PROTON ENDOR FROM RANDOMLY ORIENTED NO-LIGATED HEMOGLOBIN - APPROACHING THE STRUCTURAL BASIS FOR THE R-T TRANSITION

EPR and ENDOR spectra of nitrosyl (NO)-ligated haemoglobin (HbNO) and its isolated alpha- and beta-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 degrees. 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 alpha-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 alpha-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 beta-chains owing to a smaller haem crevice.