Design and Characterization of an Enzymatically Active Amphiphilic Maquette Protein

Many questions still exist about how quinone molecules act as substrates for membrane oxidoreductase enzymes, as well as how quinones can act as a catalyst in energy conversion mechanisms. We apply our knowledge of electron tunneling and protein design towards defining the basic engineering requirements for quinone reactivity in natural membranes and heme proteins. We have synthesized and characterized a transmembrane, amphiphilic maquette protein, AP6, which extracts the basic structural components from Complex III necessary to perform transmembrane proton-coupled electron transfer. We have shown that our AP6 peptide assembles as a four-helix bundle protein and can potentially bind up to six bis-histidine ligated hemes tightly across a membrane interface. Given its sequence and heme binding capabilities, our AP6 design could accomplish a variety of potential functions, including: transmembrane electron transfer, electron transfer with aqueous proteins, proton-coupled electron transfer, or combining these, quinol-cytochrome c oxidoreductase activity. Through standard Complex III activity assays, we have demonstrated that AP6 has quinol-cytochrome c oxidoreductase activity in detergent micelles that is within two orders of magnitude of the activity of natural Complex III purified from R. capsulatus. This activity can be generated with a variety of reduced quinone substrates, and is dependent on the concentration of cytochrome c present. With no obvious quinone-binding site included in our protein design, AP6 provides clear evidence that a specific quinone-binding site within a membrane protein is not essential for generating significant quinol-cytochrome c oxidoreductase enzymatic activity from a heme protein.