II Comparison of mobility Indices A and B with rotational

In order to investigate the protein conformation in the vicinity of the heme and the spin states of the heme-iron in dissolved hemoglobins, a nitroxide spin label was covalently attached to one of the propionic acid groups of the porphyrin ring. The optical spectra of the heme-spin labeled ferrihemoglobins were identical to those of native hemoglobins indicating that the spin labeling did not affect significantly the electronic structure of the prosthetic group. The EPR spectra of the nitroxide moiety in the fluoride, cyanide, and azide derivatives of the heme-spin-labeled hemoglobin and of the corresponding acid methemoglobin in solution were not identical, suggesting that the protein conformation in the vicinity of the label is different in each of these hemoglobin derivatives. The resonance amplitude of the nitroxide in heme-spinlabeled hemoglobin was sensitively influenced by the high spin heme-iron located in the center of the porphyrin ring due to the magnetic dipolar interactions between them. The degree of the dipolar interaction depended on the magnetic moment and electron-spin relaxation time of the heme-iron, as well as the distance between the nitroxide and heme-iron. From the strength of this interaction, the distance between the heme-iron and the nitroxide radical was estimated as 11.8 A. Since the spin label attached to heme is sensitive to changes in the magnetic moment of the iron, the heme-spinlabeled ferrihemoglobin can also be used for studying the thermal equilibrium between high and low spin electronic states of the heme-iron. By comparing the nitroxide resonance amplitudes of the hydroxide form with those of the high spin acid met form and the low spin cyanide form of hemoglobin, the ratio of high to low spin components of the hydroxide was calculated as 53:47 at 0”. This ratio was increased at higher temperatures due to the shift of the equilibrium composition in favor of the high spin form. Optical and EPR spectra of free spin-labeled protohemin were also investigated. EPR spectra of protohemin were sensitive to dimer formation of hematin in aqueous alkaline solution. Two empirical indices are presented for convenient nu-