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In vitro kinetics of P700 + reduction of Thermosynechococcus elongatus trimeric Photosystem I complexes by recombinant cytochrome c 6 using a Joliot-type LED spectrophotometer

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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 c 6 (cyt c 6)/PSI ratio in Thermosynechococcus elongatus (T.e.) using quantitative Western blot analysis. This information permitted the determination of P700 + reduction kinetics via recombinant cyt c 6 in a physiologically relevant ratio (cyt c 6: PSI) with a Joliot-type, LED-driven, pump-probe spectrophotometer. Dilute PSI samples were tested under varying cyt c 6 concentration, 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 c 6 during the longer illumination regime.

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Acknowledgments

We would like to thank R. Carter, and Dr. Non Chotewutmontri for critically reading the manuscript. BDB and PDF acknowledge support from TN-SCORE, a multidisciplinary research program sponsored by NSF-EPSCoR (EPS-1004083). KN and B.D.B. acknowledge support from the UTK BCMB Department and from the Gibson Family Foundation. KN was supported as an IGERT Fellow from the National Science Foundation IGERT program (DGE-0801470 to BDB and PDF). BDB and KN also acknowledge support from the Directors Strategic Initiative, “Understanding Photo-system I as a Biomolecular Reactor for Energy Conversion” at the Army Research Laboratory, Adelphi, MD (ARL Contract No. W911NF-11-2-0029).

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Authors and Affiliations

  1. Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, 37996, USA

    Khoa Nguyen, Michael Vaughn & Barry D. Bruce

  2. Department of Chemistry, Arizona State University, Tempe, AZ, 85287, USA

    Michael Vaughn

  3. Department of Biomolecular and Chemical Engineering, University of Tennessee, Knoxville, TN, 37996, USA

    Paul Frymier & Barry D. Bruce

  4. Sustainable Energy and Education Research Center, University of Tennessee, Knoxville, TN, 37996, USA

    Paul Frymier & Barry D. Bruce

  5. Bredesen Center for Interdisciplinary Education and Research, University of Tennessee, Knoxville, TN, 37996, USA

    Paul Frymier & Barry D. Bruce

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  1. Khoa Nguyen
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  2. Michael Vaughn
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  3. Paul Frymier
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  4. Barry D. Bruce
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Correspondence to Barry D. Bruce.

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In memory of our Friend, Colleague, and Mentor, Dr. David B. Knaff.

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Nguyen, K., Vaughn, M., Frymier, P. et al. In vitro kinetics of P700 + reduction of Thermosynechococcus elongatus trimeric Photosystem I complexes by recombinant cytochrome c 6 using a Joliot-type LED spectrophotometer. Photosynth Res 131, 79–91 (2017). https://doi.org/10.1007/s11120-016-0300-8

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