title: In vitro kinetics of P700+reduction of Thermosynechococcus elongatus trimeric Photosystem I complexes by recombinant cytochrome c6using a Joliot-type LED spectrophotometer.
abstract: The reduction rate of photo-oxidized Photosystem I (PSI) with various natural and artificial electron donors have been well studied by transient absorption spectroscopy. The electron transfer rate from various donors to P700+has been measured for a wide range of photosynthetic organisms encompassing cyanobacteria, algae, and plants. PSI can be a limiting component due to tedious extraction and purification methods required for this membrane protein. In this report, we have determined the in vivo, intracellular cytochrome c6(cyt c6)/PSI ratio in Thermosynechococcus elongatus (T.e.) using quantitative Western blot analysis. This information permitted the determination of P700+reduction kinetics via recombinant cyt c6in a physiologically relevant ratio (cyt c6: PSI) with a Joliot-type, LED-driven, pump-probe spectrophotometer. Dilute PSI samples were tested under varying cyt c6concentration, temperature, pH, and ionic strength, each of which shows similar trends to the reported literature utilizing much higher PSI concentrations with laser-based spectrophotometer. Our results do however indicate kinetic differences between actinic light sources (laser vs. LED), and we have attempted to resolve these effects by varying our LED light intensity and duration. The standardized configuration of this spectrophotometer will also allow a more uniform kinetic analysis of samples in different laboratories. We can conclude that our findings from the LED-based system display an added total protein concentration effect due to multiple turnover events of P700+reduction by cyt c6during the longer illumination regime.
authors.affiliation: Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, 37996, USA.
authors.forename: Khoa
authors.initials: K
authors.lastname: Nguyen
authors.affiliation: Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, 37996, USA.|Department of Chemistry, Arizona State University, Tempe, AZ, 85287, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Vaughn
authors.affiliation: Department of Biomolecular and Chemical Engineering, University of Tennessee, Knoxville, TN, 37996, USA.|Sustainable Energy and Education Research Center, University of Tennessee, Knoxville, TN, 37996, USA.|Bredesen Center for Interdisciplinary Education and Research, University of Tennessee, Knoxville, TN, 37996, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Frymier
authors.affiliation: Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, 37996, USA. bbruce@utk.edu.|Department of Biomolecular and Chemical Engineering, University of Tennessee, Knoxville, TN, 37996, USA. bbruce@utk.edu.|Sustainable Energy and Education Research Center, University of Tennessee, Knoxville, TN, 37996, USA. bbruce@utk.edu.|Bredesen Center for Interdisciplinary Education and Research, University of Tennessee, Knoxville, TN, 37996, USA. bbruce@utk.edu.
authors.forename: Barry D
authors.initials: BD
authors.lastname: Bruce
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010084:Oxidation-Reduction; D045331:Photosystem I Protein Complex / Q000378:metabolism*; D046940:Synechococcus / Q000378:metabolism*; D013696:Temperature
chemical_list: D045331:Photosystem I Protein Complex
keywords: Cytochrome c 6; Electron transfer; Flash photolysis; JTS-10; P700; Photosystem I; Thermophilic cyanobacteria