Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respi-ration is commonly assigned to the fully reduced species of hemes a or a3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420-450 nm), alpha (560-630 nm) and red (630-700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (Delta psi m) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are Delta psi m-dependent, out of equilibrium, and therefore much slower than the Delta psi m-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.

Automated summary

The catalytic cycle of cytochrome c oxidase (COX) involves multiple intermediate states of the heme a3-CuB binuclear center. The fully reduced species of hemes a or a3, Peroxy (P), and Ferryl (F) intermediates are observed in respiration, while the R form is only detectable under nearly anoxic conditions. The reactions involving P and F species are dependent on transmembrane potential and slower than the oxidation of the R intermediate.