UC Santa Cruz

Knowledge of the mechanism of O₂ reduction and ligand access in heme-copper oxidases is essential for understanding how the protein environment modulates the function of these enzymes, which play a key role in energy production in living cells. To explore this issue, the reactions of O₂ and nitric oxide (NO) with the fully reduced wild-type Thermus thermophilus (Tt) ba₃ oxidase and selected ligand channel mutants were investigated using time-resolved optical absorption spectroscopy in the absence and presence of CO with photolabile O₂ and NO carriers. The second-order rate constant of ~1 x 10⁹ M^-1 s^-1 for O₂/NO binding in the wild-type ba₃ in the absence of carbon monoxide (CO) is 10-times faster than in the presence of CO and for the bovine enzyme in the absence and presence of CO. These results suggest inherent differences in the ligand channels between the two enzymes. The rate of O₂/NO binding was found to be ~5 times slower in the ba₃ Y133W mutant where the tryptophan constriction point residue observed in the ligand channel of bovine aa₃ was inserted into ba₃. No effect on O₂or NO binding was observed when 1) the T231 residue in ba₃ was replaced by the phenylalanine "constriction" point residue in the ligand channel of the aa₃ oxidases, 2) the A120 and A204 residues located near the presumed ligand entry/exit points of ba₃ were mutated to phenylalanine, and 3) the highly conserved G232 residue, located in the ligand channel close to the binuclear center in Tt ba₃, was mutated to the larger valine residue. These results suggest conformational freedom of the respective phenylalanine side chains in the T231F, A120F, A204F and A120F/A204F mutants and the valine residue in G232V. In contrast, mutation of the highly conserved V236 in the ligand channel of Tt ba₃ to increasing larger residue, progressively slowed down NO and O₂ access to the active site. Overall, these results suggest more open ligand channel in Tt ba₃, which may reflect the evolutionary adaptation to increase the rate of O₂ diffusion to the binuclear center, allowing the enzyme to function under microaerobic conditions.