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Open Access Publications from the University of California

Time-resolved optical absorption spectroscopic studies of cytochrome c oxidase from Rhodobacter sphaeroides and ubiquinol oxidase from Escherichia coli

  • Author(s): Cassano, Jennifer
  • Advisor(s): Einarsdottir, Olof
  • et al.
Creative Commons 'BY-NC-ND' version 4.0 license

Knowledge of the mechanism and kinetics of O2 reduction in heme-copper oxidases (HCOs) is critical for understanding the catalytic functions of these enzymes. To explore this issue, dioxygen reduction in the fully reduced wild-type fusion aa3 cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3) was investigated by multi-wavelength time-resolved optical absorption (TROA) spectroscopy, in combination with the CO flow-flash technique. The reaction of O2 with the fully reduced E286Q mutant of Rs aa3, in which a conserved glutamic acid residue was mutated to glutamine, was also investigated. In the wild-type fusion Rs aa3, the O2-bound compound AR decayed directly to the oxyferryl intermediate F, while in the bovine heart aa3 enzyme, compound AR decayed to the so-called PR ("peroxy") intermediate prior to the formation of F. However, in the E286Q Rs aa3, PM was the terminal intermediate. The detection of PR during O2 reduction in the bovine heart enzyme but not in the wild-type fusion Rs aa3 may be due to differences in the relative rates of electron and proton transfer in the two enzymes.

The reactions of O2 and NO with the fully reduced wild-type fusion Rs aa3 were investigated in the presence and absence of CO using photolabile O2- and NO-complexes and multi-wavelength TROA spectroscopy. The second-order rate constants for O2 and NO binding in the wild-type fusion Rs aa3 were the same in both the absence and presence of CO (~1×108 M‒1s‒1), suggesting that CO does not impede ligand access to the active site in the wild-type fusion Rs aa3, unlike in the Thermus thermophilus ba3 (Tt ba3) enzyme. Moreover, in the absence of CO, O2 and NO binding in the wild-type fusion Rs aa3 is 10 times slower than in Tt ba3 (~1×108 M‒1s‒1 in Rs aa3 and ~1×109 M‒1s‒1 in Tt ba3). This 10-fold kinetic difference is attributed to structural differences in the proposed ligand channels of Rs aa3 and Tt ba3.

The CO photodissociation and rebinding dynamics of the wild-type, G283L, and W172Y/F283T Rs aa3 enzymes were investigated by TROA spectroscopy. Multiple enzyme conformers of the G283L and W172Y/F282T Rs aa3 enzymes were observed. Interestingly, in the W172Y/F282T Rs aa3 enzyme, mutation of the bulky tryptophan and phenylalanine residues in the Rs aa3 ligand channel to the corresponding smaller residues in Tt ba3 resulted in ~500 times faster rate of CO recombination compared to that in the wild-type Rs aa3. The CO was at least partially trapped in the active site cavity of the Rs aa3 mutants, resulting in the fast CO recombination rate.

The reactions of the mixed-valence wild-type fusion Rs aa3 and the mixed-valence Escherichia coli bo3 ubiquinol oxidase (Ec bo3) with dioxygen were investigated by TROA spectroscopy. In Ec bo3, F is the intermediate primarily formed upon the decay of compound A in both the fully reduced and mixed-valence forms of the enzyme. However, in the fully reduced wild-type fusion Rs aa3, compound AR decays to F, while in the mixed-valence Rs aa3, AM decays to PM. If a protonable functional group donates a proton during the conversion of P to F in Rs aa3 and Ec bo3, then the detection of PM as the final intermediate during the reaction of the mixed-valence Rs aa3 with O2 suggests that this functional group may not be protonated in the mixed-valence Rs aa3 enzyme.

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