A New Approach to Understanding Biological Control of Quinone Electrochemistry

Oxidoreductases play pivotal roles in energy capturing and converting processes of life. During these processes, quinones shuttle protons and reducing equivalents between membrane-bound oxidoreductases that generate the proton motive force during oxidative phosphorylation and photophosphorylation. A key mechanistic feature of these oxidoreductases is their ability to tune the reduction potentials of the hydroquinone, semiquinone and oxidized states of their substrate quinones. This level of control allows for maximization of conversion efficiency between the energy of the quinone reducing equivalents and the proton motive force, and prevents side reactions that may be fatal to cells. A half-century of experimental study and computational modeling of the respiratory and photosynthetic complexes has revealed little information on how this mechanistic control is accomplished. To obtain mechanistic insights into the control process, it is necessary to eliminate the biological complexity intrinsic to natural quinone oxidoreductases and create experimental systems that are simplified maquettes of quinone active sites. In this work, development of a naphthoquinone amino acid (Naq), modeled after vitamin K, allowed the creation of a range of quinone peptide maquettes designed to address uncertain mechanistic details of biological quinone control. In a simple heptamer, Naq acquires properties of quinone cofactors found in the three distinct classes of active sites of membrane oxidoreductases under different experimental conditions. Study of Naq in a lanthanide ion binding EF hand peptide allowed observation of the effect of a structural transition from coil to alpha-helix on the aqueous midpoint potential of Naq and measurement of the rate of electron transfer between reduced and oxidized Naq. Naq was also incorporated into a structured miniprotein based upon the TrpCage using a combination of the SCADS computational approach and iterative redesign by hand, creating a simple scaffold for evaluating effects of changing the local environment on Naq. Finally, using expressed protein ligation, Naq was incorporated into a single chain heme-binding maquette. Studies using this multi-cofactor protein to explore electron transfer reactions to and from Naq like those critical to respiration and photosynthesis are underway