The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres

The steady-state behaviour of isolated mammalian cytochrome c oxidase was examined by increasing the rate of reduction of cytochrome c. Under these conditions the enzyme's 605 (haem a), 655 (haem a(3)/Cu-B) and 830 (Cu-A) nm spectral features behaved as if they were at near equilibrium with cytochrome c (550 nm). This has implications for non-invasive tissue measurements using visible (550, 605 and 655 nm) and near-IR (830 nm) light. The oxidized species represented by the 655 mn band is bleached by the presence of oxygen intermediates P and F (where P is characterized by an absorbance spectrum at 607 nm relative to the oxidized enzyme and F is characterized by an absorbance spectrum at 580 nm relative to the oxidized enzyme) or by reduction of haem a(3) or Cu-B. However, at these ambient oxygen levels (far above the enzyme K-m), the populations of reduced haem a(3) and the oxygen intermediates were very low (<10%). We therefore interpret 655 nm changes as reduction of the otherwise spectrally invisible Cu-B centre. We present a model where small anti-cooperative redox interactions Occur between haem a-Cu-A-Cu-B (steady-state potential ranges: Cu-A, 212-258 mV; haem a, 254-281 mV; Cu-B, 227-272 mV). Contrary to static equilibrium measurements, in the catalytic steady state there are no high potential redox centres (>300mV). We find that the overall reaction is correctly described by the classical model in which the Michaelis intermediate is a ferrocytochrome c-enzyme complex. However, the oxidation of ferrocytochrome c in this complex is not the sole rate-determining step. Turnover is instead dependent upon electron transfer from haem a to haem a(3), but the haem a potential closely matches cytochrome c at all times.