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• W2059243326 abstract "α-Nitrosyl hemoglobin, α(Fe-NO)2β(Fe)2, which is frequently observed upon reaction of deoxy hemoglobin with limited quantities of NO in vitro as well as in vivo, has been synthetically prepared, and its reaction with O2 has been investigation by EPR and thermodynamic equilibrium measurements. α-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O2 binding at the heme sites of its β-subunits. Its O2 binding is coupled to the structural/functional transition between T- (low affinity extreme) and R- (high affinity) states. This transition is linked to the reversible cleavage of the heme Fe-proximal His bonds in the α(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, α(Fe-NO)2β(Fe)2is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of α-nitrosyl hemoglobin is comparable to that of normal hemoglobin, despite the fact that the oxygenation process involves only two ligation steps. All of these structural and functional evidences have been further confirmed by examining the reactivity of the sulfhydryl group of the Cysβ93 toward 4,4′-dipyridyl disulfide of several α-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O2-carrying capacity, α-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O2 carrier with improved features that could deliver O2 to tissues effectively even after NO is sequestered at the heme sites of the α-subunits. It is concluded that the NO bound to the heme sites of the α-subunits of hemoglobin acts as a negative allosteric effector of Hb and thus might play a role in O2/CO2 transport in the blood under physiological conditions. α-Nitrosyl hemoglobin, α(Fe-NO)2β(Fe)2, which is frequently observed upon reaction of deoxy hemoglobin with limited quantities of NO in vitro as well as in vivo, has been synthetically prepared, and its reaction with O2 has been investigation by EPR and thermodynamic equilibrium measurements. α-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O2 binding at the heme sites of its β-subunits. Its O2 binding is coupled to the structural/functional transition between T- (low affinity extreme) and R- (high affinity) states. This transition is linked to the reversible cleavage of the heme Fe-proximal His bonds in the α(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, α(Fe-NO)2β(Fe)2is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of α-nitrosyl hemoglobin is comparable to that of normal hemoglobin, despite the fact that the oxygenation process involves only two ligation steps. All of these structural and functional evidences have been further confirmed by examining the reactivity of the sulfhydryl group of the Cysβ93 toward 4,4′-dipyridyl disulfide of several α-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O2-carrying capacity, α-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O2 carrier with improved features that could deliver O2 to tissues effectively even after NO is sequestered at the heme sites of the α-subunits. It is concluded that the NO bound to the heme sites of the α-subunits of hemoglobin acts as a negative allosteric effector of Hb and thus might play a role in O2/CO2 transport in the blood under physiological conditions. When deoxy hemoglobin (Hb) 1The abbreviations used are: HbhemoglobinIHPinositol hexaphosphateBPG2,3-biphosphoglyceratepMBp-hydroxymercuribenzoate4-PDS4,4′-dipyridyl disulfideαα-subunitββ-subunits(Fe)deoxy heme(porphyrin)protoporphyrin IX(Ni)nickel protoporphyrin IX(Fe-NO)nitrosyl heme(Fe-O2)oxy heme, and (Fe-CO), carbonmonoxy hemeEPRelectron paramagnetic resonance.1The abbreviations used are: HbhemoglobinIHPinositol hexaphosphateBPG2,3-biphosphoglyceratepMBp-hydroxymercuribenzoate4-PDS4,4′-dipyridyl disulfideαα-subunitββ-subunits(Fe)deoxy heme(porphyrin)protoporphyrin IX(Ni)nickel protoporphyrin IX(Fe-NO)nitrosyl heme(Fe-O2)oxy heme, and (Fe-CO), carbonmonoxy hemeEPRelectron paramagnetic resonance. is exposed to less-than-stoichiometric amounts of NO ([NO]/[heme] ≪ 0.5) in solution (1Taketa F. Antholine W.E. Chean J.Y. J. Biol. Chem. 1978; 253: 5448-5451Abstract Full Text PDF PubMed Google Scholar, 2Huang T.