cytochromes-c1 and 1-methylimidazole

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c(1) in detergent-solubilized bc(1) complex from Rhodobacter sphaeroides. Imidazole binding to cyt c(1) substantially lowers the midpoint potential of the heme and fully inhibits bc(1) complex activity. Temperature dependences showed that binding of Im (K(d) approximately 330 microM, 25 degrees C, pH 8) is enthalpically driven (DeltaH(0) = -56 kJ/mol, DeltaS(0) = -121 J/mol/K), whereas binding of MeIm is 30 times weaker (K(d) approximately 9.3 mM) and is entropically driven (DeltaH(0) = 47 kJ/mol, DeltaS(0)(o) = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c(1) triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c(2) suggested that Im binding to cyt c(1) is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c(2), and c(1), which showed hydrogen bonds formed between the N(delta)H of Im and the cyt c(1) protein, or with a water molecule sequestered with the ligand in the heme cleft.

Topics: Cytochromes c1; Electron Transport Complex III; Heme; Imidazoles; Kinetics; Ligands; Models, Molecular; Molecular Dynamics Simulation; Oxidation-Reduction; Rhodobacter sphaeroides; Spectrum Analysis; Temperature

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c(1) of detergent-solubilized bc(1) complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c(1)-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mM Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mM, the rate leveled off, indicating a rate-limiting conformational step with lifetime approximately 1 s; and (iii) at Im concentrations above 100 mM, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.

Topics: Cytochromes c1; Electron Transport Complex III; Enzyme Activation; Heme; Imidazoles; Kinetics; Ligands; Oxidation-Reduction; Rhodobacter sphaeroides; Temperature