The functional mechanism of electron transfer within photosynthetic complex I in cyanobacteria

PhD thesisPhotosynthesis relies upon a proton motive force to produce ATP. Photosynthetic complex I (PS-CI) is a member of the complex I family involved in cyclic electron flow and respiration in cyanobacteria. PS-CI couples ferredoxin (Fd) oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. This process increases the ATP:NADPH ratio in cyanobacteria, promoting survival particularly under stress conditions. However, due to its large size, amphiphilic surface and lability, the functional mechanism of PS-CI is poorly understood. PS-CI contains three iron-sulphur (FeS) clusters which facilitate the electron transfer through from ferredoxin to plastoquinone. By purifying PS-CI and utilising recent developments in EPR sensitivity, in this work it has been possible to use electron paramagnetic resonance (EPR) spectroscopy to characterise the FeS clusters within the isolated complex. The ferredoxin:plastoquinone turnover by PS-CI is also established and presents the first rate estimate of quinone turnover by isolated PS-CI. By identifying the individual paramagnetic properties of the FeS clusters of PS-CI and exploiting the dipolar interaction between intrinsic paramagnetic centres using double electron electron resonance (DEER) spectroscopy, it has been possible to unambiguously assign the individual EPR signals of PS-CI to the structure. Small volume EPR monitored potentiometric titrations could then be used to determine the reduction potential of the FeS clusters in PS-CI from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803. This fundamental property of any redox-active active centre provides information about the favourability and direction of electron transfer. The two buried, adjacent clusters N2 and N1 are isopotential and are therefore postulated to have the capacity to accept and “store” both electrons necessary for plastoquinone reduction. The solvent exposed cluster N0 was found to have a much more negative potential, making backflow from the isopotential clusters unfavourable and thus forming an energetic trap for electrons when Fd is not bound. The work described in this thesis has determined the bioenergetics of the electron transfer relay within PS-CI and delivers insights into its mechanism, providing a foundation for future work to try and understand how this important complex can fine-tune the energetic redox state of the cell, as well as investigate how conditions in the cell regulate PS-CI function