John Biggins

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.

Mechanism of the light state transition in photosynthesis. IV. Picosecond fluorescence spectroscopy of Anacystis nidulans and Porphyridium cruentum in state 1 and state 2 at 77 K

Abstract The sequential energy-transfer pathway through the phycobilin pigments to chlorophyll a was investigated as a function of the state transition in the cyanobacterium Anacystis nidulans and the red alga Porphyridium cruentum. The fluorescence decay kinetics of the phycobilin pigments and chlorophyll a were determined for cells frozen at 77 K in state 1 and state 2 using a single-photon timing fluorescence spectroscopy apparatus with picosecond resolution. Time-resolved 77 K fluorescence emission spectra were also obtained for both species in state 1 and state 2. In both A. nidulans and P. cruentum the transition to state 1 was accompanied by a large increase in the apparent fluorescent lifetime of chlorophyll a associated with PS II (emission peak at 695 nm). There were smaller increases in the lifetime of the terminal phycobilin emitter (685 nm) in both species and no change in phycocyanin (645 nm) or allophycocyanin (660 nm). Time-resolved spectra showed sequential emission from phycocyanin, allophycocyanin, the terminal phycobilin emitter and chlorophyll a. Spectral red shifts were observed with time for all emission peaks with the exception of the terminal phycobilin emitter. In A. nidulans this peak showed a small blue shift with time. The results are interpreted as evidence for an effective uncoupling of PS II chlorophyll a from subsequent energy transfer to PS I chlorophyll a upon transition to state 1. Our recently proposed model for the mechanism of the state transition in phycobilisome-containing organisms is discussed in terms of a decrease in the energy transfer overlap between PS II chlorophyll a and PS I chlorophyll a in state 1.

Mechanism of the light state transition in photosynthesis. II. Analysis of phosphorylated polypeptides in the red alga, Porphyridium cruentum

Abstract The possible involvement of a reversible protein phosphorylation event in the regulation of excitation energy distribution was studied in the red alga Porphyridium cruentum . Whole cells were incubated in phosphate-depleted growth medium containing carrier-free [ 32 P]orthophosphate for several hours to label the intracellular phosphate pools, and they were then converted to State 1 or State 2 by illumination using blue or green light, respectively. The successful transition to State 1 or State 2 was verified by 77 K fluorescence spectroscopy of the chlorophyll emission and the cells were then denatured using either acetone, trichloroacetic acid or boiling detergent. The whole cell lysates were solubilized, treated with RNAase, and analyzed for phosphoproteins by SDS-polyacrylamide gel electrophoresis. At least twelve polypeptides were found to be phosphorylated but no changes in specific radioactivity of the polypeptides were detected when samples from cells in State 1 and State 2 were compared. We conclude that a reversible protein phosphorylation event is not implicated in the state transition in P. cruentum . A model is presented for the mechanism of the light state transition in organisms that contain phycobilisomes which is different from the mechanism of energy distribution proposed for higher plants.

Mechanism of the light-state transition in photosynthesis: V. 77 K linear dichroism of Anacystis nidulans in State 1 and State 2

Abstract Linear-dichroism spectra of Anacystis nidulans at 77 K were determined for whole cells chemically fixed in light State 1 and light State 2. Whole cells were oriented by the squeezed gel technique using 5% gelatin 2.2 M sucrose gels. Peaks with positive dichroism were observed at 638 nm and 688 nm with shoulders at approx. 650 nm and 700 nm. The amplitude of the 650 nm shoulder was greater for cells in State 2 than those in State 1, and the State-2-minus-State 1 difference spectrum had a single peak at 656 nm. The linear dichroism spectrum of phycobilisomes isolated from A. nidulans showed peaks at 635 nm (phycocyanin) and 656 nm (allophycocyanin). The spectrum for thylakoid membranes free of phycobilisomes had one peak at 685 nm with a shoulder at 698 nm. We suggest that the change in dichroism at 656 nm between cells in State 1 and State 2 results from a change in orientation of the allophycocyanin core of the phycobilisome. This result is discussed in the context of our model for the light-state transition in phycobilisome-containing organisms.

Light-induced charge separation in photosystem I at low temperature is not influenced by vitamin K-1

The photoreduction of iron-sulfur centers was studied at low temperature in Photosystem I particles from spinach and the cyanobacterium Synechocystis 6803, which contain various amounts of vitamin K-1 (recently tentatively identified as the acceptor A1). The irreversible charge separation that was progressively induced at low temperature between P-700 and FA (or FB) by successive laser flashes was studied at 15 K. Its maximum amount after a large number of flashes was shown to be fairly independent of the number (0, 1 or 2) of vitamins K-1 per reaction center. Moreover, the first flash yield of this charge separation was diminished by only about 50% when vitamin K-1 was completely absent from the particles by comparison with particles containing one or two vitamin K-1 per reaction center. When FA and FB were prereduced, the iron-sulfur center FX was also reversibly photoreduced at 9 K in the absence of vitamin K-1. The implications of these results for the electron pathways of Photosystem I are discussed and it is proposed that a direct electron transfer from A0- to the iron-sulfur centers is highly efficient at low temperature.