-H. J. Biol. Chem. 1979; 254: 11467-11474Abstract Full Text PDF PubMed Google Scholar, 3Hille R. Olson J.S. Palmer G. J. Biol. Chem. 1979; 254: 12110-12120Abstract Full Text PDF PubMed Google Scholar) and in the erythrocytes (4Kruszyna R. Kruszyna H. Smith R.P. Wilcox D.E. Toxicol. Appl. Pharmacol. 1988; 94: 458-465Crossref PubMed Scopus (23) Google Scholar, 5Eriksson L.E.G. Biochem. Biophys. Res. Commun. 1994; 203: 176-181Crossref PubMed Scopus (15) Google Scholar, 6Tsuneshige A. Yonetani T. Biophys. J. 1998; 74: A81Google Scholar), the predominant species formed upon equilibrium are α-nitrosyl Hbs, i.e.α(Fe-NO)α(Fe)β(Fe)2 or α(Fe-NO)2β(Fe)2. Such compounds were readily identified by their EPR spectra with a set of sharp triplet14N hyperfine structures (Az = 17 Gauss) around gz = 2.009, which is derived from the 5-coordinate nitrosyl hemes in the α-subunits. When rats or mice had been exposed to doses of lipopolyscaccaride, tumor necrosis factor, nitroglycerin, nitrite, or NO (7Oda H. Kusumoto S. Nakajima T. Arch. Environ. Health. 1975; 30: 453-456Crossref PubMed Scopus (67) Google Scholar, 8Wang Q. Jacobs J. DeLeo J. Kruszyna H. Kruszyna R. Smith R. Wilcox D.E. Life Sci. 1991; 49: PL55-PL60Crossref PubMed Scopus (72) Google Scholar, 9Cantilena Jr., L.R. Smith R.P. Frasur S. Kruszyna H. Kruszyna R. Wilcox D.E. J. Lab. Clin. Med. 1992; 120: 902-907PubMed Google Scholar, 10Huot A.E. Kruszyna H. Kruszyna R. Smith R.P. Hacker M.P. Biochem. Biophys. Res. Commun. 1992; 182: 151-157Crossref PubMed Scopus (12) Google Scholar, 11Chamulitret W. Jordan S.J. Nason R.P. Mol. Pharmacol. 1994; 46: 391-397PubMed Google Scholar, 12Kosaka H. Sawai Y. Sakaguchi H. Kumura E. Harada N. Watanabe M. Shiga T. Am. J. Physiol. 1994; 266: C1400-C1405Crossref PubMed Google Scholar), their plasma concentration of NO was known to increase. Venous bloods from the treated animals invariably exhibited EPR spectra with distinct triplet hyperfine signals. Such EPR spectra cannot be expected from tetranitrosyl Hb, α(Fe-NO)2β(Fe-NO)2, in the absence of IHP at a physiological pH of 7.4 (13Rein H. Ristau O. Scheller W. FEBS Lett. 1972; 24: 24-26Crossref PubMed Scopus (95) Google Scholar). Therefore, it is obvious that the primary nitrosyl products formed upon reaction of deoxy Hb with NO under physiological conditions, where [NO] ≪ [heme], are α-nitrosyl Hbs (6Tsuneshige A. Yonetani T. Biophys. J. 1998; 74: A81Google Scholar, 12Kosaka H. Sawai Y. Sakaguchi H. Kumura E. Harada N. Watanabe M. Shiga T. Am. J. Physiol. 1994; 266: C1400-C1405Crossref PubMed Google Scholar, 14Henry Y. Banerjee R. J. Mol. Biol. 1973; 73: 469-482Crossref PubMed Scopus (92) Google Scholar, 15Nagai K. Hori H. Yoshida S. Sakamoto H. Morimoto H. Biochim. Biophys. Acta. 1978; 532: 17-28Crossref PubMed Scopus (62) Google Scholar). In order to assess physiological roles of such compounds, we have investigated its O2binding properties of α(Fe-NO)2β(Fe)2 by EPR and O2 binding measurements as well as the reactivity of Cysβ93 toward 4-PDS as a probe for the quaternary structure. We have found that its oxygenation characteristics and allosteric functions make α(Fe-NO)2β(Fe)2 a unique cooperative low affinity O2 carrier with full allosteric sensitivity that could deliver O2 to