title: Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus.
abstract: Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P(+)QB(-) yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P(+)QB(-) are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 μs. The resulting ranking of mutants for their yield of P(+)QB(-) from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P(+)HB(-)→P(+)QB(-) electron transfer or initial P*→P(+)HB(-) conversion highlight unmet challenges of optimizing both processes simultaneously.
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130, United States.
authors.forename: Kaitlyn M
authors.initials: KM
authors.lastname: Faries
authors.affiliation: Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, United States.
authors.forename: Lucas L
authors.initials: LL
authors.lastname: Kressel
authors.affiliation: Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, United States.
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Dylla
authors.affiliation: Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, United States.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Wander
authors.affiliation: Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, United States.
authors.forename: Deborah K
authors.initials: DK
authors.lastname: Hanson
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130, United States.
authors.forename: Dewey
authors.initials: D
authors.lastname: Holten
authors.affiliation: Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, United States.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Laible
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130, United States. Electronic address: kirmaier@wustl.edu.
authors.forename: Christine
authors.initials: C
authors.lastname: Kirmaier
mesh_terms: D020816:Amino Acid Motifs; D001429:Bacteriochlorophylls / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D015870:Gene Expression; D006860:Hydrogen Bonding; D007700:Kinetics; D008027:Light; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010674:Pheophytins / Q000737:chemistry* / Q000378:metabolism; D010788:Photosynthesis / Q000502:physiology* / Q000528:radiation effects; D016354:Rhodobacter capsulatus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D055672:Static Electricity; D013329:Structure-Activity Relationship; D014451:Ubiquinone / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001429:Bacteriochlorophylls; D045342:Light-Harvesting Protein Complexes; D010674:Pheophytins; D014451:Ubiquinone; C006678:bacteriopheophytin
keywords: Asymmetry; Charge recombination; Directionality; Membrane; Picosecond; Saturation mutagenesis