Proton NMR study of myoglobin reconstituted with 3, 7-diethyl-2, 8-dimethyl iron porphyrin: remarkable influence of peripheral substitution on heme rotation.

The iron complex of 3,7-diethyl-2,8-dimethylporphyrin was incorporated into horse heart apomyoglobin to investigate the influence of peripheral substitution on artificial heme rotation. The hyperfine-shifted 1H NMR spectrum of the reconstituted deoxymyoglobin (rMb) revealed the proximal imidazole N-H resonance at 82.5 ppm to indicate the formation of the Fe--N (His93) bond. The pyrrole-protons of the hemin of myoglobin in the absence of external ligand appeared as four resonances between -10 and -18 ppm, indicating a mainly low-spin ferric hemin, with a ligated distal histidine (His64). This also indicates the lost of the symmetry of the hemin, according to an absence of free rotation of the prosthetic group. The 1H NMR spectrum of reconstituted rMbCO revealed a set of four pyrrole-protons and a set of four meso-protons. Accordingly, the prosthetic group without acid side chains interacts specifically with the surrounding globin showing a unique heme orientation in the 1H NMR time-scale, despite the presence of only four alkyl substituents on the porphine ring. This also suggests that two ethyl groups are large enough to avoid the free rotation movement of the heme.