tissues efficiently under physiological conditions. This study has also provided a new insight into the molecular mechanism of cooperativity and allostery in Hb, particularly the major role of the α-heme Fe-F helix linkage in the quaternary structural transition, and the mode interaction of Hb with NO. hemoglobin inositol hexaphosphate 2,3-biphosphoglycerate p-hydroxymercuribenzoate 4,4′-dipyridyl disulfide α-subunit β-subunits deoxy heme protoporphyrin IX nickel protoporphyrin IX nitrosyl heme oxy heme, and (Fe-CO), carbonmonoxy heme electron paramagnetic resonance. hemoglobin inositol hexaphosphate 2,3-biphosphoglycerate p-hydroxymercuribenzoate 4,4′-dipyridyl disulfide α-subunit β-subunits deoxy heme protoporphyrin IX nickel protoporphyrin IX nitrosyl heme oxy heme, and (Fe-CO), carbonmonoxy heme electron paramagnetic resonance. 2,3-Biphosphoglycerate, IHP, pMB, 4-PDS, Tris, bis-Tris, bis-Tris propane, dithiothreitol, catalase, superoxide dismutase (Sigma), and argon (grade 5 gas; BOC gases, Murray Hill, NJ) were used without further purification. Nitric oxide (99.00% pure; MG Industries, Malvern, PA) was purified by passing through a series of gas-bubble washing bottles containing 1 m NaOH and deoxygenated distilled water and another bottle containing deoxygenated distilled water. Purified NO gas was used under strict anaerobic conditions. Anaerobic conditions were obtained by removing O2 from the media with repeated evacuation and flushing with water-saturated argon gas or by continuous flushing with water-saturated argon gas over the surface of stirred reaction media. The use of dithionite as reductant was avoided as much as possible to prevent inducing unknown side reactions. All preparation procedures were carried out at 4 °C. Freshly outdated adult human Hb was obtained from a local branch of the American Red Cross; it was purified according to the method of Drabkin (16Drabkin D.L. J. Biol. Chem. 1946; 164: 703-723Abstract Full Text PDF PubMed Google Scholar) and stripped from organic phosphates by the method of Berman et al. (17Berman M. Benesch R. Benesch R.E. Arch. Biochem. Biophys. 1971; 145: 236-239Crossref PubMed Scopus (118) Google Scholar). The Hb solution was stored in the CO form, and no further attempt was made to strip Hb from its minor components. α- and β-chains from Hb in the CO form were separated according to the method of Bucci and Fronticelli (18Bucci E. Fronticelli C. J. Biol. Chem. 1965; 240: PC551-PC552Abstract Full Text PDF PubMed Google Scholar) using the pMB treatment with modifications. After overnight incubation, thepMB-treated Hb solution was passed through a Sephadex G-25 with 20 mm Tris buffer, pH 8.5, and the eluent was loaded onto a Macro-Prep HighQ (Bio-Rad) column equilibrated with the same buffer. Elution of isolated chains was achieved by applying a salt gradient using a ConSep LC 100 liquid chromatography system (Millipore Corp., Bedford, MA). The pMB-treated α-chains were eluted first, followed by unreacted Hb and finally by thepMB-treated β-chains. The α- and β-chains were reconstituted to their native sulfhydryl forms after incubation for 2 h with dithiothreitol in a concentration of 3 mg per ml of chain solution, in the presence of 5 μm of catalase. Subsequently, reconstituted Hb chains were passed through Sephadex G-25 fine in 10 mm bis-Tris buffer, pH 7.4, flushed with CO, concentrated by ultrafiltration through a disc membrane, Omega 10K (Filtron, Clinton, MA) if necessary, and kept on ice for further use. The quality of chains was checked by spectrophotometry, cellulose acetate electrophoresis, and the ability to reconstitute tetrameric Hb. α-Nitrosyl chains were prepared according to Henry