Cytochrome aa 3 Oxygen Reductase Utilizes the Tunnel Observed in the Crystal Structures To Deliver O 2 for Catalysis.

Cytochrome aa ₃ is the terminal respiratory enzyme of all eukaryotes and many bacteria and archaea, reducing O ₂ to water and harnessing the free energy from the reaction to generate the transmembrane electrochemical potential. The diffusion of O ₂ 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 ₃ , a homologous enzyme of cytochrome aa ₃ in Thermus thermophilus, demonstrated that O ₂ 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 ₃ , this putative pathway appears partially occluded between the entrances and the reduction site. Also, the experimentally determined second-order rate constant for O ₂ delivery in cytochrome aa ₃ (∼10 ⁸ M ^-1 s ^-1 ) is 10 times slower than that in cytochrome ba ₃ (∼10 ⁹ M ^-1 s ^-1 ). A question to be addressed is whether cytochrome aa ₃ utilizes this X-ray-inferred tunnel as the primary pathway for O ₂ delivery. Using complementary computational methods, including multiple independent flooding MD simulations and implicit ligand sampling calculations, we probe the O ₂ delivery pathways in cytochrome aa ₃ of Rhodobacter sphaeroides. All of the O ₂ 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 ₂ delivery pathway in cytochrome aa ₃ . The rate constant for O ₂ delivery in cytochrome aa ₃ , approximated using the simulation results, is 10 times slower than in cytochrome ba ₃ , in agreement with the experimentally determined rate constants.