Molecular Organization of Cytochrome c2 near the Binding Domain of Cytochrome bc1 Studied by Electron Spin−Lattice Relaxation Enhancement

ABSTRACT: Measurements of specific interactions between proteins are challenging. In redox systems, interactions involve surfaces near the attachment sites of cofactors engaged in interprotein electron transfer (ET). Here we analyzed binding of cytochrome c2 to cytochrome bc1 by measuring para-magnetic relaxation enhancement (PRE) of spin label (SL) attached to cytochrome c2. PRE was exclusively induced by the iron atom of heme c1 of cytochrome bc1, which guaranteed that only the configurations with SL to heme c1 distances up to ∼30 Å were detected. Changes in PRE were used to qualitatively and quantitatively characterize the binding. Our data suggest that at low ionic strength and under an excess of cytochrome c2 over cytochrome bc1, several cytochrome c2 molecules gather near the binding domain forming a “cloud ” of molecules. When the cytochrome bc1 concentration increases, the cloud disperses to populate additional available binding domains. An increase in ionic strength weakens the attractive forces and the average distance between cytochrome c2 an