). The oxy form of the heme complex of heme oxygenase has several unique features relevant to HO catalytic function (20Takahashi S. Ishikawa K. Takeuchi N. Yoshida T. Ikeda-Saito M. Rousseau D.L. J. Am. Chem. Soc. 1995; 117: 6002-6006Crossref Scopus (103) Google Scholar, 21Migita C.T. Mansfield Matera K. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 22Fujii H. Dou Y. Zhou H. Yoshida T. Ikeda-Saito J. Am. Chem. Soc. 1998; 120: 8251-8252Crossref Scopus (29) Google Scholar). O2 binds to the complex with high oxygen affinity due to a very small oxygen dissociation rate constant (21Migita C.T. Mansfield Matera K. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This is one of the reasons why HO catalysis is not severely inhibited by its product, CO, in air-saturated buffer. The cause for the small O2 dissociation rate is assumed to involve hydrogen bonding between the bound ligand and its adjacent protein environment; but, until this report, this assumption has not been confirmed by direct structure determination (21Migita C.T. Mansfield Matera K. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 22Fujii H. Dou Y. Zhou H. Yoshida T. Ikeda-Saito J. Am. Chem. Soc. 1998; 120: 8251-8252Crossref Scopus (29) Google Scholar, 23Sugishima M. Sakamoto H. Kakuta Y. Omata Y. Hayashi S. Noguchi M. Fukuyama K. Biochemistry. 2002; 41: 7293-7300Crossref PubMed Scopus (62) Google Scholar). Analysis of the Fe–O2 vibration modes predicts that the Fe–O–O unit is highly bent by steric interactions between the bound O2 and the residues in the distal pocket (20Takahashi S. Ishikawa K. Takeuchi N. Yoshida T. Ikeda-Saito M. Rousseau D.L. J. Am. Chem. Soc. 1995; 117: 6002-6006Crossref Scopus (103) Google Scholar). These results, which indicated both steric restriction and hydrogen bonding, led to the proposal that the terminal oxygen is “held” in a position adjacent to the α-meso-carbon of the porphyrin ring, facilitating a highly regiospecific oxygenation (14Hirotsu S. Chu G.C. Unno M. Lee D.-S. Yoshida T. Park S.-Y. Shiro Y. Ikeda-Saito M. J. Biol. Chem. 2004; 279: 11937-11947Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Takahashi S. Ishikawa K. Takeuchi N. Yoshida T. Ikeda-Saito M. Rousseau D.L. J. Am. Chem. Soc. 1995; 117: 6002-6006Crossref Scopus (103) Google Scholar, 24Schuller D.J. Wilks A. Ortiz de Montellano P.R. Poulos T.L. Nat. Struct. Biol. 1999; 6: 860-867Crossref PubMed Scopus (312) Google Scholar); but, again, this orientation has not been confirmed directly by x-ray crystallography. A hydrogen bond with an adjacent exchangeable proton from a distal pocket water molecule has also been proposed (13Chu G.C. Tomita T. Sonnichsen F.D. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 24490-24496Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22Fujii H. Dou Y. Zhou H. Yoshida T. Ikeda-Saito J. Am. Chem. Soc. 1998; 120: 8251-8252Crossref Scopus (29) Google Scholar), and this proton is thought to become the hydroperoxo proton after a one-electron reduction of the oxy form (17Davydov R.M. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10656-10657Crossref Scopus (147) Google Scholar, 25Davydov R.M. Kofman V. Fujii H. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 2002; 124: 1798-1808Crossref PubMed Scopus (147) Google Scholar). This hydrogen bonding proposal was based on electron paramagnetic resonance studies with HmuO and HO containing oxy cobalt porphyrin and also requires direct structural confirmation. Until this report, a crystal structure of the oxy form of heme oxygenase had not been reported because of the meta-stable nature of the Fe–O2 intermediate. The ferrous NO complex has been used as a mimic of the oxy form in recent structural studies and has provided useful information about the active site of HO (23Sugishima M. Sakamoto H. Kakuta Y. Omata Y. Hayashi S. Noguchi M. Fukuyama K. Biochemistry. 