Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: II. Kinetics and mechanism of binding.
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.
Kokhan
Oleksandr
O
Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801, USA.
Shinkarev
Vladimir P
VP
Wraight
Colin A
CA
D003575:Cytochromes c1 / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors*; D004789:Enzyme Activation / Q000187:drug effects; D006418:Heme / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism* / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction / Q000187:drug effects; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013696:Temperature
D007093:Imidazoles; D008024:Ligands; D006418:Heme; C029899:imidazole; D003575:Cytochromes c1; D014450:Electron Transport Complex III; C018100:1-methylimidazole
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