NMR study of Galeorhinus japonicus myoglobin. 1H-NMR evidence for a structural alteration on the active site of G. japonicus myoglobin upon azide ion binding.
The heme molecular structure of the met-azido form of the myoglobin from the shark Galeorhinus japonicus has been investigated by 1H NMR. A nuclear Overhauser effect (NOE) was clearly observed among the heme peripheral side-chain proton signals of this complex, which undergoes thermal spin equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states, and the NOE connectivities provided the assignment of the resonances from the heme C13(1)H2 and C17(1)H2 protons. Chemical shift inequivalence of these proton resonances not only provided information about the orientation of these methylene protons with respect to the heme plane, but also allowed characterization of the time-dependent build-up of the NOE between them, which yields the correlation time for the internal motion of the inter-proton vector. The relatively large mobility found for the C17(1)H2 group suggests that the carboxyl oxygen of the heme C17 propionate is not anchored to the apo-protein by a salt bridge. It has been shown that the ferric high-spin form of G. japonicus Mb possesses a penta-coordinated heme [Suzuki, T. (1987) Biochim. Biophys. Acta 914, 170-176; Yamamoto, Y., Osawa, A., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 192, 225-229] and that the conformation of both heme propionate groups is fixed with respect to the heme, as well as the apo-protein, by a salt bridge [Yamamoto, Y., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 189, 567-573]. Therefore the weakening or interruption of the interaction between the C17 propionate and His FG3 upon the changes of the coordination and spin state of the heme iron, during azide ion binding to ferric high-spin G. japonicus Mb, is attributed to the displacement of the FG corner of the apoprotein away from the heme C17 propionate group. A similar structural alteration has been revealed by X-ray structural analyses of unliganded and liganded forms of ferrous hemoproteins [Baldwin, J. & Chothia, C. (1979) J. Mol. Biol. 129, 175-220; Phillips, S.E.V. (1980) J. Mol. Biol. 142, 531-554].
Yamamoto
Y
Y
Department of Biomolecular Engineering, Tokyo Institute of Technology, Japan.
Chûjô
R
R
Suzuki
T
T
D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D006418:Heme / Q000378:metabolism; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009132:Muscles / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012754:Sharks
D001386:Azides; D009211:Myoglobin; D006418:Heme; D006859:Hydrogen