Identification of hydrogen bonding network for proton transfer at the quinol oxidation site of Rhodobacter capsulatus cytochrome $bc_{1}$

Cytochrome $bc_{1}$ catalyzes electron transfer from quinol ($QH_{2}$) to cytochrome c in reactions coupled to proton translocation across the energy-conserving membrane. Energetic efficiency of the catalytic cycle is secured by a two-electron, two-proton bifurcation reaction leading to oxidation of $QH_{2}$ and reduction of the Rieske cluster and heme $QH_{2}$. The proton paths associated with this reaction remain elusive. Here we used site-directed mutagenesis and quantum mechanical (QM) calculations to analyze the contribution of protonable side chains located at the heme $QH_{2}$ side of the quinol oxidation site ($Q_{o}$) in Rhodobacter capsulatus cytochrome $bc_{1}$. We observe that the proton path is effectively switched off when H276 and E295 are simultaneously mutated to the non-protonable residues in the H276F/E295V double mutant. The two single mutants, H276F or E295V, are less efficient, but still transfer protons at functionally-relevant rates. Natural selection exposed two single mutations, N279S and M154T, that restored the functional proton transfers in H276F/E295V. QM calculations indicated that H276F/E295V traps the side chain of Y147 in a position distant from $QH_{2}$, while either N279S or M154T induce local changes releasing Y147 from that position. This shortens the distance between the protonable groups of Y147 and D278 and/or increases mobility of the Y147 side chain, which makes Y147 efficient in transferring protons from $QH_{2}$ toward D278 in H276F/E295V. Overall, our study identified an extended hydrogen bonding network, build up by E295, H276, D278 and Y147, involved in efficient removal of the proton from $QH_{2}$ at the heme $b_{L}$ side of $Q_{o}$.