title: Trans-membrane Signaling in Photosynthetic State Transitions: REDOX- AND STRUCTURE-DEPENDENT INTERACTION IN VITRO BETWEEN STT7 KINASE AND THE CYTOCHROME b6f COMPLEX.
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.
authors.affiliation: From the Department of Biological Sciences and.
authors.forename: Sandeep K
authors.initials: SK
authors.lastname: Singh
authors.affiliation: From the Department of Biological Sciences and.
authors.forename: S Saif
authors.initials: SS
authors.lastname: Hasan
authors.affiliation: From the Department of Biological Sciences and.
authors.forename: Stanislav D
authors.initials: SD
authors.lastname: Zakharov
authors.affiliation: From the Department of Biological Sciences and.
authors.forename: Sejuti
authors.initials: S
authors.lastname: Naurin
authors.affiliation: the Pasarow Mass Spectrometry Laboratory, NPI-Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, California 90024.
authors.forename: Whitaker
authors.initials: W
authors.lastname: Cohn
authors.affiliation: Biophysical Analysis Laboratory, Bindley Bioscience Center,Purdue University, West Lafayette, Indiana 47907 and.
authors.forename: Jia
authors.initials: J
authors.lastname: Ma
authors.affiliation: the Pasarow Mass Spectrometry Laboratory, NPI-Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, California 90024.
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.affiliation: From the Department of Biological Sciences and waclab@purdue.edu.
authors.forename: William A
authors.initials: WA
authors.lastname: Cramer
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D045346:Cytochrome b6f Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D013329:Structure-Activity Relationship
chemical_list: D045342:Light-Harvesting Protein Complexes; D045346:Cytochrome b6f Complex; D017346:Protein Serine-Threonine Kinases
keywords: Stt7; cytochrome; cytochrome b6f; light-harvesting complex (antenna complex); redox signaling; serine/threonine protein kinase; signal transduction; state transitions