2002; 41: 7293-7300Crossref PubMed Scopus (62) Google Scholar, 26Lad L. Wang J. Li H. Friedman J. Bhaskar B. Ortiz de Montellano P.R. Poulos T.L. J. Mol. Biol. 2003; 330: 527-538Crossref PubMed Scopus (73) Google Scholar). However, the structure of the oxy complex is needed to more accurately delineate the mode of oxygen binding. The heme oxygenase from C. diphtheriae (HmuO) has been thoroughly characterized structurally and enzymatically (12Chu G.C. Katakura K. Zhang X. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 21319-21325Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 13Chu G.C. Tomita T. Sonnichsen F.D. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 24490-24496Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Hirotsu S. Chu G.C. Unno M. Lee D.-S. Yoshida T. Park S.-Y. Shiro Y. Ikeda-Saito M. J. Biol. Chem. 2004; 279: 11937-11947Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and we have now been able to obtain crystals of the oxy form of this enzyme. Oxygenated crystals were obtained by using strictly anaerobic conditions for reduction of ferric crystals, carefully removing excess reducing agent, exposing the one-electron reduced sample to mother liquor equilibrated with 1 atm of O2 for 10 min, and then flash freezing the final oxygenated samples. These crystals were used to determine the structure of oxy HmuO to 1.85 Å resolution. We have also examined O2 and CO binding to the ferrous heme-HmuO complex and characterized the resonance Raman spectrum of the oxy complex. The crystal structure shows that bound oxygen is tightly confined by two Gly residues, which direct the O–O axis toward the heme α-meso-carbon atom. Bound O2 also forms a hydrogen bond with a solvent-accessible distal pocket water molecule that both slows oxygen dissociation and provides protons for the formation of the hydroperoxo intermediate, which hydroxylates the porphyrin ring. Expression, purification, and reconstitution of the recombinant HmuO with heme were carried out as described previously (12Chu G.C. Katakura K. Zhang X. Yoshida T. Ikeda-Saito M. J. Biol. Chem. 1999; 274: 21319-21325Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Ligand binding reactions were measured using stopped flow and flash photolysis techniques as described previously (27Rohlfs R.J. Mathews A.J. Carver T.E. Olson J.S. Springer B.A. Egeberg K.D. Sligar S.G. J. Biol. Chem. 1990; 265: 3168-3176Abstract Full Text PDF PubMed Google Scholar). The CO form of the heme-HmuO complex was prepared by diluting the ferrous forms of the complex into the anaerobic buffer solutions containing known CO concentrations. The oxygenated form was made as follows. First, the ferric heme-HmuO complex was reduced by sodium dithionite in deoxygenated buffer. Then, the reduced form of the complex was loaded on to a column of Sephadex G-25 and eluted with buffer containing the known oxygen concentrations. Formation of the complex and sample integrity were confirmed by the optical absorption spectra recorded before and after the flash photolysis measurements. All measurements were carried out in 0.1 m phosphate buffer, pH 7, at 20 °C. Resonance Raman spectra of the oxy form were obtained by the use of a continuous flow rapid mixing apparatus as described previously (28Takahashi S. Yeh S.-R. Das T.K. Chan C.-K. Gottfried D.S. Rousseau D.L. Nat. Struct. Biol. 1997; 4: 44-50Crossref PubMed Scopus (216) Google Scholar, 29Couture M. Stuehr D.J. Rousseau D.L. J. Biol. Chem. 2000; 275: 3201-3205Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Continuous laser illumination caused oxidation of the oxy form of the heme-HmuO complex, so only the flow method yielded usable Raman spectra of the oxy form of HmuO. The deoxy form of the heme-HmuO complex was mixed with either 16O2 - or 18O2-saturated buffer (0.1 m phosphate, pH 7, containing 1 mm EDTA) with a flow rate of 1.2 μs/μm. The Raman data were taken at 25 ms after mixing at 4 °C with excitation by the 413.1-nm line of a krypton laser (40 milliwatts). The Raman shifts were calibrated by using indene as standard. Optical absorption spectra of the collected sample were taken after Raman measurements to confirm formation of the O2-bound heme-HmuO. The crystals of the oxy form of the heme-HmuO complex were prepared as follows. The crystals of the ferric heme-HmuO complex were obtained by a hanging drop vapor diffusion method as described previously (14Hirotsu S. Chu G.C. Unno M. Lee D.