STRUCTURE AND REACTIVITY OF RECONSTITUTED MYOGLOBINS - INTERACTION BETWEEN PROTEIN AND POLAR SIDE-CHAIN OF CHEMICALLY-MODIFIED HEMIN

We prepared the C-2-symmetrical hemin, 1,2,3,4-tetraethyl-5,8-dimethyl -6,7-dipropionate porphyrin iron complex (1), and its derivatives 2-4 conjugated with glycine, valine or phenylalanine at the terminal of on e of two peripheral propionates through amide linkage. These hemins we re incorporated into apomyoglobin from horse heart and the reconstitut ed proteins were characterized by H-1 NMR spectroscopy. Replacement of one propionate in the hemin with propionamide conjugated with amino a cid has no serious effect on the structure of the heme pocket, and the chemical shifts of heme 5-CH3 and Ile99 protons in the cyanometmyoglo bins are comparable with those in reference myoglobin with 1. Upon rec onstitution with 2-4, H-1 NMR spectra show two-set heme orientations i n the heme pocket in a ratio of 1:0.17 similar to 1:0.40 at equilibriu m. Thermodynamic analysis based on NMR results indicates that propiona mide-Lys45 pairing is more stable by similar to 0.2 kcal mol(-1) than Ser92/His97 pairing, whereas propionate-Lys45 pairing is less stable b y similar to 0.6 kcal mol(-1) than Ser92/His97 pairing. The autoxidati on rates of oxymyoglobin reconstituted with 24 are comparable with tha t of reference oxymyoglobin with 1. Furthermore, the kinetic analysis suggests that the oxyform of 6-propionamide-7-methyl-heme 5 is more st able than that of 6-propionate-7-methyl-heme 6 in myoglobin owing to t he different interaction of polar side chain with protein. (C) 1998 El sevier Science S.A. All rights reserved.