Mutational Analysis of Photosystem I Function

Iowa State University, Ames IA

Investigators

Abstract

0078264 Chitnis Energy generation in a cell requires electron transfer across membranes, which is typically mediated through multiprotein enzymes, such as cytochrome oxidase of respiration and photosystems of photosynthesis. Photosystem I is one of the two membrane-bound reaction centers of the photosynthetic electron transfer chain in cyanobacteria and chloroplasts. It functions as the light-driven plastocyanin-ferredoxin oxidoreductase and is a heteromultimeric pigment-protein complex. In this project, the PI is attempting to determine the function of photosystem I proteins through site-directed mutagenesis and targeted gene inactivation. A photosystem I complex contains two phylloquinone molecules, one (or both) of which serves as the redox center A1. The PI proposes to introduce cysteinyl residues near the phylloquinone-binding site by mutagenesis and to attach spin labels to them. Paramagnetic interactions between the label and A1 signal will allow identification of the redox-active phylloquinone(s) and consequently the active branch of electron transfer pathway. The PI has generated phylloquinone-less mutants that contain plastoquinone in their photosystem I complexes. The PI proposes to use those mutants to recruit foreign quinones in the A1 site in vivo. The quinones that compete successfully with the native plastoquinone in binding to the A1 site will be useful in determining structural features of the quinones that are important for their function at an extremely low redox potential. The quinones that are unable to substitute function of phylloquinone will be used for directed evolution of the binding site to accommodate and to use these quinones. In addition to studies on phylloquinones in photosystem I, the PI will investigate the role of b-carotene molecules in the excitation energy transfer in photosystem I. The PI recently discovered that several photosystem I proteins are modified post-translationally. He proposes to identify these modifications and examine their functional significance. Electron transfer reactions are key steps in photosynthesis, respiration, drug metabolism, and many other biochemical pathways. Photosynthesis and respiration contain membrane-bound electron transfer chains of general significance. The electron-transfer proteins in energy-transducing complexes provide structural stability, recognize reaction partners, and accurately orient cofactors. We expect that the outcome of the proposed research will provide information about how the protein environment determines functional properties of cofactors. In addition, the results will suggest ways to modify proteins for accommodating and using foreign cofactors and to engineer better biomimetic photoconversion systems. Oxygenic photosynthesis in plants, algae and cyanobacteria is the primary source of energy and oxygen for the life on our planet. Therefore, better understanding of this process is necessary to address the environmental and food challenges of the future.