EPR and ENDOR studies of the distal site coordination of shark heart myoglobin

The coordination of small molecules and ions at the sixth position of ferric myoglobins (Mb) from the heart muscle of two species of shark, Lamna ditropis (LD) and Prionace glauca (PG), was studied in frozen solution by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy. The results were compared with those of horse heart Mb (EQMb). From our preliminary studies of the absorption spectra and partial amino acid sequences of PGMb and LDMb, we expected PGMb to have glutamine (Gin E7) in place of the distal His E7 that is highly conserved in most animals and present in LDMb. Here, we have used proton ENDOR to show that a water molecule coordinates to the ferric ion in both LDMb and PGMb at pH 6.3. The observed proton hyperfine coupling constant of the coordinating water molecule of PGMb is smaller than those of LDMb and EQMb, which suggests weaker coordination of the water molecule in PGMb, consistent with the absent distal His (E7). Coordination of several exogenous ligands at the distal site was examined by EPR. PGMb showed a coordination behavior different from that of LDMb and EQMb for 1-methylimidazole, 4-methylimidazole and SCN^-, suggesting that its distal site is less sterically hindered. On the basis of the ENDOR and EPR results, we assigned the distal residues of PGMb and LDMb as Gin and His, respectively.