Isolation of yeast complex IV in native lipid nanodiscs

Abstract We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (Cyt c O) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one Cyt c O per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm × 14 nm. Based on this size we estimated that each Cyt c O was surrounded by ~ 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though Cyt c O forms a supercomplex with cytochrome bc 1 in the mitochondrial membrane, cyt. bc 1 was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated Cyt c O. The native nanodiscs displayed an O 2 -reduction activity of ~ 130 electrons Cyt c O − 1  s − 1 and the kinetics of the reaction of the fully reduced CytcO with O 2 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the Cyt c O catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.