DECONVOLUTING THE ENGINEERING AND ASSEMBLY

In respiratory systems, membrane-bound Complex III catalyzes the oxidation of ubiquinone and the reduction of a soluble cytochrome with the bioenergetic formation of a transmembrane proton gradient (∆μH+). Complex III turnover is initiated by a unique two electron oxidation of ubiquinone at the Qo site; one electron is delivered to a high potential chain containing an iron-sulfur cluster, cytochrome c1 and cytochrome c2, and a second electron is transferred to a low potential chain that terminates at the Qi site. All Complex III electron tunneling reactions are reversible, and a critical part of Complex III maintaining productive turnover is its suppression of energy-wasting reverse electron transfer reactions. The key to uncovering the controversial mechanism of Qo oxidation is determining how Complex III is regulated such that productive electron-transfer steps overwhelm unproductive steps. This thesis focuses on understanding the structural and biochemical tolerances of the redox cofactors in Complex III and applying that knowledge towards the design of a simple, but robust, amphiphilic maquette that is capable of transmembrane proton and electron transfer. In chapter two, kinetic studies of heme c1 mutants reveal that R. sphaeroides Complex III is engineered to withstand large changes in heme c1 active site residues while still preserving heme c1 midpoint potential and enzyme turnover. In chapter three, th