| The dynamics of the protein and heme ligand region of cytochrome c1 were probed through the kinetics of exogenous ligand binding. Using the detergent-solubilized bc1 complex from Rhodobacter sphaeroides and capsulatus, the binding kinetics were first order at low concentrations, but deviated at ligand concentrations >30 mM, indicating a rate-limiting conformational step that was substantially different for different ligands. Imidazole and 1-methylimidazole binding and release from the cyt c 1-ligand complexes exhibited very high activation energies and huge preexponential factors. Although qualitatively similar to previous results for typical (Class I) monoheme cytochromes c, the quantitative differences suggest more extensive structural changes triggered by ligand binding. Furthermore, differences between similar ligands and between the two bc1 complexes argue against the favored mechanism of ligand binding, in which the heme cleft opens spontaneously, providing access by small ligands. Our data strongly support mechanisms in which ligand pre-binding facilitates heme access prior to ligand exchange. This is supported by computational analyses that show the absence of ligand permeation by other pathways. We also suggest that mobility of amino acid residues adjacent to the Met residues of cyt c1, as indicated by the ligand binding behavior, can play a role in physiological function, such as in the docking of its soluble reaction partner, cyt c 2 or cyt c, and possibly in allowing access to small molecules with potential regulatory and signaling activities.;The first molecular dynamic simulations of the interaction of cyt c and yeast bc1 complex are described. Contrary to results from crystallographic studies, the simulations reveal multiple dynamic hydrogen bonds and salt bridges in the cyt c-c 1 interface. A novel structural change in cyt c is also reported, involving residues 25--30, which may be responsible for cyt c destabilization. An interaction between cyt c1 monomers is proposed to be responsible for limiting the binding of cyt c to only one molecule per bc 1 dimer by altering the affinity of the cytochrome c binding site on the second cyt c1 monomer. A mechanism is also proposed explaining changes in cyt c binding affinity related to the position of the headgroup of the iron-sulfur protein. |
