Characterization of conformational heterogeneity in the heme pocket of ferricytochrome c using high field proton nuclear magnetic resonance spectroscopy.
The proton nuclear magnetic resonance linewidth of a single hyperfine shifted heme methyl resonance in horse heart ferricytochrome c has been observed to show a strong increase upon decreasing the temperature (at neutral pH), increasing the pH (at 25 degrees C) or increasing the applied magnetic field, whereas the linewidth of an identical resonance in spectra of tuna heart ferricytochrome c is virtually independent of such changes. The mechanism producing this effect in the horse heart protein is consistent with exchange broadening due to rapid interconversion between heterogenous protein environments about this methyl. A detailed analysis of variable field and variable temperature studies indicates that the heterogeneity is highly localized about this methyl in horse heart cytochrome c and is absent in tuna heart cytochrome c. At -7 degrees C (with the addition of methanol), exchange between two environments is slow on the nmr time scale and a pH study under these conditions reveals that an amino acid with a pK of 7, previously undetected, controls the relative stability of the two environments. This methyl has been assigned to 3-CH3, indicating that the only amino acid side chain making contact with the methyl group, phenylalanine 82, must be able to exist in two nonequivalent positions. A comparison of cytochromes c from 11 species shows that those proteins having tyrosine in position 46 (such as tuna) do not exhibit field-dependent linewidths in any of the hyperfine shifted resonances, whereas those proteins with phenylalanine in position 46 all demonstrate this curious broadening.
D000818:Animals; D003574:Cytochrome c Group; D006418:Heme; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium; D011487:Protein Conformation; D013045:Species Specificity; D013696:Temperature; D014413:Tuna
D003574:Cytochrome c Group; D006418:Heme