and Banerjee (14Henry Y. Banerjee R. J. Mol. Biol. 1973; 73: 469-482Crossref PubMed Scopus (92) Google Scholar) with some modifications. About 3 ml of 3 to ∼4 mm heme of α-subunits were mixed with the same amount of 0.2 mbis-Tris buffer, 0.4 m Cl−, pH 7.4, and were converted to the oxy form by illumination under a stream of pure O2. The α-chains in oxy form were then transferred to a flask sealed with a rubber stopper. Pure argon gas, washed in water, was flushed over the surface of the continuous stirred solution in the flask. After approximately 40 min, about 0.5 ml of a solution of sodium dithionite (3 mg in 1 ml of deoxygenated distilled water) was injected with a syringe through the stopper to assure complete deoxygenation of the sample. Then, NO gas was injected into the flask. Once the reaction was completed (within a few minutes), all NO in excess was removed from the flask by purging thoroughly with argon again. The α-nitrosyl chain solution was then transferred anaerobically to a Sephadex G-25 column in 10 mm bis-Tris propane, pH 7.4, and eluted with deoxygenated buffer. From this point, the nitrosyl derivative can be exposed to air without any immediate decomposition. However, some met Hb formation was observed in samples stored at temperatures above 5 °C for prolonged periods of time. Therefore, it was used as soon as possible. The concentration of α(Fe-NO) was calculated using extinction coefficients of 13.57 and 13.83 mm−1 cm−1 at 572 and 544 nm, respectively, at pH 7.0. β(Fe-O2) chains were obtained from the CO derivative in the same manner as for the α chains, as indicated above. The α(Fe-NO)2β(Fe-O2)2 hybrid was prepared by simply mixing α(Fe-NO) with an equimolar amount of β(Fe-O2). Tetranitrosyl Hb was prepared in the same manner as α(Fe-NO) chains. The integrity of preparations was examined by spectrophotometry, acetate cellulose electrophoresis, and EPR spectroscopy. Partially nitrosylated hybrid tetramers (α(Fe-NO)2β(Fe-O2)2 and α(Fe-O2)2β(Fe-NO)2) were stored at 0 °C for short storage and at liquid nitrogen temperature for long storage. Tetranitrosyl Hb (α(Fe-NO)2β(Fe-NO)2) was stored anaerobically at low temperatures. No detectable alteration of the compounds such as heme oxidation, ligand exchange, loss of ligands, and subunit exchange occurred under such conditions of storage. To determine the optimal temperature for quantitative O2 equilibrium and EPR studies, the rates of aerobic formation of met Hb for α-nitrosyl Hb, tetranitrosyl Hb, and oxy Hb samples were measured. The reaction was followed by the absorbance increase at 630 nm of met Hb over time with a Hewlett-Packard 8452A diode array spectrophotometer (Hewlett-Packard, Palo Alto, CA). To avoid artifacts due to turbidity or water vapor condensation at low temperatures, readings were corrected with a second wavelength at 800 nm. Sample concentration was 60 μm heme in 50 mm bis-Tris-propane containing 0.1 mCl−, pH 7.4. Data were collected every second for 20 min and analyzed according to a first-order kinetic scheme. Oxygen equilibrium curves were measured by an improved version of Imai's automatic method (19Imai K. Yonetani T. Biochim. Biophys. Acta. 1977; 490: 1564-1570Google Scholar) with the following modifications. Absorbance was monitored using a computer-controlled Olis-Cary 118 spectrophotometer (Olis, Bogart, GA). Oxygen concentrations were monitored with a low noise, high response electrode (O2 Sensors, Gadwyne, PA), using a custom-made amplifier (Biomedical Instrumentation Shop, University of Pennsylvania Medical Center, Philadelphia, PA). The signal was then digitized using a 12-bit A/D converter. Absorption changes were monitored at 560 nm. Sample concentration was 120 μm heme in 50 mmbis-Tris-propane buffer, containing 0.1 mCl−, and small amounts of catalase and superoxide dismutase. Measurements were carried out at 15 °C. Analyses of oxygenation data were performed according to a two-step model, corresponding to the third and fourth O2 bindings in Hb, as reported previously (20Tsuneshige A. Zhou Y.