Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin.
Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.
Neya
S
S
Department of Physical Chemistry, Kyoto Pharmaceutical University, Japan.
Funasaki
N
N
D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009211:Myoglobin / Q000378:metabolism*; D011485:Protein Binding; D011522:Protons; D013053:Spectrophotometry; D013816:Thermodynamics; D014907:Whales
D001059:Apoproteins; D009211:Myoglobin; D011522:Protons; C010857:apomyoglobin; D006418:Heme; D006427:Hemin