VARIABLE-TEMPERATURE STUDY OF THE HEME-REORIENTATION PROCESS IN EQUINE MYOGLOBIN

The redistribution of the initially-formed myoglobin heme-insertion is omers 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, bot h of which require the rupture of the heme iron-protein bond (La Mar, G.N., Toi, H. and Krishnamoorthi, K. (1984) J. Am. Chem. Sec. 106, 639 5-6401). In this study we compared the use of nuclear magnetic resonan ce and optical spectroscopic techniques as methods for studying the re orientation of heme within myoglobin. We found that kinetics determina tions of the heme insertion isomer redistribution process in Mb by opt ical spectroscopy are quantitatively compatible with the results obtai ned 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 k cal/mol, Delta S+ = 34 cal/mol per K, and Delta G(21 degrees C)(+) = 2 1 kcal/mol. The value of Delta G(+) expected for complete dissociation of the heme from myoglobin can be estimated, from its dissociation co nstant and insertion rate, to be on the order of 23-27 kcal/mol under the same conditions as our determination. Therefore, although the mech anism for heme reorientation in Nib is likely non-dissociative, it has an activation energy which is not far from the lower bound expected f or a complete-dissociation/recombination mechanism. Our measured entro py of activation is not especially large, perhaps owing to a large con tribution by the solvent.