A. Continuous illumination. Oxidation of cytochrome c, and reaction center, and reduction of cytochrome b_H, in chromatophores from wild-type Ga strain, a strain grown so as to overproduce the bc complex, and a cytochrome c₂ overproducing strain. The kinetic traces are normalized by the extinction coefficient, and adjusted so that the deflection due to the full reaction center change was the same in each strain, to show relative concentration; an upward deflection shows oxidation of cyt c or RC, and reduction of cyt b_H. The continuous illumination was from a multiple LED array, giving sufficient intensity to turn over the reaction center in < 10 ms. The traces (timescale is in ms) show directly the different stoichiometries in the mutant strains, and show unambiguously that electron transfer between bc₁ complex, cyt c₂ and reaction center occurs with kinetics in the ms range at stoichiometries quite different from those expected from supercomplexes.

B. Flash illumination. Traces are normalized as above. An upward deflection indicates oxidation for reaction center, or reduction for cyt b and cyt c.
Note various consequences of the diffusional role of cyt c₂. These effects are NOT expected in the supercomplex hypothesis:

• the amplitudes of cyt c_{(1 + 2)} or heme b_H changes reflected the stoichiometry, and do not follow the fixed stoichiometry expected in supercomplexes;
• the kinetics in strains with excess cyt c₂ or bc₁ complex show no indication of the biphasic pattern expected if there was an impediment to cyt c₂ diffusion outside a supercomplex;
• the kinetics of cyt c₁ oxidation (see panel C below) are more rapid when an excess of ferricyt c₂ is generated in the cyt c₂ overproducer;
• reaction center re-reduction was more rapid in strains with an excess of cyt c₂ or bc₁ complex.
  
  

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  C. Kinetics of cytochrome c oxidation on a single flash. Comparison of kinetics of oxidation of cyt c in wild-type and c₂-overproducer strain. Note, the time-of-flash has been offset for clarity. The traces were not normalized, and the reaction center concentration was slightly higher in the overproducer strain. At the wavelength pairs used, the kinetics of both cyt c₂ and cyt c₁ were monitored. Because of instrumental limitations (time constant was ~10 µs), the oxidation kinetics of cyt c₂ were not resolved. In the wild-type, the slower phase of cyt c₁ oxidation was clearly separated from the more rapid cyt c₂ phase. In the cyt c₂ overproducer, the slower phases, which include the cyt c₁ kinetics, were much faster, and the latter could not be so easily separated from cyt c₂. The amplitude of the combined phases was almost twice that in the wild-type (see panel B for normalized traces).