-x. Yonetani T. J. Biol. Chem. 1993; 268: 23031-23040Abstract Full Text PDF PubMed Google Scholar). This method (21Grassetti D.R. Murray Jr., J.F. Arch. Biochem. Biophys. 1967; 119: 41-49Crossref PubMed Scopus (868) Google Scholar) was carried out as described previously (22Ampulski R.S. Ayers V.E. Morell S.A. Anal. Biochem. 1969; 32: 163-169Crossref PubMed Scopus (59) Google Scholar, 23Imai K. Hamilton H.B. Miyaji T. Shibata S. Biochemistry. 1972; 11: 114-121Crossref PubMed Scopus (20) Google Scholar), with the following modifications for a quantitative measurement. A standard solution of 4-PDS was prepared by dissolving approximately 10 mg in 10 ml of deoxygenated distilled water at 60 °C. The concentration of 4-PDS was calculated using an extinction coefficient of 16.3 mm−1 cm−1 at 247 nm and pH 7.0. For oxy derivatives, 2 ml of the derivative (40 μm heme) in 50 mm bis-Tris propane buffer, containing 0.1m Cl−, was placed in a quartz cuvette thermostatted at 15 °C and containing a small stirring bar. An amount of 4-PDS equivalent to a final concentration of 160 μm was added to the solution and the reaction was monitored with a Hewlett-Packard 8452A diode array spectrophotometer by measuring the absorbance increase at 324 nm. Data points were collected at a rate of one per s. The dead time of the reaction was 2 s. For deoxy derivatives, a long-neck anaerobic quartz cuvette sealed with a stopper was used. Concentrations of Hb samples and 4-PDS were the same as for oxy derivatives. Deoxygenation was carried out at 4 °C by flushing pure argon into the cuvette. Once the deoxygenation of the sample was confirmed by spectrophotometry, the anaerobic cuvette was then transferred to the temperature-controlled cell holder of the spectrophotometer. As soon as the solutions in the cuvette reached 15 °C, the reaction was initiated by injecting with a gas-tight syringe through the stopper an aliquot of a deoxygenated 4-PDS solution. The dead time in this case was approximately 5 s. Buffers used for EPR measurements were 0.1m sodium acetate buffers, pH 4.8–5.8, and 0.1m bis-Tris propane buffers, pH 6.0-pH 9.0, containing 0.1m Cl−. Oxygen binding equilibria for EPR samples were obtained at 15 °C in a modified Imai cell (19Imai K. Yonetani T. Biochim. Biophys. Acta. 1977; 490: 1564-1570Google Scholar), in which O2 concentrations of samples were continuously monitored by a sensitive O2 electrode (O2Sensors). Aliquots of sample were taken at determined partial pressures of O2 and anaerobically transferred into EPR tubes and immediately frozen by immersion into liquid nitrogen. EPR measurements were carried out with a Varian X-band EPR spectrometer, model E109 (Varian Associates, Palo Alto, CA), integrated with the data-acquisition system (Scientific Software Services, Normal, IL). EPR samples (300 μl of 500 μm heme) in quartz EPR tubes (3-mm precision bore) were frozen by immersion into liquid nitrogen and measured at liquid nitrogen temperature. The spectrometer was operated at a microwave frequency of 9.11GHz, microwave power of 20mW, modulation frequency of 100kHz, modulation amplitude of 2.0 Gauss, magnetic field scan rate of 125 Gauss/min, and time constant of 0.2. Recorded EPR data were manipulated with the EPR software (Scientific Software Services) for quantitative analyses and plotted using Origin for