Doug Bruce

Regulation of excitation energy transfer in organisms containing phycobilins.

The mechanism of excitation energy redistribution (state transition) in organisms containing phycobilins is reviewed. Recent measurements using time-resolved fluorescence spectroscopy in the picosecond range confirm that the state transition in cyanobacteria and red algae is controlled by changes in the kinetics of energy transfer from PS 2 to PS 1 (spillover) rather than by physical dislocation of the phycobilisome and reassociation between the two photosystems (mobile antenna model). Contrary to the analogous situation in higher plants, there is no compelling evidence for the involvement of a protein phosphorylation event in the rapid time range of the state transition, but a variety of data indicate that a membrane conformational change occurs that might change the relative distance between, and/or orientation of the two photosystems within the thylakoid. The state transition is most probably initiated by the redox state of the intersystem electron transport chain, and the conversion to state 1 is driven by coupled PS1 cyclic electron transport. The cryptomonads also undergo wavelength dependent changes in excitation energy distribution by a mechanism very similar to that observed in the red algae and cyanobacteria. However, the changes in energy distribution in this group are most likely related to a photoprotection mechanism for PS2 rather than to a state transition.

Regulation of the distribution of chlorophyll and phycobilin-absorbed excitation energy in cyanobacteria. A structure-based model for the light state transition.

The light state transition regulates the distribution of absorbed excitation energy between the two photosystems (PSs) of photosynthesis under varying environmental conditions and/or metabolic demands. In cyanobacteria, there is evidence for the redistribution of energy absorbed by both chlorophyll (Chl) and by phycobilin pigments, and proposed mechanisms differ in the relative involvement of the two pigment types. We assayed changes in the distribution of excitation energy with 77K fluorescence emission spectroscopy determined for excitation of Chl and phycobilin pigments, in both wild-type and state transition-impaired mutant strains ofSynechococcus sp. PCC 7002 andSynechocystis sp. PCC 6803. Action spectra for the redistribution of both Chl and phycobilin pigments were very similar in both wild-type cyanobacteria. Both state transition-impaired mutants showed no redistribution of phycobilin-absorbed excitation energy, but retained changes in Chl-absorbed excitation. Action spectra for the Chl-absorbed changes in excitation in the two mutants were similar to each other and to those observed in the two wild types. Our data show that the redistribution of excitation energy absorbed by Chl is independent of the redistribution of excitation energy absorbed by phycobilin pigments and that both changes are triggered by the same environmental light conditions. We present a model for the state transition in cyanobacteria based on the x-ray structures of PSII, PSI, and allophycocyanin consistent with these results.

Excited-state dynamics in photosystem II: Insights from the x-ray crystal structure

The heart of oxygenic photosynthesis is photosystem II (PSII), a multisubunit protein complex that uses solar energy to drive the splitting of water and production of molecular oxygen. The effectiveness of the photochemical reaction center of PSII depends on the efficient transfer of excitation energy from the surrounding antenna chlorophylls. A kinetic model for PSII, based on the x-ray crystal structure coordinates of 37 antenna and reaction center pigment molecules, allows us to map the major energy transfer routes from the antenna chlorophylls to the reaction center chromophores. The model shows that energy transfer to the reaction center is slow compared with the rate of primary electron transport and depends on a few bridging chlorophyll molecules. This unexpected energetic isolation of the reaction center in PSII is similar to that found in the bacterial photosystem, conflicts with the established view of the photophysics of PSII, and may be a functional requirement for primary photochemistry in photosynthesis. In addition, the model predicts a value for the intrinsic photochemical rate constant that is 4 times that found in bacterial reaction centers.

State transitions in a phycobilisome-less mutant of the cyanobacterium Synechococcus sp. PCC 7002.

