Structural mechanism of proton pumping by ba3-type cytochrome c oxidase

Cytochrome c oxidase (CcO) utilizes the energy released as dioxygen is reduced to two water molecules to pump protons across an energy transducing biological membrane. X-ray structures of CcO have been known for three decades1-3 and all electron transfer and proton uptake and release steps have been characterized.4,5 However, it remains unclear how the reduction of dioxygen at the enzyme’s active-site achieves proton pumping. Here we apply time-resolved serial femtosecond X-ray crystallography to observe structural changes in the ba3-type CcO from Thermus thermophilus following the release of photocaged oxygen using a UV laser flash. In the resting oxidised state, chloride ions are observed to ligate CuB of the active site and the farnesyl oxygen of haem a3. Both ions are displaced when the enzyme is fully reduced. Water molecules are observed to order and disorder within the active-site 3.3 ms after photocaged oxygen is released, creating and disrupting water mediated pathways for proton transport through the enzyme. Requiring that the active-site charges sum to zero for every step of the catalytic cycle yields a step-by-step atomistic model for proton pumping by ba3-type CcOs.