Investigation Of The O2 Reduction Mechanism And Ligand Access In Cytochrome C Oxidase From Thermus Thermophilus
- Author(s): Funatogawa, Chie
- Advisor(s): Einarsdóttir, Ólöf
- et al.
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.