Effects of amino acid residue substitutions on bicarbonate function in the plastoquinone reductase in cyanobacteria
Cao, Jiancheng
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https://hdl.handle.net/2142/22447
Description
Title
Effects of amino acid residue substitutions on bicarbonate function in the plastoquinone reductase in cyanobacteria
Author(s)
Cao, Jiancheng
Issue Date
1992
Doctoral Committee Chair(s)
Govindjee
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Language
eng
Abstract
The electron transfer at the Q$\sb{\rm A}$FeQ$\sb{\rm B}$ complex is significantly and reversibly inhibited in chloroplasts of higher plants and algae depleted of bicarbonate while no such inhibition has been observed in photosynthetic bacteria. It was observed in this study that a more than four fold stimulation of the Hill reaction by 5 mM bicarbonate and a significant reversible slowing of oxidation of Q$\sb{\rm A}\sp{-}$ in bicarbonate-depleted cells and thylakoids of Synechocystis sp. PCC 6803, showing the existence of the bicarbonate effect in cyanobacteria. Thus, this effect is present in all Photosystem II (PSII) reaction centers.
Oligonucleotide-directed mutagenesis was used to construct Synechocystis 6803 mutants carrying mutations in arginine residues in the D2 protein. Measurements of oxygen evolution showed that the D2 mutants D2-R233Q (arginine-233 $\to$ glutamine) and D2-R251S (arginine-251 $\to$ serine) were ten-fold more sensitive to formate, which displaces bicarbonate, than the wild type. Measurements of oxygen evolution in single-turnover flashes and chlorophyll (Chl) a fluorescence decay kinetics confirmed that D2-R251S and D2-R233Q are more sensitive than the wild type. It is suggested that the D2 protein is involved in the bicarbonate effect in PSII and the two arginine residues are important for the stabilization of bicarbonate binding in PSII.
The D1 protein of PSII is known to be involved in the bicarbonate effect. To examine the changes in the binding affinity of bicarbonate and formate in site-selected herbicide-resistant D1 mutants in Synechococcus sp. PCC 7942, a rapid equilibrium model involving activator-inhibitor interactions was used to estimate the dissociation constant for bicarbonate and for formate. The data indicated that these D1 mutations increase the dissociation constants for bicarbonate while those for formate are virtually unchanged. A working hypothesis is proposed in which bicarbonate forms a bidentate ligand to Fe$\sp{2+}$ and participates in the proton transfer pathway to the formation of plastoquinol Q$\sb{\rm B}$H$\sb2$ at the plastoquinone reductase.
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