Electron paramagnetic resonance and oxygen binding studies of alpha-Nitrosyl hemoglobin. A novel oxygen carrier having no-assisted allosteric functions.
alpha-Nitrosyl hemoglobin, alpha(Fe-NO)2beta(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. alpha-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O2 binding at the heme sites of its beta-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 alpha(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, alpha(Fe-NO)2beta(Fe)2 is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of alpha-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 Cysbeta93 toward 4, 4'-dipyridyl disulfide of several alpha-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O2-carrying capacity, alpha-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 alpha-subunits. It is concluded that the NO bound to the heme sites of the alpha-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.
Yonetani
T
T
Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19194-6089, USA. yontant@mail.med.upenn.edu
Tsuneshige
A
A
Zhou
Y
Y
Chen
X
X
D019794:2,3-Diphosphoglycerate / Q000494:pharmacology; D000494:Allosteric Regulation / Q000502:physiology; D004220:Disulfides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009569:Nitric Oxide / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011725:Pyridines / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D004220:Disulfides; D006454:Hemoglobins; D011725:Pyridines; D013438:Sulfhydryl Compounds; C008657:nitrosyl hemoglobin; D019794:2,3-Diphosphoglycerate; C007542:4,4'-dipyridyl disulfide; D009569:Nitric Oxide; D006418:Heme; D010100:Oxygen