Non-Equivalence and Inverse Allosteric Response of the a and ß Chains in Haemoglobins. An Electron Spin Resonance Study of NO-Ligated Hb Kansas

The electron spin resonance (ESR) spectra of 15NOand 14NO-ligated Hb Kansas have been measured at 77 K in the range of pH 5 to 10. At low pH the ESR spectrum is the composite of a type I and a type II spectrum which changes to another composite of a type I and type II spec­ trum at high pH. For the definition of type I and type II spectra and the correlation of these types with two tertiary conformation states see Overkamp et al., Z. Naturforsch. 3 1 c , 524 [1976]. Both, the type I and the type II spectra observed at low and high pH respectively are different with regard to (/-tensors and hyperfine-splitting constants. Therefore at intermediate pH values the ESR spectra of NO-Hb Kansas are the composites of four spectral components. The assignments of the four spectral components to the a and the ß chains are arrived at from the comparison of the ESR spectra of the aXmetß%° and of the <x5INO/?2° species of Hb M Iwate. oc and ß chains are both characterized by a pH-dependent spectral transition from a type I to a type II spectrum. The chains are non-equivalent with regard to both the type I and the type II spectra. The type I spectra assigned to the a and the ß chains are characterized by gzz = 2 .0 0 9 5 with a hyperfine splitting of a zz (15NO) = 2 .3 6 mT and <7zz = 2.0085 with a hyperfine splitting of aZz(15NO) = 2 .41 mT respectively. The type II spectra assigned to the a and the ß chains are characterized by g*z = 2 .0 0 5 and a hyperfine splitting of a**' (15NO) = 3 .0 7 mT and <7^=2.005 and a hyperfine splitting of a'zz (15NO) = 3 .3 1 mT. The change of the hyperfine splitting at gzz during the transition from type I to type II cor­ responds to an increase of the spin density at the NO by about 25% in both types of chains. Com­ parison of type I spectra of the NO-ligated ex. and ß chains respectively demonstrates that the spin density at the NO is larger in the ß chains than in the oc chains. The spectral types are correlated with functional states defined by the kinetics of NO-binding. Binding of inositol hexaphosphate has no influence on the ESR spectra in the whole range of pH as it is expected if NO-Hb Kansas is in the quaternary T structure.