Conformation-Controlled Trans-Effect of the Proximal Histidine in Haemoglobins. An Electron Spin Resonance Study of Monomeric Nitrosyl-57Fe-Haemoglobins

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Abstract
A monomeric allosteric Hb from Chironomus thummi thummi was reconstituted with 57Fe-heme. This reconstituted Hb was identical to the non-reconstituted material with regard to the O2-binding properties and the visible spectra. The 270 MHz proton magnetic resonance of the bis(cyano)-57Fe-hemin showed that the reconstituted heme was identical to the non-reconstituted heme. PMR studies showed that in Chironomus Hb, the prosthetic group was proto-heme IX. The ESR spectrum of the native nitrosyl Hb demonstrated rhombic symmetry of the heme Fe (gxx = 2.086, gyy = 1.981, gzz = 2.005) and hyperfine structures at gyy (aN.epsilon. = 1.35 mT) and at gzz (a15NO = 3.05 mT, a14NO = 2.19 mT, aN.epsilon. = 0.715 mT, a57Fe = 0.38 mT). The spectrum was independent of pH and could be classified as a type II spectrum following the classification of ref. 2. NO-binding obviously stabilizes the tertiary structure of this Hb in a tense conformation with a relatively strong .sigma. bond of the 5th ligand (N.epsilon. of imidazole) and a relatively weak .sigma. bond of the 6th ligand (NO). Reaction of this Hb with anionic, cationic and non-ionic detergents, respecively, leads to a transformation of the NO-ligated form into a relaxed conformation with a stretched or broken .sigma. bond of the 5th ligand (N.epsilon. of imidazole) and a strong .sigma. bond of the 6th ligand (NO). The ESR spectrum of this modified NO-Hb shows again a rhombic symmetry of the heme Fe (gxx = 2.10, gyy = 2.06, gzz = 2.01), but dramatically changes in the g tensors (low field shift), hyperfine structures and hyperfine splitting constants (a15NO = 2.32 mT, a14NO = 1.66 mT, a57Fe = 0.48 mT). The hyperfine splitting is isotropic. Transition from the tense conformation to the relaxed conformation corresponds with an increase of the spin density at the iron atom by 26% and a decrease of the spin density at the NO ligand by 25%. The spin density at the N.epsilon. of imidazole strongly decreases in the relaxed conformation, so that a hyperfine splitting of this ligand is not resolved. The trans-effect of the proximal imidazole which in Hb controls the binding properties of the external ligand in trans-position is demonstrated.

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