Proton NMR investigation of the reconstitution of equine myoglobin with hemin dicyanide. Evidence for late formation of the proximal His93F8-iron bond.

Reconstitution of apoequine myoglobin (apoEqMb) with hemin dicyanide (FePPIX(CN)2) was monitored by 1H NMR spectroscopy to gain information about the sequence of events leading to metEqMbCN. At least one step in the pathway is slow enough to allow us to follow the time-dependence of formation of the product, a mixture of heme-insertion isomers characterized by others (Jue, T., Krishnamoorthi, R. and La Mar, G.N. (1983) J. Am. Chem. Soc. 105, 5701-5703; Lecomte, J.T.J., Johnson, R.D. and La Mar, G.N. (1985) Biochim. Biophys. Acta 829, 268-274). However, in contrast to all previously reported Mb-FePPIX reconstitutions, we find that the initial ratio of heme-insertion isomers is not 1:1. This mixture is, instead, found to be enhanced in the isomer which dominates at equilibrium. This is taken as evidence for a '[FePPIX(CN)2.EqMb]' intermediate which lacks the proximal His93F8-Fe bond and which proceeds quickly toward an equilibrium ratio of heme-insertion isomers. Therefore the heme-insertion isomer ratio is frozen only when the proximal His93F8-Fe bond is formed. The difference in this ratio of heme-insertion isomers between EqMb (4.5:1) and SwMb (2.5:1) likely reflects the amino acid substitution: Lys----Arg45CD3 (EqMb----SwMb).