-S. Yoshida T. Park S.-Y. Shiro Y. Ikeda-Saito M. J. Biol. Chem. 2004; 279: 11937-11947Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The ferric HmuO crystals were transferred into an N2-bubbled reservoir solution containing 50 mm MES, pH 6.1, 2.2 m ammonium sulfate, and 5% (w/v) sucrose. The crystals were brought into a nitrogen glove box, where they were transferred into the reservoir solution containing 50 mm sodium dithionite and 5% sucrose. The crystals were soaked for 10 min to ensure complete conversion to the ferrous form. To remove dithionite and its oxidized products, the ferrous crystals were then transferred into another reservoir solution containing 15% sucrose without dithionite and washed by repeated pumping with a micropipette. The crystals were further transferred into another reservoir solution that contained 20% sucrose without dithionite and washed as before. After washing, the crystals were taken out of the globe box and transferred into the reservoir solution containing 25% sucrose, which had been equilibrated thoroughly with 1 atm of pure O2 prior to soaking the crystals. After soaking for 10 min, the crystals were picked up by nylon loops and flash frozen with liquid N2. Formation of the oxy complex was confirmed by recording light absorption spectra of the crystals by a microspectrophotometer (4DX Systems AB, Uppsala, Sweden) in the visible region. During the absorption measurements, the crystals were kept at 100 K by an Oxford liquid nitrogen flow cryostat. The crystals used for the x-ray diffraction experiments were mostly in the oxy form, with the remainder being the ferric form (∼5 to 10%). X-ray diffraction data sets were collected with a Rigaku R-Axis V imaging plate using 1 Å synchrotron radiation at BL41XU of SPring-8. The temperature around the crystals was maintained at 100 K throughout data collection. The oscillation angle, camera length, and exposure time were 1°, 200 mm, and 5 s, respectively. The incident x-ray intensity was attenuated by use of a 0.6-mm thick aluminum film to reduce radiation damage to the crystals. Data were integrated, merged, and processed with HKL-2000 (30Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Diffraction data with 100% completeness was obtained from 180 frames. The crystals belonged to the space group P21 with unit-cell parameters of a = 54.0 Å, b = 63.0 Å, c = 107.5 Å, and β = 101° and contained three HmuO molecules per one asymmetric unit. Diffraction statistics are summarized in Table I.Table IStatistics of data collection and structure refinementData collectionResolution range (Å)50.0-1.85 (1.92-1.85)Total no. of reflections231,185No. of unique reflections60,627Mosaicity (°)0.18I/σ22 (4.1)Completeness (%)100 (100)Rmergea%Rmerge = Σ|I - 〈I〉|/ΣI0.071 (0.343)Structure refinementResolution range (Å)40-1.85No. of non-hydrogen protein atoms5,686No. of water molecules449R-factorb%R = Σ|Fobs| - |Fcalc|/Σ|Fobs|. The Rfree is the R calculated on the 10% reflections excluded for refinement0.153Rfreeb%R = Σ|Fobs| - |Fcalc|/Σ|Fobs|. The Rfree is the R calculated on the 10% reflections excluded for refinement0.192Mean B-values (Å2)All atoms13.85Proteins11.98Hemes10.44Waters28.67R.m.s. deviations from ideal geometryc%r.m.s. is root mean squareBond lengths (Å)0.020Bond angles (°)1.785a Rmerge = Σ|I - 〈I〉|/ΣIb R = Σ|Fobs| - |Fcalc|/Σ|Fobs|. The Rfree is the R calculated on the 10% reflections excluded for refinementc r.m.s. is root mean square Open table in a new tab Rigid body refinement up to 3 Å was performed by CNS (31Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Reak R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) using the structure of ferrous HmuO (Ref. 14Hirotsu S. Chu G.C. Unno M. Lee D.