Parts I and II (Perutz et al. (1974), Biochemistry 13, 2163, 2174) presented evidence indicating that inositol hexaphosphate (IHP) is capable of switching the quaternary structure of certain high-spin ferrous and ferric hemoglobins from the oxy (R) to the deoxy (T) state. Here we analyze the resulting changes in visible and near-infrared absorption spectra, paramagnetic susceptibility, and paramagnetically shifted proton and electron spin resonances. In aquo- and hydroxymethemoglobin where components of high and low spin coexist in thermal equilibrium, IHP causes changes in the absorption spectra and paramagnetically shifted proton resonances indicative of a shift of the equilibrium to higher spin. This is confirmed by a stoichiometric rise in the paramagnetic susceptibility of aquomethemoglobin solutions by up to 6.4 % at concentrations of 1 mol of IHP/mol of tetramer. Other spectral changes produced by IHP include a rise in intensity of the Soret band and red shifts of all high-spin bands. The spectral changes are identical with those observed by Perutz ((1972), Nature (London) 237, 495) on dissociation of carbon monoxide from valency hybrids. In fluoromethemoglobin which is probably pure high spin, IHP causes red shifts of all bands, a rise in intensity of the Soret band and a fall in all others. In iron-porphyrin complexes which are in thermal equilibrium between two different spin states, a shift toward higher spin is stereochemically equivalent to a lengthening of the distances between the iron atom and its electronegative ligands. Such a lengthening would diminish electrostatic repulsion of electronic charge on the iron, thus lessening the energy needed to transfer charge from the porphyrin to the iron and raising the energy required for the transfer of charge from the iron to the porphyrin. Qualitatively this would account for the blue shifts of charge-transfer bands in deoxyhemoglobin (part I) and for their red shifts in high-spin methemoglobins. A mechanism explaining the observed intensity changes is also proposed. It was shown in part I that in the R structure the hemes appear to be in the same state as in free α and β subunits. The lengthening of the ironnitrogen bond distances observed in the T structure implies that the globin exercises a tension on the heme which pulls the iron atoms further from the planes of the porphyrin rings. Such a tension would oppose the transition to the low-spin state that is needed for combination with oxygen and thereby lower the oxygen affinity. Since heme-heme interaction is observed only when reaction with ligands is accompanied by a change of quaternary structure, our results imply that it is coupled to a change of tension at the heme, transmitted by a change in quaternary structure of the globin.
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