Investigating the Nature of the Interaction between the Inner Membrane Quinol Dehydrogenase CymA and the Periplasmic Fumarate Reductase FccA of Shewanella oneidensis MR-1

Shewanella oneidensis exhibits a diverse respiratory system, able to use over 20 terminal electron acceptors, including solid metal oxides. CymA is located in the inner membrane, facing the periplasmic space, and it is responsible for the re-oxidation of the quinol/quinone pool. A plethora of proteins re-oxidise CymA, such as the periplasmic fumarate reductase FccA. This thesis investigates the nature of the interaction between CymA and FccA with a variety of biochemical and biophysical techniques. Chapter 3 describes a new approach in purifying recombinant form of CymA as a pure monodispersed solution that was developed with the reconstruction of the expression vector. The purity was >90% with a yield of 2 mg L-1 of culture. The biochemical and electrocatalytic properties of the recombinant CymA remained unaltered. Chapter 4 presents for the first time the purification of a soluble version of CymA, CymAsol [CymA-Δ(1-31)], at >95% purity, but with a lower yield of 0.3 mg L-1 of culture due to limitations from the TEV proteolytic cleavage reaction for tag removal. Nonetheless, the first crystallisation attempt was achieved with CymAsol. In total, 2,016 conditions were assayed, from which PEG 3350 seemed to promote atypical protein precipitation, although no crystals were formed. Chapter 5 presents a systematic investigation of the CymA: FccA interaction, where no strong interaction was observed with cyclic voltammetry using a CymA-containing tethered bilayer lipid membrane system, or with QCM-D using a CymA-containing solid supported bilayer lipid membrane system. Dissolved oxygen alone was the only condition that replicated the previously published electrocatalytic data under the same system. This was further validated with isothermal titration calorimetry with CymAsol, while, in a separate experiment, it was shown that CymA is necessary for the FccA-specific fumarate reduction in vitro with an initial rate of 3.3 (±0.3, n=3) µM NADH oxidised min-1. Lastly, QCM-D showed that CymAsol binds to a positively charged lipid bilayer, while the MBP-tag of the whole construct binds to a negatively charged lipid bilayer. Thus, the new evidence presented herein points towards a transient nature of weak interaction between CymA and FccA. The contributions of this work are in advancing the understanding of the anaerobic respiratory system of S. oneidensis.