State transitions were investigated in the cyanobacterium Synechococcus sp. PCC 7002 in both wild-type cells and mutant cells lacking phycobilisomes. Preillumination in the presence of DCMU induced State 1 and dark-adaptation induced State 2 in both wild-type and mutant cells as determined by 77 K fluorescence emission spectroscopy. Light-induced transitions were observed in the wild-type after preferential excitation of phycocyanin (State 2) or preferential excitation of Chl a (State 1). Light-induced transitions were also observed in the phycobilisome-less mutant after preferential excitation of short-wavelength Chl a (State 2) or carotenoids and long-wavelength Chl a (State 1). We conclude that the mechanism of the light-state transition in cyanobacteria does not require the presence of the phycobilisome. Our results contradict proposed models for the state transition, which require phosphorylation of, and an active role for, the phycobilisome.

Structural-functional role of chloride in photosystem II.

Chloride binding in photosystem II (PSII) is essential for photosynthetic water oxidation. However, the functional roles of chloride and possible binding sites, during oxygen evolution, remain controversial. This paper examines the functions of chloride based on its binding site revealed in the X-ray crystal structure of PSII at 1.9 Å resolution. We find that chloride depletion induces formation of a salt bridge between D2-K317 and D1-D61 that could suppress the transfer of protons to the lumen.

Photoprotection in the Lichen Parmelia sulcata : The Origins of Desiccation-Induced Fluorescence Quenching

Lichens, a symbiotic relationship between a fungus (mycobiont) and a photosynthetic green algae or cyanobacteria (photobiont), belong to an elite group of survivalist organisms termed resurrection species. When lichens are desiccated, they are photosynthetically inactive, but upon rehydration they can perform photosynthesis within seconds. Desiccation is correlated with both a loss of variable chlorophyll a fluorescence and a decrease in overall fluorescence yield. The fluorescence quenching likely reflects photoprotection mechanisms that may be based on desiccation-induced changes in lichen structure that limit light exposure to the photobiont (sunshade effect) and/or active quenching of excitation energy absorbed by the photosynthetic apparatus. To separate and quantify these possible mechanisms, we have investigated the origins of fluorescence quenching in desiccated lichens with steady-state, low temperature, and time-resolved chlorophyll fluorescence spectroscopy. We found the most dramatic target of quenching to be photosystem II (PSII), which produces negligible levels of fluorescence in desiccated lichens. We show that fluorescence decay in desiccated lichens was dominated by a short lifetime, long-wavelength component energetically coupled to PSII. Remaining fluorescence was primarily from PSI and although diminished in amplitude, PSI decay kinetics were unaffected by desiccation. The long-wavelength-quenching species was responsible for most (about 80%) of the fluorescence quenching observed in desiccated lichens; the rest of the quenching was attributed to the sunshade effect induced by structural changes in the lichen thallus.

Diverse mechanisms for photoprotection in photosynthesis. Dynamic regulation of photosystem II excitation in response to rapid environmental change.

Photosystem II (PSII) of photosynthesis catalyzes one of the most challenging reactions in nature, the light driven oxidation of water and release of molecular oxygen. PSII couples the sequential four step oxidation of water and two step reduction of plastoquinone to single photon photochemistry with charge accumulation centers on both its electron donor and acceptor sides. Photon capture, excitation energy transfer, and trapping occur on a much faster time scale than the subsequent electron transfer and charge accumulation steps. A balance between excitation of PSII and the use of the absorbed energy to drive electron transport is essential. If the absorption of light energy increases and/or the sink capacity for photosynthetically derived electrons decreases, potentially deleterious side reactions may occur, including the production of reactive oxygen species. In response, a myriad of fast (second to minutes timescale) and reversible photoprotective mechanisms are observed to regulate PSII excitation when the environment changes more quickly than can be acclimated to by gene expression. This review compares the diverse photoprotective mechanisms that are used to dissipate (quench) PSII excitation within the antenna systems of higher land plants, green algae, diatoms, and cyanobacteria. The molecular bases of how PSII excitation pressure is sensed by the antenna system and how the antenna then reconfigures itself from a light harvesting to an energy dissipative mode are discussed.

