This review focuses on the type A cytochrome c oxidases (CcO), which are found in all mitochondria and also in several aerobic bacteria. CcO catalyzes the respiratory reduction of dioxygen (O₂) to water by an intriguing mechanism, the details of which are fairly well understood today as a result of research for over four decades. Perhaps even more intriguingly, the membrane-bound CcO couples the O₂ reduction chemistry to translocation of protons across the membrane, thus contributing to generation of the electrochemical proton gradient that is used to drive the synthesis of ATP as catalyzed by the rotary ATP synthase in the same membrane. After reviewing the structure of the core subunits of CcO, the active site, and the transfer paths of electrons, protons, oxygen, and water, we describe the states of the catalytic cycle and point out the few remaining uncertainties. Finally, we discuss the mechanism of proton translocation and the controversies in that area that still prevail.

Membrane-bound cytochrome c oxidase catalyzes cell respiration in aerobic organisms and is a primary energy transducer in biology. The two halves of the catalytic cycle may be studied separately: in an oxidative phase, the enzyme is oxidized by O ₂ , and in a reductive phase, the oxidized enzyme is reduced before binding the next O ₂ molecule. Here we show by time-resolved membrane potential and pH measurements with cytochrome oxidase liposomes that, with both phases in succession, two protons are translocated during each phase, one during each individual electron transfer step. However, when the reductive phase is not immediately preceded by oxidation, it follows a different reaction pathway no longer coupled to proton pumping. Metastable states with altered redox properties of the metal centers are accessed during turnover and relax when external electron donors are exhausted but recover after enzyme reduction and reoxidation by O ₂ . The efficiency of ATP synthesis might be regulated by switching between the two catalytic pathways.

Electron transfer in complex I from Escherichia coli was investigated by an ultrafast freeze-quench approach. The reaction of complex I with NADH was stopped in the time domain from 90 μs to 8 ms and analyzed by electron paramagnetic resonance (EPR) spectroscopy at low temperatures. The data show that after binding of the first molecule of NADH, two electrons move via the FMN cofactor to the iron–sulfur (Fe/S) centers N1a and N2 with an apparent time constant of ≈90 μs, implying that these two centers should have the highest redox potential in the enzyme. The rate of reduction of center N2 (the last center in the electron transfer sequence) is close to that predicted by electron transfer theory, which argues for the absence of coupled proton transfer or conformational changes during electron transfer from FMN to N2. After fast reduction of N1a and N2, we observe a slow, ≈1-ms component of reduction of other Fe/S clusters. Because all elementary electron transfer rates between clusters are several orders of magnitude higher than this observed rate, we conclude that the millisecond component is limited by a single process corresponding to dissociation of the oxidized NAD ⁺ molecule from its binding site, where it prevents entry of the next NADH molecule. Despite the presence of approximately one ubiquinone per enzyme molecule, no transient semiquinone formation was observed, which has mechanistic implications, suggesting a high thermodynamic barrier for ubiquinone reduction to the semiquinone radical. Possible consequences of these findings for the proton translocation mechanism are discussed.