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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 goodmore » 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.« less

Authors:
 [1];  [2]; ORCiD logo [2];  [2];  [2];  [1];  [2];  [1]
  1. Washington Univ., St. Louis, MO (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1248732
DOE Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochimica et Biophysica Acta - Bioenergetics; Journal Volume: 1857; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; asymmetry; charge recombination; directionality; membrane; picosecond; saturation mutagenesis

Citation Formats

Faries, Kaitlyn M., Kressel, Lucas L., Dylla, Nicholas P., Wander, Marc J., Hanson, Deborah K., Holten, Dewey, Laible, Philip D., and Kirmaier, Christine. Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus. United States: N. p., 2016. Web. doi:10.1016/j.bbabio.2015.11.013.
Faries, Kaitlyn M., Kressel, Lucas L., Dylla, Nicholas P., Wander, Marc J., Hanson, Deborah K., Holten, Dewey, Laible, Philip D., & Kirmaier, Christine. Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus. United States. doi:10.1016/j.bbabio.2015.11.013.
Faries, Kaitlyn M., Kressel, Lucas L., Dylla, Nicholas P., Wander, Marc J., Hanson, Deborah K., Holten, Dewey, Laible, Philip D., and Kirmaier, Christine. Mon . "Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus". United States. doi:10.1016/j.bbabio.2015.11.013.
@article{osti_1248732,
title = {Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus},
author = {Faries, Kaitlyn M. and Kressel, Lucas L. and Dylla, Nicholas P. and Wander, Marc J. and Hanson, Deborah K. and Holten, Dewey and Laible, Philip D. and Kirmaier, Christine},
abstractNote = {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.},
doi = {10.1016/j.bbabio.2015.11.013},
journal = {Biochimica et Biophysica Acta - Bioenergetics},
number = 2,
volume = 1857,
place = {United States},
year = {Mon Feb 01 00:00:00 EST 2016},
month = {Mon Feb 01 00:00:00 EST 2016}
}