Resonance Raman (RR) spectra are reported for the deoxy, oxy, and metcyano species of sperm whale myoglobin (Mb) reconstituted with deuteroheme, 2-acetyldeuteroheme, 4-acetyldeuteroheme, and 2,4-diacetyldeuteroheme. The functional inequivalence of the 2- and 4-positions of the heme moiety is manifested in the vibrational frequencies of the carbonyl and certain heme skeletal modes. The RR data indicate that the protein influences the porphyrin pi-system primarily through the 2-substituent. In the deoxy Mbs, the 2-acetyl group is more conjugated into the pi-macrocycle than the 4-acetyl group. In the ligated Mbs, the extent of conjugation of the 2-group is less than in the deoxy forms. This result indicates that ligand binding differentially alters the pi-electronic structure for 2- vs 4-substituted systems. The proximal histidine-iron stretching vibration of all four deoxy Mbs occurs at 222 +/- 1 cm-1, which indicates that the substituent-induced changes in the pi-electronic structure do not result in large changes of the electron density in the axial ligand bonds. RR spectra recorded immediately after heme reconstitution indicate the presence of two inequivalent forms of the protein. 1H NMR spectroscopy indicates that these two forms are the normal and reversed heme orientational isomers previously described by La Mar and co-workers [La Mar, G.N., Budd, D.L., Viscio, D.B., Smith, K.M., & Langry, K.C. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5755-5759]. The RR spectra demonstrate that in the deoxy species the 2-acetyl group of the normal form is conjugated into the macrocycle to approximately the same extent as the 4-acetyl group of the reversed form and vice versa.(ABSTRACT TRUNCATED AT 250 WORDS)
Deuterium Heme