Conserved glycine 232 in the ligand channel of ba3 cytochrome oxidase from Thermus thermophilus.

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O-2 and NO with the fully reduced G232V mutant of ba(3) cytochrome c oxidase from Thermus thermophilus (Tt ba(3)) in which a conserved glycine residue in the O-2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa(3) cytochrome c oxidase suggested that the valine completely blocked the access of O-2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617-11621]. Using photolabile O-2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O-2 and NO binding are not significantly affected in the Tt ba(3) G232V mutant. Classical molecular dynamics simulations of diffusion of O-2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O-2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.