Interactions between the Quaternary Structure of the Globin and the Spin State of the Heme in Ferric Mixed Spin Derivatives of Hemoglobin

We have studied the effect of the P6-inositol (IHP)-induced change from the quaternary oxy (R) to the deoxy (T) structure in derivatives of human, trout IV, and carp methemoglobins. Addition of IHP to human fluoro- and aquomethemoglobin leads to the appearance of the slowly exchanging proton resonance at about  10 ppm from HDO diagnostic of the T structure. This experiment, and the crystallization of aquomethemoglobin + IHP by G. Fermi & M. F. Perutz ((1977) J. Mol. Biol. 114, 421) confirmed that the spectral change in the UV which IHP induces in these compounds can be used as a reliable indicator of the R→T transition. Judged by this spectral change, IHP converts all derivatives of carp hemoglobin from the R to the T structure. The pH at which the midpoint of the IHP-induced transition occurs increases with rising spin, being lowest in cyano, intermediate in azido, and highest in thiocyanate and aquomethemoglobin of carp. Conversely the replacement of water by fluoride or thiocyanate as the sixth ligand is unaffected by IHP because all three derivatives are predominantly high spin, but the affinity of azide for carp aquomethemoglobin is reduced 2.7-fold and that of cyanide 3.3-fold by IHP, corresponding to changes in the free energy of binding of 600 and 700 cal/mol heme. Conversion to the T structure of all carp methemoglobin derivatives except the cyanide one is accompanied by large changes in the visible absorption spectra, the most spectacular being that of the nitrite derivative whose color is changed from red to brown. IHP converts all human methemoglobin derivatives except the azide and cyanide ones from the R to the T structure. The conversion is accompanied by similar, but smaller, spectral changes than those in the corresponding carp derivatives. The change in paramagnetic susceptibility on addition of IHP to some of the derivatives was measured by NMR. Human aquo- and cyanate methemoglobins showed no changes. Human thiocyanate methemoglobin showed an 11%, imidazole methemoglobin a 5%, and ydroxy methemoglobin approximately 45% rise. The discovery of a 2.5-fold rise in azide methemoglobin of trout IV led to the detailed study of the magnetic properties of the closely related carp hemoglobin reported in the accompanying paper (Messana C., et al. (1978) Biochemistry 17 (following paper in this issue)). The high- and low-spin components of azide methemoglobin show distinct N3- stretching frequencies in the infrared. We have used the relative optical density of the two infrared bands to study the spin equilibria of human and carp azide methemoglobins. Addition of IHP to those derivatives which are converted from the R to the T structure caused a change in the equilibrium corresponding to a free-energy change of about 1 kcal/mol heme; in derivatives in which the quaternary structure remains unchanged on addition of IHP, the free-energy change amounts to only 0.1-0.2 kcal/mol. We have also tried to measure the relaxation time of the spin equilibrium in the azide derivatives and found it to be 260 ns for the human but faster than 100 ns for the carp derivative and for myoglobin.