Proton NMR study of yellowfin tuna myoglobin in whole muscle and solution. Evidence for functional metastable protein forms involving heme orientational disorder.

Proton NMR spectra of met-aquo-myoglobin have been recorded in whole dark muscle from yellowfin tuna for the first time; in addition, spectra of the met-aquo, met-cyano, and deoxy forms were recorded in solution. The number of resolved methyl resonances in the met-aquo and met-cyano derivatives of the purified protein indicates a molecular heterogeneity, with the two species present in a ratio of approximately 3:2. These same two species, in very similar environments, are found in whole muscle. Upon reconstitution of the protein, the ratio of the two species is approximately 1:1. Specific isotope labeling in the met-cyano form reveals an interchange of 5-methyl and 8-methyl environments that is characteristic of heme orientational disorder about the alpha, gamma-meso axis. Incubation of the met-cyano protein at 37 degrees C shows that the two species are interconvertible, with the minor component declining to about one-eighth of that of the major component. This indicates that one of the two orientations is favored thermodynamically. The rate of equilibration for the met-aquo protein is exceedingly slow (half-life greater than 30 days), some 10(2) slower than for the analogous sperm whale derivative. The "reversed" heme orientation is therefore present in muscle as a kinetically trapped or metastable species, suggesting that the last step in the biosynthesis of myoglobin is similar to that of in vitro reconstitution. The presence of both heme orientations in myoglobin in whole muscle proves that heme orientational disorder is a physiological phenomenon.