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 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.

Photo-induced dynamics of the heme centers in cytochrome bc1

“…Our results indeed coincide with the fact that electron transfer can be readily initiated from the b-heme without the need for close contact with an external electron acceptor such as quinones or molecular oxygen. (6) It was also reported that the axial histidylimidazole ligands of the b L heme have a distinct ionic character, 40 which would favour electron transfer processes. This electronegative character of at least one of the two His ligands was explained by the presence of nearby arginine residues.…”

“…1 The cyt bc 1 complex is a dimer whose monomer comprises four key elements that are directly involved in the electronic pathway (Fig. 1): (1) the heme c 1 is located near the intermembrane-space side of the membrane; it is situated near the cyt c docking interface and serves as an electron donor for the reduction of cyt c; 2,[5][6][7] (2) the Rieske iron-sulfur cluster mediates electron transfer between the quinol at the Q o site and the heme c 1 via a series of conformational changes; 1 (3) and (4) the hemes b L and b H that mediate electron transfer between the two quinone binding sites, Q o and Q i sites. 2 Depending on the reaction stage, the three iron centers of the hemes are found in either their ferric (Fe(III)) or ferrous forms (Fe(II)) and are all characterized by a 6-coordinated (6-c) lowspin state, therefore minimizing the reorganization energy required to undergo the electron transfer.…”

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…At ionic strength typical of physiological conditions, the complexes are very short-lived and the stationary concentration of bound cytochrome c is low. This implicates a mechanism in which under physiological conditions cytochrome c does not form stable, longlived complexes but rather constantly collides with the surface of cytochrome bc 1 and electron transfer takes place coincidentally with one of such collision (117,206,221).…”

“…The transient binding of cytochrome c to cytochrome bc 1 in solution was inferred from the analysis of electron transfer between these proteins (175) and from the measurements based on the techniques that were independent of electron transfer, such as plasmon wave-guide resonance spectroscopy (75) or EPR (206,221,224).…”

Effect of H bond removal and changes in the position of the iron–sulphur head domain on the spin–lattice relaxation properties of the [2Fe–2S]2+Rieske cluster in cytochrome bc1

“…EPR spectroscopy was performed on a Bruker Elexsys E580 spectrometer operating at X- and Q-bands using similar equipment and settings as described previously in ref. 26 and 35. CW spectra were recorded at the X-band over the temperature range 20–120 K using a non-saturating microwave power.…”