Site-directed mutagenesis of the bacterial photosynthetic reaction center

This effort concentrates on understanding the structure/function relationships in a model transmembrane protein-cofactor complex, the bacterial photosynthetic reaction center. The project involves construction and characterization of the effects of site-specific mutations of the reaction centers of Rhodobacter capsulatus and Rhodobacter shpaeroides. By understanding the effects of these mutations, we expect to gain insight into the protein’s role in tuning the redox and electronic properties of its cofactors. Structure-based rational design is used to target specific residues for mutagenesis as a means to change the environments or chemical properties of the cofactors, introduce cavities into the complex, probe the involvement of the iron-ligand complex in electron and proton transfer, and determine the structural basis for coupling light-induced electron transfer with proton transfer. The effects of the mutations on reaction center function are assessed by spectroscopic measurements of changes in:

• directionality of electron transfer
• proton uptake
• rates and yields of electron transfer steps
• stabilities of charge-separated states

The advantages of studying this particular pigment-protein complex to probe specific cofactor-protein interactions lie in the availability of its three-dimensional structure (known to nearly atomic resolution), the ease by which the structure can be modified specifically by mutagenesis, and its amenability to a large variety of types of spectroscopies. This project also encompasses the selection and characterization of phenotypic revertants of any non-photosynthetic mutants in the above categories. Many of these phenotypic revertants carry compensatory mutations which have revealed long-distance interactions that were not apparent even though the structure of the complex was known.