Observation of deuterium-labeled diacetyldeuteroporphyrin incorporated in cyanoferrimyoglobin by deuterium nuclear magnetic resonance

Sperm whale apomyoglobin was reconstituted with selectively deuterated D 6-2,4-diacetyldeuterohemin in which the 2H label was confined to the methyl groups of the acetyl moieties. A single resonance was observed in 2H NMR of the cyanoferrimyoglobin derivative, with a chemical shift 0.80 ppm downfield of external D 12-TMS at pH 6.7. The corresponding chemical shift of D 6-2,4-diacetyldeuterohemin-OMe as the cyanide complex in pyridine-water was 0.96 ppm downfield of external D 12-TMS. The prominent HOD peak was well separated at 4.4 ppm downfield. The line width of the porphyrin 2H resonances in both the protein and free solvent environments yields evidence of considerable rotational freedom of the -CD3 groups about their axes.