-S. Yoshida T. Park S.-Y. Shiro Y. Ikeda-Saito M. J. Biol. Chem. 2004; 279: 11937-11947Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar; Protein Data Bank code 1IW1) as a starting model. The phases were extended to 1.85 Å by density modifications, including solvent flattening, non-crystallographic symmetry (NCS) averaging between three molecules, and histogram matching with program DM from the CCP4 suites (32CCP4: Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-767Crossref PubMed Scopus (19797) Google Scholar). The structure was refined to fit to the observed structure factors using simulated annealing (T = 4000 K) for the 40–1.85 Å resolution data by CNS. The crystallographic refinements with simulated annealing and individual B-factor refinement with CNS were performed to calculate an unbiased model. At this stage, the non-crystallographic symmetry restraint was removed because each of the three molecules in the asymmetric unit had slightly different conformations, as was the case for the ferric and ferrous heme-complexes of HmuO (14Hirotsu S. Chu G.C. Unno M. Lee D.-S. Yoshida T. Park S.-Y. Shiro Y. Ikeda-Saito M. J. Biol. Chem. 2004; 279: 11937-11947Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). After several refinement cycles, water molecules were added to the model by CNS and moved and/or removed by manual inspection in the σA-weighted 2Fo - Fc and Fo - Fc maps with the program O (33Jones T.A. Zou J.-Y. Cowan S.E. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 100-119Google Scholar). The model was further refined using the maximum-likelihood target with the program REFMAC5 (34Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (691) Google Scholar). The O2 ligand was modeled into a rugby ball-shaped density next to the iron at the final stages of refinement. After the introduction of alternative conformations for several residues and the translation-liberation-screw refinement (35Schomaker V. Trueblood K.N. Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 1968; 24: 63-76Crossref Google Scholar), the final R and Rfree factors dropped to 0.153 and 0.192, respectively. The final refinement statistics are summarized in Table I. Throughout the model building and refinement process, 10% of the reflections were excluded to monitor the Rfree value. Several residues located in N- and C-terminal regions were not visible in the electron density map, probably due to their disorder. Drawings were made by MOLSCRIPT (36Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), and BOBSCRIPT (37Esnouf R.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 938-940Crossref PubMed Scopus (850) Google Scholar). Association and dissociation rate constants for O2 binding to the ferrous form of the heme-HmuO complex are compared with those of rat HO-1 and wild-type and V68N pig Mb in Table II. Bimolecular rebinding of O2 to the heme-HmuO complex after flash photolysis is monophasic, and the observed rate depends linearly on the O2 concentration. The association rate constant for O2 binding to the heme-HmuO complex, k′O2 = 5 μm-1 s-1, is 3-fold smaller than that of mammalian Mbs (Table II) (38Springer B.A. Sligar S.G. Olson J.S. Phillips Jr., G.N. Chem. Rev. 1994; 94: 699-714Crossref Scopus (728) Google Scholar). However, the O2 dissociation rate constant (kO2) for the oxy heme-HmuO complex is ∼0.22 s-1, which is ∼70 times smaller than that of Mb (∼15 s-1). The oxygen equilibrium constant (KO2) of HmuO is 21 μm-1, which is ∼20 times greater than that of MbTable IIParameters for O2 binding to the HmuO and rat HO-1 heme complexes, pig Mb, and Ascaris Hb in 0.1 m phosphate, pH 7, 20 °C The rate constants for rat HO-1 are taken from Migita et al. (21Migita C.T. Mansfield Matera K. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), those for pig wild-type and V68N Mb are from Krzywda et al. (39Krzywda S. Mushudov G.N. Brzozowski A.M. Jaskowlski M. Scott E.E. Klizas S.A. Gibson Q.H. Olson J.S. Wilkinson A.J. Biochemistry. 