Binding of imidazole to the heme of cytochrome c1and inhibition of the bc1 complex from Rhodobacter sphaeroides: I. equilibrium and modeling studies

Abstract

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c1 in detergent-solubilized bc1 complex from Rhodobacter sphaeroides. Imidazole binding to cyt c1 substantially lowers the midpoint potential of the heme and fully inhibits bc1 complex activity. Temperature dependences showed that binding of Im (Kd ≈ 330 μM, 25 °C, pH 8) is enthalpically driven (ΔH0 = -56 kJ/mol, ΔS0 = -121 J/mol/K), whereas binding of MeIm is 30 times weaker (Kd ≈ 9.3mM) and is entropically driven (ΔH0 = 47 kJ/mol, ΔS0° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c1 triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c2 suggested that Im binding to cyt c1 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, c2, and c1, which showed hydrogen bonds formed between the NδH of Im and the cyt c1 protein, or with a water molecule sequestered with the ligand in the heme cleft. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.