Crystallographic investigation of the structure and mechanistic cycle of the bovine cytochrome bc(1) complex

This thesis describes the purification, crystallization, and molecular structure of the bovine mitochondrial cytochrome bc₁ complex, a bi-functional integral membrane protein complex that forms the central component of the respiratory electron transport chain. Pure and homogeneous samples attained through detergent solubilization and chromatographic purification have been used to grow crystals of the bc₁ complex in four different morphologies. Three of these crystals led to the determination of the molecular structure of the bc₁ complex through x-ray crystallographic methods.; The structural models based on the three crystals forms reveal snapshots of this enzyme in three different conformations, with the “Rieske” iron-sulfur protein subunit showing the most significant differences between the three conformations. These differences provide insights into the mechanism of electron transfer by the bc₁ complex, including an explanation for the bifurcation of the electron flow during the mechanistic cycle of the enzyme. Other features of the structure, such as the docking sites of the native ligands cytochrome c and ubiquinol, explain many prior observations based on biochemical and genetics studies. The structure also reveals the mitochondrial targeting presequence of the iron-sulfur protein (subunit 9) lodged between subunits 1 and 2 at the matrix side of the complex. These two subunits are related to the matrix processing peptidase, and the structure unveils the mechanism of mitochondrial targeting presequence recognition and provides the first structural model of a matrix processing peptidase.