Deconvoluting the Engineering and Assembly Instructions for Complex Iii Activity

Thumbnail Image
Degree type
Doctor of Philosophy (PhD)
Graduate group
Biochemistry & Molecular Biophysics
Complex III
electron transfer
protein design
Biochemistry, Biophysics, and Structural Biology
Grant number
Copyright date
Related resources

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, the maquette approach was applied toward developing a simple model protein (AP6) that retained the minimum engineering requirements for Complex III electron and proton transfer reactions but lacked the complexity found in the natural system. The AP6 peptide assembles as a four-α-helix bundle protein and can potentially bind up to six hemes tightly across a membrane interface. Chapter four demonstrates that AP6 successfully performs quinol-cytochrome c oxidoreductase activity in hundreds of milliseconds. AP6 is the first example of a synthetic enzyme capable of near-natural turnover rates. Chapter five focuses on defining the thermodynamic limit for maquette activity. This work supports the further development of simple model proteins to study aspects of Complex III mechanism.

P. Leslie Dutton
Date of degree
Date Range for Data Collection (Start Date)
Date Range for Data Collection (End Date)
Digital Object Identifier
Series name and number
Volume number
Issue number
Publisher DOI
Journal Issue
Recommended citation