Identification of hydrogen bonding network for proton transfer at the quinol oxidation site of Rhodobacter capsulatus cytochrome bc1

Abstract

Cytochrome bc₁ catalyzes electron transfer from quinol (QH₂) 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 and two-proton bifurcation reaction leading to oxidation of QH₂ and reduction of the Rieske cluster and heme b_L. The proton paths associated with this reaction remain elusive. Here, we used site-directed mutagenesis and quantum mechanical calculations to analyze the contribution of protonable side chains located at the heme b_L side of the QH₂ oxidation site in Rhodobacter capsulatus cytochrome bc₁. We observe that the proton path is effectively switched off when H276 and E295 are simultaneously mutated to the nonprotonable 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. Quantum mechanical calculations indicated that H276F/E295V traps the side chain of Y147 in a position distant from QH₂, whereas 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 QH₂ 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 proton removal from QH₂ at the heme b_L side of QH₂ oxidation site.

Keywords: Rhodobacter capsulatus; cytochrome bc(1); enzymatic activity; proton transfer; quantum mechanical calculations; quinol oxidation.