Site-directed mutagenesis: Directionality of primary electron transfer in the bacterial photosynthetic reaction center

The initial steps in the conversion of light energy to chemical energy are performed by the photosynthetic reaction center. This protein-cofactor complex, one of a very few membrane proteins whose structure is known to nearly atomic resolution, serves as an important model for other membrane-bound energy-transducing proteins. The structures of the bacterial reaction center reveal two apparently identical symmetry-related branches of cofactors that should be competent for electron transfer, yet spectroscopic evidence indicates that only one of them is active. In wild-type reaction centers, the A-branch of cofactors is the exclusive pathway for light-initiated electron transfer, resulting in very high yields of the charge-separated states involving this set of cofactors. Many molecular and biochemical approaches have been applied to determine the basis for the observed unidirectional electron flow, but the factors which optimize this pathway are not yet understood, even though the structure of the complex is known.

It is clear that the protein modulates the energy levels of its cofactors. The specifics of how the side chains of particular residues can tune the redox potentials of individual cofactors are beginning to be understood both in energetic and/or structural terms. The short-range objective of this research project – performed in collaboration with the laboratory of Dewey Holten and Christine Kirmaier at Washington University – is to rationally redesign the pathway for electron transfer in the reaction center, combining mutations which will redirect and optimize electron transfer to the B-branch cofactors and inactivate the A-branch pathway. In the long-term, the results of this work will provide a clearer picture of the general rules governing protein-cofactor interactions in energy-transducing proteins. An understanding of these types of structure/function relationships is ultimately necessary for de novo protein design.