Molecular organization of cytochrome c2 near the binding domain of cytochrome bc1 studied by electron spin-lattice relaxation enhancement.
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 paramagnetic 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 and cytochrome bc1 increases. The spatial arrangement of the protein complex at various ionic strengths is different. Above 150 mM NaCl the lifetime of the complexes becomes so short that they are undetectable. All together the results indicate that cytochrome c2 molecules, over the range of salt concentration encompassing physiological ionic strength, do not form stable, long-lived complexes but rather constantly collide with the surface of cytochrome bc1 and ET takes place coincidentally with one of these collisions.
Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University , 30-387 Kraków, Poland.
Rafał
R
Pietras
Marcin
M
Sarewicz
Artur
A
Osyczka
D045362:Cytochromes c2 / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism; D004583:Electrons; D007700:Kinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016354:Rhodobacter capsulatus / Q000378:metabolism; D013113:Spin Labels
D013113:Spin Labels; D045362:Cytochromes c2; D014450:Electron Transport Complex III