Abstract
Although ligand binding in c-type cytochromes is not directly related to their physiological function, it has the potential to provide valuable information on protein stability and dynamics, particularly in the region of the methionine sixth heme ligand and the nearby peptide chain that has been implicated in electron transfer. Thus, we have measured the equilibrium and kinetics of binding of imidazole to eight mutants of Rhodobacter capsulatus cytochrome c2 that differ in overall protein stability. We found that imidazole binding affinity varies 70-fold, but does not correlate with overall protein stability. Instead, each mutant exerts an effect at the local level, with the largest change due to mutant G95E (glycine substituted by glutamate), which shows 30-fold stronger binding as compared with the wild-type protein. The kinetics of imidazole binding are monophasic and reach saturation at high ligand concentrations for all the mutants and wild-type protein, which is attributed to a rate-limiting conformational change leading to breakage of the iron-methionine bond and providing a binding site for imidazole. The mutants show as much as an 18-fold variation in the first-order rate constant for the conformational change, with the largest effect found with mutant G95E. The kinetics also show a lack of correlation with overall protein stability, but are consistent with localized effects on the dynamics of hinge region 88-102 of the protein, which changes conformation to permit ligand binding. These results are consistent with R. capsulatus cytochrome c2 stabilizing the complex through hydrogen bonding to the imidazole. The larger effects of mutant G95E on equilibrium and kinetics are likely to be due to its location within the hinge region adjacent to heme ligand methionine 96, which is displaced by imidazole.
Highlights
In a preceding study,[1] a detailed equilibrium and kinetic analysis of the binding of imidazole to the oxidized form of horse mitochondrial cytochrome c and two c2-type cytochromes from the purple phototrophic bacteria (Rhodobacter capsulatus and Rhodobacter sphaeroides) was performed
We have measured the equilibrium and kinetics of binding of imidazole to eight mutants of Rhodobacter capsulatus cytochrome c2 that differ in overall protein stability
We have proposed that the rate-limiting conformational change is a consequence of a movement of peptide region 88 – 102 (R. capsulatus numbering), which we have termed the hinge region based upon the crystal structure of the R. sphaeroides cytochrome c2-imidazole complex (3)
Summary
In a preceding study,[1] a detailed equilibrium and kinetic analysis of the binding of imidazole to the oxidized form of horse mitochondrial cytochrome c and two c2-type cytochromes from the purple phototrophic bacteria (Rhodobacter capsulatus and Rhodobacter sphaeroides) was performed. The rate-limiting step at high ligand concentrations for all three proteins This is attributed to a conformational change (occurring with a rate constant of ϳ35 Ϯ 10 sϪ1) leading to a breakage of the iron–methionine bond and providing a binding site for imidazole as previously proposed for horse cytochrome c (2). Horse cytochrome c is much more stable to guanidine denaturation than are the c2-type cytochromes, and at least some of the difference in imidazole binding may be related to the disparity in overall protein stability. We have studied the equilibrium and kinetics of imidazole binding to a variety of R. capsulatus cytochrome c2 mutants that differ in overall stability to guanidine denaturation. Vide information on the role of overall protein stability in the movement of the hinge region and on more localized factors, which influence the immediate heme environment and electronic properties
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