Recombinant expression and band-gap engineering of the bacterial photosynthetic reaction centre

The Rhodobacter sphaeroides photosynthetic reaction centre (RC) is a pigment-protein complex that efficiently captures and converts photon energy into a charge-separated state. Given the conversion efficiency and the high electric potential of the electron, the major focus of my project was to deliver/extract electrons to/from various cofactors along the charge-separation pathway in the RC, including the special pair of bacteriochlorophylls (P), the bacteriopheophytin (HA), the primary quinone (QA), and the secondary quinone (QB). An over-expression system was created to produce RCs, using the R. sphaeroides RCx strain, pIND4 plasmid, a modified culture medium, and changes to growth conditions. These changes resulted in a 35-fold increase in protein levels compared to the previous system. To extract electrons from the quinone region of the RC, this region was made more accessible to the solvent by deleting portions of the H subunit cytoplasmic globular domain. The results indicated that the truncated RC mutants assembled stably and thereby reduced the electron transfer distance between the quinone and an external electron acceptor. Photochronoamperometry measurements on mutant RCs designed to test the feasibility of delivering electrons from an electrode to P showed photocurrent generation and direction that were consistent with the binding of the RC P-side to the electrode surface. Similar experiments on the feasibility of extracting electrons from HA, QA and QB, for delivery to highly ordered pyrolytic graphite (HOPG) or gold electrodes, also showed photocurrent generation and direction consistent with the binding of the RC HA-side or Q-side to the electrode surface. Finally, the thermal stability of complexes was studied by in vivo addition of light harvesting complex 1 (LH1) from the thermophile Thermochromatium tepidum to the RC. A hybrid core complex consisting of an R. sphaeroides RC surrounded by T. tepidum TLH1 conferred greater tolerance to thermal energies, compared to the analogous R. sphaeroides RC-LH1 core complex, at temperatures up to 70 °C. The combination of these results show that, in principle, the RC can be modified to extract electrons at different energy levels, or band gaps, with possible applications in heat-stabile biohybrid solar cell technologies.