EPR spectral changes of nitrosyl hemes and their relation to the hemoglobin T-R transition

Abstract

EPR spectra of nitrosyl hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25°C. After the equilibration of NO among the α and β subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO-β-hemes and the other two arising from NO-α-hemes of molecules in the high- and low-affinity conformations. The fractional amounts of α subunits exhibiting the high-affinity spectrum fitted the two-state model (Edelstein, S.J. (1974) Biochemistry 13, 4998–5002) with the allosteric constant L = 7 · 10⁶ and relative affinities c_NO^α and c_NO^β approx. 0.01. Hemoglobin has been marked with nitric oxide at one chain using low-saturation amounts of nitric oxide. The EPR spectra were studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high-affinity spectrum fitted the two-state model with L = 7 · 10⁶, c_O2 = 0.0033 and c_NO^α = 0.08, instead of c_NO^α = 0.01. Thus, the two-state model is not adequate to describe the conformational transition of these hybrids. The results present evidence of the non-equivalence between oxygen and nitric oxide as ligands.