title: High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers.
abstract: Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.
authors.affiliation: Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
authors.forename: Kaitlyn M
authors.initials: KM
authors.lastname: Faries
authors.forename: Lucas L
authors.initials: LL
authors.lastname: Kressel
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Wander
authors.forename: Dewey
authors.initials: D
authors.lastname: Holten
authors.forename: Philip D
authors.initials: PD
authors.lastname: Laible
authors.forename: Christine
authors.initials: C
authors.lastname: Kirmaier
authors.forename: Deborah K
authors.initials: DK
authors.lastname: Hanson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D045322:Photosynthetic Reaction Center Complex Proteins