Cytochrome aa Oxygen Reductase Utilizes the Tunnel Observed in the Crystal Structures To Deliver O for Catalysis.

Cytochrome aa is the terminal respiratory enzyme of all eukaryotes and many bacteria and archaea, reducing O to water and harnessing the free energy from the reaction to generate the transmembrane electrochemical potential. The diffusion of O 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 ba, a homologous enzyme of cytochrome aa in Thermus thermophilus, demonstrated that O 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 aa, this putative pathway appears partially occluded between the entrances and the reduction site. Also, the experimentally determined second-order rate constant for O delivery in cytochrome aa (∼10 M s) is 10 times slower than that in cytochrome ba (∼10 M s). A question to be addressed is whether cytochrome aa utilizes this X-ray-inferred tunnel as the primary pathway for O delivery. Using complementary computational methods, including multiple independent flooding MD simulations and implicit ligand sampling calculations, we probe the O delivery pathways in cytochrome aa of Rhodobacter sphaeroides. All of the O 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 O delivery pathway in cytochrome aa. The rate constant for O delivery in cytochrome aa, approximated using the simulation results, is 10 times slower than in cytochrome ba, in agreement with the experimentally determined rate constants.