1998; 37: 15896-15907Crossref PubMed Scopus (31) Google Scholar), and that for Ascaris Hb are from Gibson and Smith (40Gibson Q.H. Smith M.H. Proc. R. Soc. Lond. B Biol. Sci. 1965; 163: 206-214Crossref PubMed Scopus (66) Google Scholar) and Draghi et al. (41Draghi F. Miele A.E. Travaglini-Allocatelli C. Vallone B. Brunori M. Gibson Q.H. Olson J.S. J. Biol. Chem. 2002; 277: 7509-7519Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Errors for HO-1 were obtained from three different independent experimental determinations (different samples and different days) and indicate that there are no significant differences between the ligand binding parameters for HmuO and HO-1. The errors in the myoglobin rate constants are ∼ ±20%, as has been observed for sperm whale myoglobin (38Springer B.A. Sligar S.G. Olson J.S. Phillips Jr., G.N. Chem. Rev. 1994; 94: 699-714Crossref Scopus (728) Google Scholar).ProteinO2 BindingCO BindingRatio KCO/KO2k′O2kO2KO2k′COkCOKCOμm-1 s-1s-1μm-1μm-1 s-1s-1μm-1HmuO4.70.2221∼0.6a%The time courses for CO rebinding to HmuO and HO-1 were moderately biphasic and could be fitted to a two-exponential expression with roughly equal amplitudes. The fast and slow phase bimolecular rate constants for CO binding were 0.96 μm-1 s-1 and 0.29 μm-1 s-1 for HmuO and 1.3 μm-1 s-1 and 0.64 μm-1 s-1 for HO-1. The values reported in the table were obtained by fitting the time courses to a single exponential expression at several different CO concentration0.0041507.1HO-16.9 ± 1.50.25 ± 0.1028 ± 13∼ 0.9 ± 0.2a%The time courses for CO rebinding to HmuO and HO-1 were moderately biphasic and could be fitted to a two-exponential expression with roughly equal amplitudes. The fast and slow phase bimolecular rate constants for CO binding were 0.96 μm-1 s-1 and 0.29 μm-1 s-1 for HmuO and 1.3 μm-1 s-1 and 0.64 μm-1 s-1 for HO-1. The values reported in the table were obtained by fitting the time courses to a single exponential expression at several different CO concentration0.009100 ± 253.6 ± 1.9Pig Mb17 ± 314 ± 31.2 ± 0.30.78 ± 0.160.019 ± 0.00341 ± 1034 ± 12Pig V68N Mb3.00.674.50.120.011112.4Ascaris Hb1.50.0043700.210.018120.03a The time courses for CO rebinding to HmuO and HO-1 were moderately biphasic and could be fitted to a two-exponential expression with roughly equal amplitudes. The fast and slow phase bimolecular rate constants for CO binding were 0.96 μm-1 s-1 and 0.29 μm-1 s-1 for HmuO and 1.3 μm-1 s-1 and 0.64 μm-1 s-1 for HO-1. The values reported in the table were obtained by fitting the time courses to a single exponential expression at several different CO concentration Open table in a new tab As shown in Table II, the O2 and CO binding parameters of the heme-HmuO complex are very similar to those of the HO-1 complex (21Migita C.T. Mansfield Matera K. Ikeda-Saito M. Olson J.S. Fujii H. Yoshimura T. Zhou H. Yoshida T. J. Biol. Chem. 1998; 273: 945-949Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Both heme oxygenases have O2 association equilibrium constants that are 20–30-fold greater than those for mammalian Mbs, but the HO-1 and HmuO affinities for CO are only 2.5–4-fold greater than those for Mbs. As result, the heme oxygenases show very low M values (KCO/KO2 ≈ 5). The high KO2 and low M values are reminiscent of mutant and naturally occurring heme proteins with multiple hydrogen bonds to bound dioxygen (39Krzywda S. Mushudov G.N. Brzozowski A.M. Jaskowlski M. Scott E.E. Klizas S.A. Gibson Q.H. Olson J.S. Wilkinson A.J. Bioch" @default.
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• W2168427863 date "2004-05-01" @default.
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• W2168427863 title "Crystal Structure of the Dioxygen-bound Heme Oxygenase from Corynebacterium diphtheriae" @default.
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• W2168427863 doi "https://doi.org/10.1074/jbc.m400491200" @default.
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