All the O₂ Consumed by Thermus thermophilus Cytochrome ba₃ Is Delivered to the Active Site through a Long, Open Hydrophobic Tunnel with Entrances within the Lipid Bilayer

Cytochrome ba₃ is a proton-pumping heme-copper oxygen reductase from the extreme thermophile Thermus thermophilus. Despite the fact that the enzyme's active site is buried deep within the protein, the apparent second order rate constant for the initial binding of O₂ to the active-site heme has been experimentally found to be 10⁹ M^-1 s^-1 at 298 K, at or near the diffusion limit, and 2 orders of magnitude faster than for O₂ binding to myoglobin. To provide quantitative and microscopic descriptions of the O₂ delivery pathway and mechanism in cytochrome ba₃, extensive molecular dynamics simulations of the enzyme in its membrane-embedded form have been performed, including different protocols of explicit ligand sampling (flooding) simulations with O₂, implicit ligand sampling analysis, and in silico mutagenesis. The results show that O₂ diffuses to the active site exclusively via a Y-shaped hydrophobic tunnel with two 25-Å long membrane-accessible branches that coincide with the pathway previously suggested by the crystallographically identified xenon binding sites. The two entrances of the bifurcated tunnel of cytochrome ba₃ are located within the lipid bilayer, where O₂ is preferentially partitioned from the aqueous phase. The largest barrier to O₂ migration within the tunnel is estimated to be only 1.5 kcal/mol, allowing O₂ to reach the enzyme active site virtually impeded by one-dimensional diffusion once it reaches a tunnel entrance at the protein surface. Unlike other O₂-utilizing proteins, the tunnel is "open" with no transient barriers observed due to protein dynamics. This unique low-barrier passage through the protein ensures that O₂ transit through the protein is never rate-limiting. (Figure Presented).