Windows, Version 5.0 (Microcal, Northampton, MA). Native oxy Hb, α(Fe-NO)2β(Fe-O2)2, and tetranitrosyl Hb were slowly oxidized to respective met forms under aerobic conditions. Both nitrosyl Hb derivatives were less stable than native oxy Hb, and tetranitrosyl Hb was the least stable of the species. Half-life times at 37 °C were 15 h, 2 h, and 41 min for oxy Hb, α(Fe-NO)2β(Fe-O2)2, and tetranitrosyl Hb, respectively. However, these values increased to 42 h, 22 h, and 16 h, respectively, at 15 °C. Accordingly, all experiments were conducted at this temperature. Isolated α(Fe-NO) and β(Fe-NO) subunits exhibited EPR spectra around g = 2.0 of the 6-coordinate nitrosyl hemes at pH 7.4, as shown in Fig. 1 A, in agreement with previous reports (14Henry Y. Banerjee R. J. Mol. Biol. 1973; 73: 469-482Crossref PubMed Scopus (92) Google Scholar, 15Nagai K. Hori H. Yoshida S. Sakamoto H. Morimoto H. Biochim. Biophys. Acta. 1978; 532: 17-28Crossref PubMed Scopus (62) Google Scholar, 24Shiga T. Huang K.-J. Tyuma I. Biochemistry. 1969; 8: 378-383Crossref PubMed Scopus (64) Google Scholar). However, the overall line shape of their EPR spectra was distinctly different from one to the other: the isolated α(Fe-NO) subunits (Fig. 1 A,broken line) showed a more rhombically distorted line shape than the isolated β(Fe-NO) subunits (dotted line). These spectra were independent of pH in a range from pH 6.0 to pH 9.0. The EPR spectrum of tetranitrosyl Hb (solid line) was essentially a sum of those of the isolated α(Fe-NO) and β(Fe-NO) subunits, as previously reported (14Henry Y. Banerjee R. J. Mol. Biol. 1973; 73: 469-482Crossref PubMed Scopus (92) Google Scholar, 15Nagai K. Hori H. Yoshida S. Sakamoto H. Morimoto H. Biochim. Biophys. Acta. 1978; 532: 17-28Crossref PubMed Scopus (62) Google Scholar, 24Shiga T. Huang K.-J. Tyuma I. Biochemistry. 1969; 8: 378-383Crossref PubMed Scopus (64) Google Scholar) and did not change significantly over a pH range from 7.0 to 9.0. The EPR spectrum of the α-deoxy, β-nitrosyl hybrid, α(Fe)2β(Fe-NO)2 was reported to be practically identical with that of isolated β(Fe-NO) subunits (spectrum B in Fig. 1 A) (14Henry Y. Banerjee R. J. Mol. Biol. 1973; 73: 469-482Crossref PubMed Scopus (92) Google Scholar, 24Shiga T. Huang K.-J. Tyuma I. Biochemistry. 1969; 8: 378-383Crossref PubMed Scopus (64) Google Scholar) and was pH-independent over a pH range from 6.0 to 9.0. The α-nitrosyl, β-deoxy hybrid, α(Fe-NO)2β(Fe)2, on the other hand, exhibited an EPR spectrum of mixed 5- and 6-coordinate nitrosyl hemes at pH 7.4, as indicated by the appearance of a sharp triplet hyperfine structure (Az = 17 Gauss at gz = 2.009) of the 5-coordinate nitrosyl heme (spectrum B in Fig.1 B). This implied that the α-heme Fe-His (F8) bonds were partially cleaved in α(Fe-NO)2β(Fe)2 under these conditions (15Nagai K. Hori H. Yoshida S. Sakamoto H. Morimoto H. Biochim. Biophys. Acta. 1978; 532: 17-28Crossref PubMed Scopus (62) Google Scholar, 25Wayland B.B. Olson L.W. J. Am. Chem. Soc. 1974; 96: 6037-6041Crossref PubMed Scopus (310) Google Scholar, 26Kon H. Biochim. Biophys Acta. 1975; 379: 103-113Crossref PubMed Scopus (73) Google Scholar, 27Szabo A. Perutz M.F. Biochemistry. 