Zinc inhibition of bacterial cytochrome bc₁ reveals the role of cytochrome b E295 in proton release at the Q_o 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 bc ₁ 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 K_m values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K _I values for Zn²⁺ are 8.3 and 38.5 μM, respectively. The calorimetry-based K_D values for the high-affinity site of cyt bc₁ are on the same order of magnitude as the K_I 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⁺. 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.