Mechanism of the light state transition in photosynthesis. I. Analysis of the kinetics of cytochrome f oxidation in State 1 and State 2 in the red alga, Porphyridium cruentum

Abstract The kinetics of photooxidation and reduction of cytochrome f were examined spectrophotometrically in the red alga Porphyridium cruentum in light State 1 and light State 2. Experiments were performed on intact cells that had been chemically fixed and stabilized in the light states. The cytochrome f turnover was measured during conditions of linear electron transport driven by both photosystems and during several cyclic reactions mediated by the long-wavelength Photosystem (PS) I. The data show that the rate of photooxidation of cytochrome f increased in State 2 when the cells were activated by subsaturating intensities of green light absorbed primarily by the phycobilisome. No differences in kinetics were found between algae in State 1 or State 2 when they were activated by light absorbed primarily by the chlorophyll of PS I. The results confirm that changes in energy distribution between the two photosystems occur as a result of the light state transition and verify that the redistribution of excitation results in the predicted changes in electron transport.

Reconstitution and exchange of quinones in the A1 site of Photosystem I. An electron spin polarization electron paramagnetic resonance study

Abstract The electron spin polarized (ESP) electron paramagnetic resonance (EPR) signal observed in spinach Photosystem I (PS I) particles was examined in preparations depleted of vitamin K 1 by solvent extraction, followed by reconstitution with a series of quinones and quinone analogues. The ESP EPR signal was previously attributed to a radicalpair that included vitaim K 1 − (Rustandi, R.R., et al. (1990) Biochemistry 29, 8030–8032) and, in addition, vitamin K 1 was assigned as the secondary acceptor A 1 in PS I (Snyder, S.W., et al. (1991) Proc. Natl. Acad. Sci. USA, 88, 9895–9896). The ESP EPR signal was observed in untreated PS I preparations, was not detected in the solvent-extracted PS I samples and was restored upon reconstitution using certain quinones. The ability to restore the ESP EPR signal was dependent upon two properties of the reconstituted acceptor. First, the solution reduction potential of the reconstituted acceptor must be more positive than about −750 mV where the solution reduction potential of vitamin K 1 is −710 mV. Second, the structure of the reconstituted acceptor requires either a minimum of two aromatic rings (i.e., naphthoquinone) or a benzoquinone (or larger) derivative substituted with an alkyl tail. A model was developed to describe both the requirements for electron transfer to A 1 and also previous results for electron transfer from A 1 − to the iron-sulfur centers (Biggins, J. (1990) Biochemistry 29, 7259–7264). When untreated PS I preparations were incubated with perdeuterated vitamin K 1 (DK 1 ) the endogenous K 1 was observed to exchange with DK 1 . The replacement rate was strongly dependent upon temperature (h-days at 4°C, min at 37°C) and upon illumination (min). Naphthoquinones lacking a long alkyl tail were unable to exchange with endogenous vitamin K 1 . Although no known physiological role exists for vitamin K 1 ejection from its A 1 binding site, the quinone appears to be somewhat labile. Direct exchange of vitamin K 1 with exogenously supplied quinones indicates that the PS I A 1 site might be a target for the design of new herbicides in green plants.

Mechanism of the light state transition in photosynthesis. III. Kinetics of the state transition in Porphyridium cruentum

Abstract The kinetics of the light state transition in the red alga Porphyridium cruentum were studied in low intensities of initiating light and in saturating flashes brief enough to elicit single turnovers of the photochemical apparatus. We confirm that the state transition is dose-dependent, but also found that the transition to state 2 was biphasic. The slow phase was correlated with the induction of photosynthesis and was eliminated if the preceding time spent in state 1 was very short. The full transition to state 1 developed following a minimum of 15 turnovers of Photosystem I, and the optimal frequency for the flash sequence was determined to be 2.5 Hz. In contrast, the turnover time required for the transition to state 2 was found to be smaller than 30 ms. The data are consistent with a mechanism we have recently proposed for the state transition in organisms that contain phycobilisomes. The mechanism proposed involves a small conformational change within the thylakoid that is brought about by localized differences in electrochemical potential. A Photosystem-I-generated potential difference of H + is prerequisite for the initiation of state 1 and, under certain conditions, a localized electric-field generated by Photosystem II may play a significant role in the transition to state 2.

Thylakoid conformational changes accompanying membrane protein phosphorylation

Abstract The effect of reversible membrane phosphorylation on the room temperature linear dichroism signal of magneto-oriented pea thylakoids was investigated. Membrane phosphorylation, induced by photoreduction of the plastoquinone pool, resulted in a change in the linear dichroism signal in the region of the red absorption band of chlorophyll. The optical change was due to modifications in selective polarized light scattering which have been shown to be indicative of alterations in the degree of membrane stacking. No changes in linear dichroism due to a reorientation of pigments were observed. It is concluded that phosphorylation of the membrane results in about a 10% destacking of the thylakoids and that this conformational change is implicated in energy redistribution between the two photosystems.

The kinetic behavior of P-700 during the induction of photosynthesis in algae

The kinetics of P-700 were examined spectrophotometrically during the induction of photosynthesis in algae. A pronounced oscillation was observed in the redox level of P-700 upon illumination of dark-adapted cells. The dark adaptation required approximately 1 min. The oscillation may be described as an initial rapid oxidation reaching a peak at approx. 50 ms followed by complete reduction of the pool of P-700. A subsequent slower oxidation resulted in attainment of the final state around 1 s. The main features of the oscillation were qualitatively the same in a wide variety of algae. The modulation in redox level of P-700 required high intensity activation of both photosystems and was eliminated by pre-illumination of the cells with weak short wavelength light but not by longer wavelengths absorbed primarily by Photosystem I. We propose that the P-700 modulation is directly related to the fast redox changes in Photosystem II which occur during the induction of photosynthesis. Cells incubated with methyl viologen did not show the P-700 oscillation confirming the suggestion previously advanced that exhaustion of Photosystem I acceptor and kinetic limitations in the carbon reduction cycle partially control the fast phase of photosynthetic induction.