1976; 15: 4427-4428Crossref PubMed Scopus (113) Google Scholar). The EPR spectrum of α(Fe-NO)2β(Fe)2 was pH-dependent. The 6-coordinate state was favored at higher pH, whereas the proportion of the 5-coordinate state increased at acidic pH (Fig. 2, open rectangles). Upon addition of stoichiometric amounts of CO or saturating concentrations of O2, the 5- ↔ 6-coordination equilibrium of the α-nitrosyl hemes of α(Fe-NO)2β(Fe)2 shifted in favor of the 6-coordinate state as a function of pH (Fig. 2, closed rectangles). The α-nitrosyl hemes of α(Fe-NO)2β(Fe-CO)2 (not shown) and α(Fe-NO)2β(Fe-O2)2 (Fig.1 B, spectrum C, and Fig. 2, closed rectangle at pH 9.0) were estimated to be essentially 100% 6-coordinate at pH 9.0. The EPR spectral changes, which were induced by the oxygenation at the β-hemes, were smaller at acidic extremes and most pronounced at around pH 7.4. Inositol hexaphosphate shifted the 5- ↔ 6-coordination equilibrium of the α-nitrosyl hemes of α(Fe-NO)2β(Fe)2 in favor of the 5-coordinate state (Fig. 2). The EPR spectral changes, induced by the oxygenation at the β-hemes in the presence of IHP, were larger at higher pH values. Its EPR spectrum indicated that its α-nitrosyl hemes were essentially 100% 5-coordinate at and below pH 5.0 in the presence of IHP and the absence of O2.(Fig. 1 B, spectrum A, and Fig. 2, open rectangle at pH 4.9). The effect of BPG on the coordination equilibrium of the α-nitrosyl hemes of α(Fe-NO)2β(Fe)2 and α(Fe-NO)2β(Fe-O2)2, as measured by EPR (not shown), were also in favor of the 5-coordinate state, but less pronounced than that observed with IHP (Fig. 2). The degree of saturation with O2 of the β-hemes in α(Fe-NO)2β(Fe)2 was readily controlled by adjusting the concentration of O2 in the medium, which was in turn regulated by the atmospheric partial pressure of O2.(pO2). A series of EPR spectra of α(Fe-NO)2β(Fe)2 at numbers of fixed pO2 values at 15 °C during the course of deoxygenation at pH 7.4 (Fig. 1 C) shows reasonable isosbestic points, indicating that the observed spectral changes were derived from the two-component system of the 5- ↔ 6-coordination equilibrium in the α-nitrosyl hemes. This further indicated that undesirable side reactions, such as formation of met heme, transfer of NO from α- to β-subunits, and release of NO from Hb, were practically negligible during the course of measurements. It should be pointed out that these spectra changes were reversible upon reoxygenation. The 5- ↔ 6-coordination equilibrium of the α-nitrosyl hemes of α(Fe-NO)2β(Fe)2 was shifted in a predictable manner toward the 5-coordinated state upon deoxygenation as a function of pH (Fig. 2). The apparent pH dependence of the midpoints of the EPR spectral transition (Fig. 2, open andclosed circles) is a measure of the Bohr effect of the O2 binding in α(Fe-NO)2β(Fe)2. Its O2 affinity decreases (or its P50 value increases) continuously at lower pHs, even below neutral pH, where the Bohr effect of O2 binding of native Hb is leveled off. However, because EPR data obtained at 77 K may not represent true pH and O2 equilibrium values of α(Fe-NO)2β(Fe)2 measured at 15 °C, caution is warranted in interpreting the EPR data too quantitatively. Results obtained from spectrophotometric O2 equilibrium measurements are shown in Fig. 3. At pH 5.8 (Fig. 3 A), α-nitrosyl Hb showed a strikingly diminished O2 affinity, virtually absent cooperativity, and decreased effect of BPG and IHP. In the absence of organic phosphates, the lower asymptote of the Hill plot for α-nitrosyl Hb matched that for Hb, indicating that despite α-nitrosyl Hb having both the α-subunits ligated with NO, the O2 affinity of the complementary β-subunits remained as low as at initial ligation stages of native Hb. In the presence of IHP, the lower asymptote for the α-nitrosyl Hb was also similar to that of Hb under the same conditions. On the other hand, at pH 8.2 (Fig. 3 C), the upper asymptote of the curve for α-nitrosyl Hb approached that for Hb. This indicates that the affinity for O2 of this derivative increased with pH, while showing a trend of being comparable but not completely equaling the O2 affinity at the last oxygenation steps of Hb under similar