Protein Dynamics: Imidazole Binding to Class I C-Type Cytochromes

The oxidized cytochrome c2 from the purple phototrophic bacteria, Rhodobacter sphaeroides and Rhodobacter capsulatus, bind the neutral species of imidazole (Ka = 1440 ± 40 M−1) 50 times more strongly than does horse mitochondrial cytochrome c (Ka = 30 ± 1 M−1). The kinetics of imidazole binding are consistent with a change in rate-limiting step at high ligand concentrations for all three proteins. This is attributed to a conformational change leading to breakage of the iron–methionine bond which precedes imidazole binding. The three-dimensional structure of the Rb. sphaeroides cytochrome c2 imidazole complex (Axelrod et al., Acta Crystalogr. D50, 596–602) supports the view that the conformational changes are essentially localized to approximately seven residues on either side of the ligated methionine and there is a hydrogen bond between the Phe 102 carbonyl, an internal water, and the bound imidazole. Insertions and deletions in this region of cytochrome c2, the presence of a proline near the methionine, and the smaller size of the dynamic region of horse cytochrome c suggest that the stabilizing hydrogen bond is not present in horse cytochrome c, hence, the dramatic difference in affinity for imidazole. The kinetics of ligand binding do not correlate with either the strength of the iron–methionine bond as measured by the pK of the 695-nm absorption band or the overall stability of the cytochromes studied. However, the very similar imidazole binding properties of the two cytochromes c2 indicate that the Rb. sphaeroides cytochrome c2–imidazole complex structure is an excellent model for the corresponding Rb. capsulatus cytochrome c2 complex. It is notable that the movement of the peptide chain in the vicinity of the ligated methionine has been preserved throughout evolution and suggests a role in the function of c-type cytochromes.