Proton ENDOR from randomly oriented NO-ligated haemoglobin: approaching the structural basis for the R–T transition

Abstract

EPR and ENDOR spectra of nitrosyl (NO)-ligated haemoglobin (HbNO) and its isolated α- and β-chains are presented. In comparison with the NO-ligated Fe-tetraphenylporphyrin as a model compound, with imidazole as the sixth ligand, the temperature dependence of the EPR spectra is analysed in terms of two different stereochemistries of the N(imidazole)—Fe—N(NO) bonds, with respect to the porphyrin plane. One represents an axial EPR spectrum with the bond direction coinciding with the intermediate g factor which is parallel to the normal porphyrin plane. The other comprises a rhombic EPR spectrum in which that direction is tilted from the plane normal by 30–40°. These two structures were deduced from analysis of the ENDOR response in the region of weakly coupled protons (10–20 MHz) by theoretical simulations using the low-temperature X-ray structure of met-myoglobin as the backbone. The R–T transition in HbNO was reported to involve α-chain conformational changes only comprising a partial loss of the proximal histidine in the isolated subunits and in the tetrameric HbNO. The strong tilt of the N(imidazole)—Fe—N(NO) in the α-chains with decreasing temperatures is proposed to be the precursor to the loss of the proximal histidine. It involves a sideward movement of the F-helix which is not possible in β-chains owing to a smaller haem crevice.