3] Structure of beef heart cytochrome-c oxidase obtained by combining studies of two-dimensional crystals with biochemical experiments

Abstract

Publisher Summary This chapter describes the mammalian cytochrome- c oxidase by combining the data of biochemical studies with a low-resolution structure provided by electron microscopy and image reconstruction. Cytochrome- c oxidase is the terminal enzyme in the mitochondrial electron transfer chain, catalyzing the four electron reduction of molecular oxygen and coupling this reaction to the generation of a proton gradient across the mitochondrial inner membrane. As purified under certain conditions, the beef heart enzyme is arranged in 2D crystals, and this allows for detailed structural analysis. 2D crystals of beef heart cytochrome- c oxidase are made by three different procedures that involve (1) purifying the enzyme in Triton X-114 followed by Triton X-100, (2) using deoxycholate alone as the detergent, and (3) using cholate followed by deoxycholate. The electron microscopy done using ice rather than phosphotungstate or uranylacetate as the contrast stain for water against protein, allows higher resolution imaging of the protein and allows direct visualization of a cytochrome c -cytochrome- c oxidase complex.