Reorientation of cytochrome c2 upon interaction with oppositely charged macromolecules probed by SR EPR: implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer

Abstract

The reaction of water-soluble cytochrome c (c₂) with its physiological redox partners is facilitated by electrostatic attractions between the two protein surfaces. Using spin-labeled cytochrome c₂ from Rhodobacter capsulatus and pulse electron paramagnetic resonance (EPR) measurements we compared spatial orientation of cytochrome c₂ upon its binding to surfaces of opposite charge. We observed that cytochrome c₂ can use its negatively charged “back” side when exposed to interact with positively charged surfaces (DEAE resin) which is the opposite to the use of its positively charged “front” side in physiological interaction with negatively charged binding domain of cytochromebc₁. The later orientation is also adopted upon non-physiological binding of cytochrome c₂ to negatively charged carboxymethyl cellulose resin. These results directly demonstrate how the electric dipolar nature of cytochrome c₂ influences its orientation in interactions with charged surfaces, which may facilitate collisions with other redox proteins in a proper orientation to support physiologically-competent electron transfer. Saturation recovery EPR provides an attractive tool for monitoring spatial orientation of proteins in their interaction with surfaces in liquid phase. It is particularly valuable for metalloproteins engaged in redox reactions as a means to monitor the geometry and dynamics of formation of protein complexes in measurements that are independent of electron transfer processes.