Probing the Paracoccus denitrificans Cytochrome c1-Cytochrome c552 Interaction by Mutagenesis and Fast Kinetics.

Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c1 and cytochrome c552 was studied using the soluble redox fragments cyt c1CF and cyt c552F. A new ruthenium cyt c552F derivative labeled at C23 (Ruz-23-c552F) was designed to measure rapid electron transfer with cyt c1CF in the physiological direction using flash photolysis. The bimolecular rate constant k12 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 <35 mM with the ruthenium photoexcitation technique in which k12 became very rapid (3 × 109 M−1 s−1) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ruz-23-c552F and c1CF was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was >30 μM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ruz-39-Cc formed a tight 1:1 complex with cyt c1CF at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s−1. A group of cyt c1CF variants in the presumed docking site were generated on the basis of information from the yeast cyt bc1−cyt c cocrystal structure. Kinetic analysis of cyt c1CF mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c552F and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions.