Cytochrome aa₃ Oxygen Reductase Utilizes the Tunnel Observed in the Crystal Structures To Deliver O₂ for Catalysis

Cytochrome aa₃ is the terminal respiratory enzyme of all eukaryotes and many bacteria and archaea, reducing O₂ to water and harnessing the free energy from the reaction to generate the transmembrane electrochemical potential. The diffusion of O₂ to the heme-copper catalytic site, which is buried deep inside the enzyme, is the initiation step of the reaction chemistry. Our previous molecular dynamics (MD) study with cytochrome ba₃, a homologous enzyme of cytochrome aa₃ in Thermus thermophilus, demonstrated that O₂ diffuses from the lipid bilayer to its reduction site through a 25 Å long tunnel inferred by Xe binding sites detected by X-ray crystallography [Mahinthichaichan, P., Gennis, R., and Tajkhorshid, E. (2016) Biochemistry 55, 1265-1278]. Although a similar tunnel is observed in cytochrome aa₃, this putative pathway appears partially occluded between the entrances and the reduction site. Also, the experimentally determined second-order rate constant for O₂ delivery in cytochrome aa₃ (∼10⁸ M^-1 s^-1) is 10 times slower than that in cytochrome ba₃ (∼10⁹ M^-1 s^-1). A question to be addressed is whether cytochrome aa₃ utilizes this X-ray-inferred tunnel as the primary pathway for O₂ delivery. Using complementary computational methods, including multiple independent flooding MD simulations and implicit ligand sampling calculations, we probe the O₂ delivery pathways in cytochrome aa₃ of Rhodobacter sphaeroides. All of the O₂ molecules that arrived in the reduction site during the simulations were found to diffuse through the X-ray-observed tunnel, despite its apparent constriction, supporting its role as the main O₂ delivery pathway in cytochrome aa₃. The rate constant for O₂ delivery in cytochrome aa₃, approximated using the simulation results, is 10 times slower than in cytochrome ba₃, in agreement with the experimentally determined rate constants.