Research ArticleBIOCHEMISTRY

Splitting of the O–O bond at the heme-copper catalytic site of respiratory oxidases

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Science Advances  16 Jun 2017:
Vol. 3, no. 6, e1700279
DOI: 10.1126/sciadv.1700279

Figures

  • Fig. 1 Structure of the catalytic site.

    The redox-active cofactors of cyt. ba3. The low-spin heme b and heme a3 are depicted in cyan; CuB and the dinuclear CuA are indicated by yellow spheres. The cross-linked residues His233 and Tyr237, located in proximity of the catalytic site, are shown. The image was prepared using the software PyMOL [Protein Data Bank code 3S8F (20)]. The dashed line is the approximate position of the membrane surface on the positive (p) side of the membrane.

  • Fig. 2 Absorbance changes during reaction of the fully reduced ba3 CytcO with O2.

    (A and B) pH 7 at 10° and 45°C, respectively. (C and D) pH 10 at 10° and 45°C, respectively. At 610 and 560 nm, the absorbance changes are mainly attributed to redox changes at heme a3 and heme b, respectively. The black lines are fits of the data with a model that is described by a sum of three exponential functions. The rate constants obtained from the fit are given in the text and in Table 1. The difference between the fit and the data (that is, the residuals) is shown below each panel. In addition, in (A), we show the residuals of a fit with a single rate constant (5 × 104 s−1) for electron transfer from heme b to the catalytic site and PR formation (gray lines). Experimental conditions after mixing: 0.6 to 0.8 μM CytcO (scaled to 1 μM), 0.05% DDM, 90 mM Hepes (pH 7) or 90 mM CAPS (pH 10), and ~1 mM O2. The cuvette path length was 1.00 cm. The 560-nm traces are shifted up by 1.7 × 10−3 units for clarity.

  • Fig. 3 Temperature dependence of the rate constants.

    The rates of O2 binding (Arrhenius plots), heme b oxidation, and PR formation are shown as indicated in the graphs for (A) pH 7 and (B) pH 10. Conditions were the same as in Fig. 2.

  • Fig. 4 Kinetics of the reaction of mixed-valence CytcO with O2.

    The reaction was monitored at 610 nm. Experimental conditions after mixing: 0.6 to 0.8 μM CytcO (scaled to 1 μM), 0.05% DDM, and 90 mM Hepes (pH 7.4). A laser artifact at t = 0 has been truncated for clarity.

  • Fig. 5 Mechanism of the initial steps of O2 reduction catalyzed by the ba3 CytcO.

    Energy profiles for the initial reaction steps after binding of O2 to heme a3, reflecting the approximate rate constants observed at pH 7 and 10°C. The reduced CytcO (state R) binds O2 to state A. In state IP, a peroxy state is formed upon oxidation of the heme a3 iron and either CuB or Tyr237. The peroxy state is stabilized by a proton from Tyr237. The reaction sequence below the diagram illustrates the specific electron transfers within the ba3 CytcO. Two possible configurations that are in resonance are shown for state IP. It is a geometric single minimum with an electronic structure that is a mixture (resonance) of two main electronic configurations.

Tables

  • Table 1 Rate constants for the early steps of O2 reduction at low and high temperature for pH 7 and 10, respectively.

    Errors are the SDs for n = 3 (pH 10 data) or n = 4 (pH 7 data) measurements.

    Rate constant (s−1) (Time constant) (μs)
    T (°C)pH 7pH 10
    O2 binding (610-nm decay)10(2.8 ± 0.8) × 105 (3.6)(2.9 ± 0.1) × 105 (3.4)
    45(2.7 ± 0.8) × 105 (3.7)(4 ± 1) × 105 (2.5)
    Heme b oxidation (kb) (560-nm decay)10(9 ± 4) × 104 (11)(2.6 ± 0.6) × 104 (38)
    45(1.9 ± 0.2) × 105 (5.3)(1.9 ± 0.2) × 105 (5.3)
    PR formation (kP) (610-nm increase)10(9 ± 1) × 103 (110)(8.7 ± 0.3) × 103 (120)
    45(1.8 ± 0.5) × 105 (5.5)(1.5 ± 0.5) × 105 (6.7)
    Ratio (kb/kP)10103
    451.11.3
  • Table 2 Thermodynamic parameters.

    The parameters were determined by fitting the data in Fig. 3 with the expression Embedded Image. The range of A values was 30 to 40% of the values in the table (when taking into account the error bars in Fig. 3).

    pH 7pH 10
    Ea (kJ/mol)ln AEa (kJ/mol)ln A
    Heme b oxidation
    (560-nm first decay)
    17 ± 21840 ± 427
    PR formation
    (610-nm increase)
    60 ± 53460 ± 534

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