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Abstract
Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation.
Highlights
Tetrameric Organization of Stt7—It is significant to note that, previously, the Stt[7] kinase pool was reported to be partitioned between the soluble and thylakoid membrane fractions (6)
Summary of New Information Regarding the Properties of Stt7—This study provides a molecular characterization of purified Stt[7], the enzyme that senses changes in trans-membrane redox poise across the thylakoid membrane and responds to quinol oxidation within the cyt b6f complex to activate photosynthetic state transitions by catalyzing the phosphorylation of the light-harvesting LHCII proteins
Purified full-length Stt[7] was found to be enzymatically active in catalyzing the hydrolysis of ATP to ADP (Fig. 5A), indicating that the enzyme was properly folded and retained its native state
Summary
Tetrameric Organization of Stt7—It is significant to note that, previously, the Stt[7] kinase pool was reported to be partitioned between the soluble and thylakoid membrane fractions (6). Summary of New Information Regarding the Properties of Stt7—This study provides a molecular characterization of purified Stt[7], the enzyme that senses changes in trans-membrane redox poise across the thylakoid membrane and responds to quinol oxidation within the cyt b6f complex to activate photosynthetic state transitions by catalyzing the phosphorylation of the light-harvesting LHCII proteins.
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