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 10(9) 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.