Molecular organization of cytochrome c_{2} near the binding domain of cytochrome bc_{1} studied by electron spin-lattice relaxation enhancement

[Image: see text] 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 c(2) to cytochrome bc(1) by measuring paramagnetic relaxation enhancement (PRE) of spin label (SL) attached to cytochrome c(2). PRE was exclusively induced by the iron atom of heme c(1) of cytochrome bc(1), which guaranteed that only the configurations with SL to heme c(1) 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 c(2) over cytochrome bc(1), several cytochrome c(2) molecules gather near the binding domain forming a “cloud” of molecules. When the cytochrome bc(1) 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 c(2) and cytochrome bc(1) 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 c(2) 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 bc(1) and ET takes place coincidentally with one of these collisions

Zinc Inhibition of Bacterial Cytochrome bc1 Reveals the Role of Cytochrome b E295 in Proton Release at the Qo Site

The cytochrome (cyt) bc1 complex (cyt bc1) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc1 is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn2+-induced inhibition of Rhodobacter capsulatus cyt bc1 using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn2+ binding mechanism and its inhibitory effect on cyt bc1 function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn2+ on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn2+ binding affinity. The kinetic study showed that wild-type cyt bc1 and its E295V mutant have similar levels of apparent Km values for decylbenzohydroquinone as a substrate (4.9 +/- 0.2 and 3.1 +/- 0.4 μM, respectively), whereas their KI values for Zn2+ are 8.3 and 38.5 μM, respectively. The calorimetry-based KD values for the high-affinity site of cyt bc1 are on the same order of magnitude as the KI values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn2+, whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn2\ufe. Our overall results indicate that the proton-active E295 residue near the Q o site of cyt bc1 can bind directly to Zn2+, resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q o site of cyt bc1