Flexibility and Hydration of the Q Site Determine Multiple Pathways for Proton Transfer in Cytochrome .

The detailed catalytic activity of cytochrome (or respiratory complex III) and the molecular mechanism of the Q cycle remain elusive. At the Q site, the cycle begins with oxidation of the coenzyme-Q substrate (quinol form) in a bifurcated two-electron transfer to the iron-sulfur (FeS) cluster and the heme center. The release of two protons during quinol oxidation and their transfer is less understood, with one proton likely delivered to the histidine side chain attached to the FeS cluster. Here, we present extensive molecular dynamics simulations with enhanced sampling of side-chain torsions at the Q site and analyze available sequences and structures of several homologs to probe the interactions of quinol with potential proton acceptors and identify viable pathways for proton transfer. Our findings reveal that side chains at the Q site are highly flexible and can adopt multiple conformations. Consequently, the quinol head is also flexible, adopting three distinct binding modes. Two of these modes are proximal to the heme and represent reactive conformations capable of electron and proton transfer, while the third, more distal mode likely, represents a prereactive state, consistent with recent cryo-EM structures of with bound coenzyme-Q. The Q site is highly hydrated, with several water molecules bridging interactions between the quinol head and the conserved side chains Tyr147, Glu295, and Tyr297 in cytochrome (numbering according to ), facilitating proton transfer. A hydrogen bond network and at least five distinct proton wires are established and possibly transport protons via a Grotthuss mechanism. Asp278 and propionate-A of heme in cytochrome are in direct contact with external water and are proposed as the final proton acceptors. The intervening water molecules in these proton wires exhibit low mobility, and some have been resolved in recent experimental structures. These results help to elucidate the intricate molecular mechanism of the Q-cycle and pave the way to a detailed understanding of chemical proton transport in several bioenergetic enzymes that catalyze coenzyme-Q redox reactions.