Variable-temperature study of the heme-reorientation process in equine myoglobin.

The redistribution of the initially-formed myoglobin heme-insertion isomers from the initially formed 50/50 mixture to the equilibrium ratio of 90/10 has long been assumed to occur by one of two mechanisms, both of which require the rupture of the heme iron-protein bond (La Mar, G.N., Toi, H. and Krishnamoorthi, K. (1984) J. Am. Chem. Soc. 106, 6395-6401). In this study we compared the use of nuclear magnetic resonance and optical spectroscopic techniques as methods for studying the reorientation of heme within myoglobin. We found that kinetics determinations of the heme insertion isomer redistribution process in Mb by optical spectroscopy are quantitatively compatible with the results obtained by nuclear magnetic resonance spectroscopy. A variable-temperature analysis for horse myoglobin using the optical method at pH 8.4 +/- 0.1 yielded the following activation energy parameters: delta H++ = 31 kcal/mol, delta S++ = 34 cal/mol per K, and delta G++21 degrees C = 21 kcal/mol. The value of delta G++ expected for complete dissociation of the heme from myoglobin can be estimated, from its dissociation constant and insertion rate, to be on the order of 23-27 kcal/mol under the same conditions as our determination. Therefore, although the mechanism for heme reorientation in Mb is likely non-dissociative, it has an activation energy which is not far from the lower bound expected for a complete-dissociation/recombination mechanism. Our measured entropy of activation is not especially large, perhaps owing to a large contribution by the solvent.