Probing the Paracoccus denitrificans Cytochrome c(1)-Cytochrome c(552) Interaction by Mutagenesis and Fast Kinetics
by Janzon, Julia; Yuan, Quan; Malatesta, Francesco; Hellwig, Petra; Ludwig, Bernd; Durham, Bill; Millett, Francis Spencer
Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1cf) and cyt c(552F). A new ruthenium cyt c(552F) derivative labeled at C23 (Ru-z-23-c(552F)) was designed to measure rapid electron transfer with cyt c(1CF) in the physiological direction using flash photolysis. The bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected at an ionic strength of 30 mu M. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ru-z-39-Cc formed a tight 1:1 complex with cyt c1(CF) at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s-1. A group Of cyt c1(CF) variants in the presumed docking site were generated on the basis of information from the yeast cyt bc(1)-cyt c cocrystal structure. Kinetic analysis Of cyt c(1CF) mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c(552F) and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions.
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- 1520-4995; 0006-2960