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.
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