Functional analysis of the iron-stress induced CP 43' polypeptide of PS II in the cyanobacterium Synechococcus sp. PCC 7942.

Under conditions of iron-stress, the Photosystem II associated chlorophyll a protein complex designated CP 43′, which is encoded by the isiA gene, becomes the major pigment-protein complex in Synechococcus sp. PCC 7942. The isiB gene, which is located immediately downstream of isiA, encodes the protein flavodoxin, which can functionally replace ferredoxin under conditions of iron stress. We have constructed two cyanobacterial insertion mutants which are lacking (i) the CP 43′ apoprotein (designated isiA−) and (ii) flavodoxin (designated isiB−). The function of CP 43′ was studied by comparing the cell characteristics, PS II functional absorption cross-sections and Chl a fluorescence parameters from the wild-type, isiA− and isiB− strains grown under iron-stressed conditions. In all strains grown under iron deprivation, the cell number doubling time was maintained despite marked changes in pigment composition and other cell characteristics. This indicates that iron-starved cells remained viable and that their altered phenotype suggests an adequate acclimation to low iron even in absence of CP 43′ and/or flavodoxin. Under both iron conditions, no differences were detected between the three strains in the functional absorption crossection of PS II determined from single turnover flash saturation curves of Chl a fluorescence. This demonstrates that CP 43′ is not part of the functional light-harvesting antenna for PS II. In the wild-type and the isiB− strain grown under iron-deficient conditions, CP 43′ was present in the thylakoid membrane as an uncoupled Chl-protein complex. This was indicated by (1) an increase of the yield of prompt Chl a fluorescence (Fo) and (2) the persistence after PS II trap closure of a fast fluorescence decay component showing a maximum at 685 nm.

Exploring the energetics of water permeation in photosystem II by multiple steered molecular dynamics simulations.

The Mn(4)Ca cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the Mn(4)Ca cluster by moving through protein. Molecular dynamics simulations were used to determine the energetic barriers for water permeation though PSII extrinsic proteins. Potentials of mean force (PMFs) for water were derived by using the technique of multiple steered molecular dynamics (MSMD). Calculation of free energy profiles for water permeation allowed us to characterize previously identified water channels, and discover new pathways for water movement toward the Mn(4)Ca cluster. Our results identify the main constriction sites in these pathways which may serve as selectivity filters that restrict both the access of solutes detrimental to the water oxidation reaction and loss of Ca(2+) and Cl(-) from the active site.

Regulation of photosynthetic light harvesting by nitrogen assimilation in the green alga Selenastrum minutum

The interaction of whole cell metabolism with the distribution of excitation energy between photosystem 2 (PS2) and photosystem 1 (PS1), the light state transition, was investigated in vivo in the green alga Selenastrum minutum. Nitrogen limited cells of S. minutum were presented with a pulse of either NH4 + or NO3 − in the light. As shown previously, CO2 fixation is inhibited and high rates of N assimilation ensue [(1986) Plant Physiol. 81, 273–279]. NH4 + assimilation has a much higher requirement ratio for ATP/NADPH than either CO2 or NO3 − assimilation and thus drastically increases the demand for ATP relative to reducing power. Room temperature chlorophyll a fluorescence kinetic measurements showed that a reversible non‐photochemical quenching of PS2 fluorescence accompanied the assimilation of NH4 + but not the assimilation of NO3 − or CO2. 77K fluorescence emission spectra taken from samples removed at regular intervals during NH+ 4assimilation showed that the non‐photochemical quenching of PS2 was accompanied by a complementary increase in the fluorescence yield of PS1, characteristic of a transition to state 2. Our data suggests that S. minutum responds to the increased demand for ATP/NADPH during NH4 assimilation by inducing the light state transition to direct more excitation energy to PS1 at the expense of PS2 to increase the production of ATP by cyclic electron transport.