Electron Paramagnetic Resonance and Oxygen Binding Studies of α-Nitrosyl Hemoglobin

Abstract

Nitrosyl hemoglobin, (Fe-NO) 2 (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 O 2 has been investigation by EPR and thermodynamic equilibrium measurements.-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O 2 binding at the heme sites of its-sub-units. Its O 2 binding is coupled to the structural/func-tional 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 (Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, (Fe-NO) 2 (Fe) 2 is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of-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 Cys 93 toward 4,4-dipyridyl disulfide of several-nitrosyl he-moglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O 2-carrying capacity,-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O 2 carrier with improved features that could deliver O 2 to tissues effectively even after NO is sequestered at the heme sites of the-sub-units. It is concluded that the NO bound to the heme sites of the-subunits of hemoglobin acts as a negative allos-teric effector of Hb and thus might play a role in O 2 /CO 2 transport in the blood under physiological conditions. When deoxy hemoglobin (Hb) 1 is exposed to less-than-stoi-chiometric amounts of NO ([NO]/[heme] 0.5) in solution (1-3) and in the erythrocytes (4-6), the predominant species formed upon equilibrium are-nitrosyl Hbs, i.e. (Fe-NO)(Fe)(Fe) 2 or (Fe-NO) 2 (Fe) 2. Such compounds were readily identified by their EPR spectra with a set of sharp triplet 14 N hyperfine structures (A z 17 Gauss) around g z 2.009, which is derived from the 5-coordinate nitrosyl hemes in the-subunits. When rats or mice had been exposed to doses of lipopolyscaccaride, tumor necrosis factor, nitroglycerin, nitrite, or NO (7-12), 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, (Fe-NO) 2 (Fe-NO) 2 , in the absence of IHP at a physiological pH of 7.4 (13). Therefore, it is obvious that the primary nitrosyl products formed upon reaction of deoxy Hb with NO under physiological conditions, where [NO] [heme], are-nitrosyl Hbs (6, 12, 14, 15). In order to assess physiological roles of such compounds, we have investigated its O 2 binding properties of (Fe-NO) 2 (Fe) 2 by EPR and O 2 binding measurements as well as the reactivity of Cys 93 toward 4-PDS as a probe for the quaternary structure. We have found that its oxygenation characteristics and allosteric functions make (Fe-NO) 2 (Fe) 2 a unique cooperative low affinity O 2 carrier with full allosteric sensitivity that could deliver O 2 to tissues efficiently under physiological conditions. This study has also provided a new insight into the molecular mechanism of cooperativity and al-lostery in Hb, particularly the major role of the-heme Fe-F helix linkage in the quaternary structural transition, and the mode interaction of Hb with NO. EXPERIMENTAL PROCEDURES Reagents-2,3-Biphosphoglycerate, IHP, pMB, 4-PDS, Tris, bis-Tris, bis-Tris propane, dithiothreitol, catalase, superoxide dismutase (Sig-ma), 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 O 2 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. Preparation of (Fe-NO) 2 (Fe-O 2) 2-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 (16) and stripped from organic phosphates by the method of Berman et al. (17). The Hb solution was stored in the CO form, and no further attempt was made to strip Hb from its minor components.-and-chains from Hb in the CO form were separated according to the method of Bucci and Fronticelli (18) using the pMB treatment with modifications. After overnight incubation, the pMB-treated Hb solution