Spectroscopic Investigation of the Roles of F282 and W172 in the Ligand Pathway of Aa3 Cytochrome C Oxidase from Rhodobacter Sphaeroides

Abstract

Knowledge of how the protein environment can tune ligand pathways and metal-based prosthetic group(s) in heme-copper oxidase is fundamental for understanding the catalytic functions of these enzymes. To establish possible relationships between the structural diversity of these enzymes and their ligand binding dynamics, we used photolabile O2 and NO complexes to compare the kinetics of molecular oxygen (O2) and nitric oxide (NO) reacting with reduced heme-copper oxidases from different species. For example, time-resolved optical absorption (TROA) measurements in our laboratory have shown that O2 and NO bind to reduced Thermos thermophilus ba3 with a superfast second-order rate constant of 1×109 M−1s−1, which is 10-times faster than observed for the aa3 oxidases.In this study, the structural and mechanistic features that moderate ligand access to the active site of aa3 cytochrome c oxidase (CcO) in Rhodobacter sphaeroidesaa3 were investigated by TROA spectroscopy. Two bulky amino acid residues (tryptophan 172 and phenylalanine 282) have been suggested to generate a putative constriction point along the ligand pathway to the active site in R. sphaeroides CcO. To investigate the possible roles of these amino acid residues in modulating ligand access, the tryptophan 172 and phenylalanine 282 were mutated to the less bulky tyrosine, and threonine residues present in Thermus thermophilusba3 oxidase. The reactions of O2 and NO with the fully reduced W172Y/F282T double mutant of the R. sphaeroides were investigated by TROA spectroscopy in combination with photolabile O2 and NO carriers. The results suggest that the amino acid residues under investigation may play an important structural role in ligand access to the active site in R. sphaeroidesaa3.