conditions. In other words, α-nitrosyl Hb at this pH exhibited characteristics of a high affinity species. Inositol hexaphosphate, as well as BPG, to a lesser degree, had the effect on this derivative of shifting the curve to the right. Cooperativity was present and comparable to that for a Hb species with two binding sites. At pH 7.4 (Fig. 3 B), the oxygenation curve for α-nitrosyl Hb shifted toward the upper asymptote of that for Hb, that is, the high affinity side. However, BPG and IHP decreased its O2 affinity by shifting the curve toward the low affinity side. A comparative view of the effect of organic phosphates on the O2 affinities of Hb and α-nitrosyl Hb at different pH values can be clearly visualized in their Bohr effects (Fig.4, A and B, respectively). In the presence of organic phosphates, α-nitrosyl Hb (Fig. 4 B) showed a greatly enhanced Bohr effect. Around pH 7, the Bohr coefficients, estimated as ΔP50/ΔpH, were −0.5 and −0.9 Bohr protons for Hb and α-nitrosyl Hb, respectively. In the presence of BPG and IHP, the Bohr effect increased in the case of Hb due to the major effect these phosphates had on lowering the O2 affinity on the acidic side, in agreement with previous works (28Imai K. Allosteric Effects on Haemoglobin. Cambridge Press, 1982Google Scholar). Bohr coefficients were −0.7 and −0.8 in presence of BPG and IHP, respectively. However, in the case of α-nitrosyl Hb, organic phosphates reduced the O2-affinity on both acidic and alkaline regions. Bohr coefficients in the presence of BPG or IHP were approximately −0.9 and did not differ from the condition without organic phosphates. The magnitude of the effects of BPG and IHP on lowering the O2 affinity can be expressed as the ratio of partial pressure of O2 at 50% saturation (P50) in the presence and absence of the organic phosphate (i.e.P50+PHOSPHATE/P50NONE). For Hb, the enhanced effect of organic phosphates on the acidic side was reflected as an increased ratio of 1.9 and 5.4 at pH 5.8versus ratios 1.2 and 1.6 at pH 9, for BPG and IHP, respectively. The maximum effects of BPG and IHP occurred between pH 7 and 7.4, and the values were 3.6 and 7.6 for BPG and IHP, respectively. In the case of α-nitrosyl Hb, these organic phosphates exerted a dramatic effect on its O2 affinity. The maximum effect was registered at pH 7.4, and the ratios were 6.6 and 23.9 for BPG and IHP, respectively. The equally effective ability of BPG and IHP to lower the O2 affinity of α-nitrosyl Hb over both sides of the pH range is reflected in a rather symmetric bell-shape curve (not shown). The pH dependence of the O2 equilibrium constants for the third and fourth binding steps, K3 andK4, in the absence and presence of IHP, is shown in Fig. 5 A. Curves were smoothed by fixing only the upper and lower asymptotes. The lowestK3 and highest K4 values were obtained by extrapolating curves for log K3in the presence of IHP at low pH values and logK4 in the absence of IHP at high pH values, respectively. Values for lowest K3 and highestK4 thus obtained were 0.011 and 7.23 mm Hg−1, respectively. The number of protons released at theith oxygenation step, ΔH+i(i = 3, 4), were then obtained from the slope of the curves in the absence and presence of IHP and are shown in Fig. 5,B and C, respectively. The total Bohr effect, expressed as ΔH+total, was calculated as the sum of ΔH+3 and ΔH+4 and is equal to the total Bohr protons released upon oxygenation of one molecule of α-nitrosyl H" @default.
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• W2059243326 title "Electron Paramagnetic Resonance and Oxygen Binding Studies of α-Nitrosyl Hemoglobin" @default.
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