Dynamic water networks in cytochrome cbb3 oxidase

Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327–330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb3. In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b3 both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b3. Protonation of Glu323 provides a mechanism to tune the redox potential of heme b3 with possible implications for proton pumping.

► Water networks in cytochrome cbb3 were studied by MD simulations.
► Water wires connected Glu323 with both P- and N-sides of the membrane.
► The proton affinity of Glu323 increased upon reduction of the active site.
► Water wires for proton translation and oxygen reduction chemistry were observed.