Kinetics of electron transfer and binding of inhibitors in the two-electron gate of chloroplasts
Taoka, Shinichi
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Permalink
https://hdl.handle.net/2142/20548
Description
Title
Kinetics of electron transfer and binding of inhibitors in the two-electron gate of chloroplasts
Author(s)
Taoka, Shinichi
Issue Date
1989
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Biophysics, General
Biology, Plant Physiology
Language
eng
Abstract
The main aim of the project is to investigate the characteristics of the secondary plastoquinone, Q$\sb{\rm B}$, and its semiquinone, Q$\sb{\rm B}\sp-$, in the Q$\sb{\rm B}$ binding site and to characterize the two electron gate in chloroplasts with thermodynamical and kinetic parameters. There are three studies involved: (1) kinetics of an electron transfer reaction between the primary plastoquinone acceptor, Q$\sb{\rm A}$, and Q$\sb{\rm B}$, associated with binding and release of a plastoquinone in the dark as well as following an actinic flash measured from the triazine-susceptible and resistant Amaranthus hybridus, (2) kinetics of inhibitor binding to the Q$\sb{\rm B}$ site after mixing an inhibitor with the dark adapted pea chloroplasts or with the chloroplasts kept in the dark 1 s following an actinic flash, using a laboratory-built stopped flow apparatus, and (3) mode of action of weak and portent inhibitors in the dark and following an actinic flash, monitored by decay kinetics of chlorophyll a fluorescence, and the measurement of the partition coefficient of the inhibitors, e.g. DCMU, atrazine, o-phenanthroline, cyanoacrylate derivatives, naphthoquinone analogues.
Decay and rise kinetics of chlorophyll a fluorescence from the thylakoid membranes were measured by the weak measuring flashes at variable times following the actinic flashes in a wide range of pH from 5.8 to 8.3, and were analyzed by the curve fitting procedures to obtain the kinetic parameters, e.g. the first order rate constant of electron transfer between the two quinones, and the on- and off-rate constant of inhibitors referring to the membrane phase. The results are interpreted basically on a previously proposed model of the two electron gate in chloroplasts under the appropriate assumptions, e.g. the concentration of plastoquinone in the thylakoid membranes is assumed to be 5 mM, and are able to expand the model in more detail including all possible protonated species in the two electron gate.
The on- and off-rate constants of the inhibitors to the Q$\sb{\rm B}$ site were also measured. (Abstract shortened with permission of author.)
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