Showing papers by "Jun Minagawa published in 2008"

Molecular Remodeling of Photosystem II during State Transitions in Chlamydomonas reinhardtii

Abstract: State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.

D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements.

Abstract: Arginine257 (R257), in the de-helix that caps the QB site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the QB site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced QB, Q B − ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q A − to QB) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 μM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q B − with S2 was reduced by 20–40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of QB/Q B − . Further, since the recombination of Q A − with S2 was unaffected, we suggest that no significant change in redox potential of QA/Q A − occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the QB site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the QB/Q B − pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.

Dissociation of Light-Harvesting Complex II from Photosystem II During State Transitions in Chlamydomonas reinhardtii

Abstract: State transition is a plant photoacclimation mechanism to regulate the equilibrium of the light-driven excitation at photosystem I (PSI) and photosystem II (PSII). It has been considered that redistribution of light-harvesting complex (LHC) II adjusts the absorption-cross section of each photosystem. Several lines of biochemical evidence for the association of LHCII to PSI were recently provided by isolating a protein supercomplex composed of PSI, LHCI, and LHCII. However, the detachment of LHCIIs from PSII, which should occur simultaneously during state transitions, still remains unclear. In this study, we established a streamlined protocol to isolate a protein supercomplex comprised of PSII and LHCII, a so-called PSII-LHCII supercomplex, to explore the changes in its structure during state transitions in Chlamydomonas reinhardtii. The results suggested that the PSII-LHCII supercomplex in state 1 formed a dimer with the molecular weight of around 1,500 kDa. We speculate that, in the course of a transition to State 2, monomerization of the PSII-LHCII supercomplex occurs first, and subsequently the detachment of LHCII from PSII takes\ place, which is accompanied by the phosphorylation of LHCIIs.

Simulation of Excitation Energy Transfer within the PSI-LHCI/II Supercomplex from Chlamydomonas reinhardtii

Abstract: State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II antenna proteins migrates from PSII to PSI. We previously identified three of those mobile proteins (CP26, CP29, and LhcbM5) in the PSI-LHCI/ II supercomplex isolated from the green alga Chlamydomonas reinhardtii placed in state 2. Here, we demonstrate the functional interaction between the chlorophylls in the mobile light-harvesting complex (LHC) II proteins and those in the PSILHCI supercomplex by examining time-resolved fluorescence spectra of (1) the “state 2-type” PSI-LHCI/II supercomplex, (2) the LHCII fraction removed from the supercomplex, and (3) the remained “state 1-type” PSI-LHCI supercomplex. The lifetime of most of the kinetic components were longer in the PSI-LHCI/II supercomplex, suggesting that the functional size of the antenna was increased by the attached LHCII. Furthermore, we propose possible energy transfer processes based on a simulation of energy migration within the PSILHCI/ II supercomplex.

Suppression of CP29 Causes Instability of the PSI-LHCI/II Supercomplex in Chlamydomonas reinhardtii Under State 2 Conditions

Abstract: The light harvesting-complexes II (LHCIIs) reversibly migrate between photosystem I (PSI) and photosystem II (PSII) during state transitions. Recently, it was reported that three minor monomeric LHCIIs (CP29, CP26 and LhcbM5) were associated with PSI and formed PSI-LHCI/II supercomplex during state 2 in the green alga Chlamydomonas reinhardtii. In this study, we generated knock-down mutants of CP29 to collect further insights on this monomeric LHCII during state transitions. In a transformant b4i-8, whose expression of CP29 was nearly completely suppressed, a PSI-LHCI/II supercomplex was not detected under state 2-promoting conditions from detergent-solubilized thylakoid membranes. However, the in vivo spectroscopic analysis showed that the PSII antenna size was reduced upon a transition from state 1 to 2 in b4i-8 cells as in wild type. These results suggest that CP29 is required for the formation of PSI-LHCI/II supercomplex, but not for the dissociation of the antenna from PSII.