Subramanyam Rajagopal

Changes in the structure of chlorophyll-protein complexes and excitation energy transfer during photoinhibitory treatment of isolated photosystem I submembrane particles

The activity of light-induced oxygen consumption, absorption spectra, low temperature (77 K) chlorophyll fluorescence emission and excitation spectra were studied in suspensions of photosystem (PS) I submembrane particles illuminated by 2000 microE m(-2) s(-1) strong white light (WL) at 4 degrees C. A significant stimulation of oxygen uptake was observed during the first 1-4 h of photoinhibitory treatment, which rapidly decreased during further light exposure. Chlorophyll (Chl) content gradually declined during the exposure of isolated PSI particles to strong light. In addition to the Chl photobleaching, pronounced changes were found in Chl absorption and fluorescence spectra. The position of the major peak in the red part of the absorption spectrum shifted from 680 nm towards shorter wavelengths in the course of strong light exposure. A 6-nm blue shift of that peak was observed after 5-h illumination. Even more pronounced changes were found in the characteristics of Chl fluorescence. The magnitude of the dominating long-wavelength emission band at 736 nm located in untreated particles was five times reduced after 2-h exposure, whereas the loss in absolute Chl contents did not exceed 10% of its initial value. The major peak in low-temperature Chl fluorescence emission spectra shifted from 736 to 721 nm after 6-h WL treatment. Individual Chl-protein complexes differed in the response of their absorption spectra to strong WL. Unlike light-harvesting complexes (LHC), LHCI-680 and LHC-730, which did not exhibit changes in the major peak position, its maximum was shifted from 678 to 671 nm in CPIa complex after PSI submembrane particles were irradiated with strong light for 6 h. The results demonstrated that excitation energy transfer represents the stage of photosynthetic utilization of absorbed quanta which is most sensitive to strong light in isolated PSI particles.

Protective effect of active oxygen scavengers on protein degradation and photochemical function in photosystem I submembrane fractions during light stress

The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illuminated (2000 µE·m−2·s−1) for various periods at 4 °C. The photochemical activity of the submembrane fractions measured as P700 photooxidation was significantly protected in the presence of histidine or n‐propyl gallate. Chlorophyll photobleaching resulting in a decrease of absorbance and fluorescence, and a blue‐shift of both absorbance and fluorescence maximum in the red region, was also greatly delayed in the presence of these scavengers. Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4. The reaction‐center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected. Addition of histidine or n‐propyl gallate lead to significant protection of reaction‐center proteins as well as Lhca against strong illumination. Circular dichroism (CD) spectra revealed that the α‐helix content decreased with increasing period of light exposure, whereas β‐strands, turns, and unordered structure increased. This unfolding was prevented with the addition of histidine or n‐propyl gallate even after 10 h of strong illumination. Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage. The specific action of histidine and n‐propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light‐induced damage in PSI submembrane fractions.

Photoinhibitory Light-induced Changes in the Composition of Chlorophyll–Protein Complexes and Photochemical Activity in Photosystem-I Submembrane Fractions¶

Abstract The effects of irradiation on photosystem (PS)-I submembrane particles using intense white light (2000 μE·m−2·s−1) at chilling temperature (4°C) were studied. PSI-dependent oxygen uptake activity was stable during the first 3 h of photoinhibitory illumination in the presence of added superoxide dismutase (SOD). Without added SOD, the oxygen uptake almost doubled during this period, presumably due to the denaturation of native membrane-bound SOD or its release from the PSI membranes. The total chlorophyll (Chl) content and the magnitude of light-induced absorbance changes at 830 nm (ΔA830) were also barely affected during the first 3–3.5 h of photoinhibitory treatment. However, further exposure to strong light markedly accelerated Chl breakdown followed by a decline in oxygen uptake rate and ΔA830. This corresponded with the disappearance of the bands attributed to PsaA/B polypeptides on electrophoretic gels. Despite the invariant maximum magnitude of ΔA830 during the first 3–3.5 h of photoinhibitory treatment, the light–response curves of P700 oxidation gradually altered, demonstrating a several-fold increase in the ability of weak actinic light to oxidize P700. The major Chl a–protein 1 (CP1) band gradually disappeared during the first 4 h of light exposure with a corresponding increase in the Chl content of a band with lower electrophoretic mobility ascribed to the formation of oligomers containing CP1, light-harvesting complex I (LHCI)-680 and LHCI-730. This aggregation of Chl–protein complexes, likely caused by photoinhibitory-induced cross-linking favoring light harvesting, is proposed to explain the enhanced capacity of weak light to oxidize P700 in photoinhibited PSI submembrane fractions compared with untreated ones.

Quenching of excited states of chlorophyll molecules in submembrane fractions of Photosystem I by exogenous quinones

The ability of three substituted quinones, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichloro-p-benzoquinone (DCBQ), and tetramethyl-p-benzoquinone (duriquinone) to quench the excited states of chlorophyll (Chl) molecules in Photosystem I (PSI) was studied. Chl fluorescence emission measured with isolated PSI submembrane fractions was reduced following the addition of exogenous quinones. This quenching progressively increased with rising concentrations of the exogenous quinones according to the Stern-Volmer law. The values of Stern-Volmer quenching coefficients were found to be 3.28 x 10(5) M(-1) (DBMIB), 1.31 x 10(4) M(-1) (DCBQ), and 3.7 x 10(3) M(-1) (duroquinone). The relative quenching capacities of the various exogenous quinones in PSI thus strictly coincided to those found for the quenching of Fo level of Chl fluorescence in isolated thylakoids, which is emitted largely by Photosystem II (PSII) [Biochim. Biophys. Acta (2003) 1604, 115-123]. Quenching of Chl excited states in PSI submembrane fractions by exogenous quinones slowed down the rate of P700, primary electron donor of PSI, photooxidation measured at limiting actinic light irradiances thus revealing a reduced photochemical capacity of absorbed quanta. The possible involvement of non-photochemical quenching of excited Chl states by oxidized phylloquinones, electron acceptors of PSI, and oxidized plastoquinones, mobile electron carriers between PSII and the cytochrome b(6)/f complex, into the control of photochemical activity of PSI is discussed.

Retardation of photo-induced changes in Photosystem I submembrane particles by glycinebetaine and sucrose

The protective role of co-solutes (glycinebetaine and sucrose) against photodamage in isolated Photosystem (PS) I submembrane particles illuminated (2000 μE m−2 s−1) for various time periods at 4 °C was studied. The photochemical activity of PS I in terms of electron transport measured as oxygen uptake and P700 photooxidation was significantly protected. A photoinduced enhancement of oxygen uptake observed during the first hours of strong light illumination attributed to denaturation or dissociation of membrane-bound superoxide dismutase [Rajagopal et al. (2003) Photochem. Photobiol 77: 284–291] was also retarded by glycinebetaine and sucrose. Chlorophyll photobleaching resulting in a decrease of absorbance and a blue-shift of the absorbance maximum in the red was greatly delayed in the presence of co-solutes. This phenomenon was also observed in the chlorophyll-protein (CP) complexes of PS I particles exposed to strong illumination separated on non-denaturing poly-acrylamide gels. In this case, a decrease in the absorbance of the CP1b band coinciding with an increase of CP1a during the course of illumination and ascribed to oxidative cross-linking (Rajagopal et al. 2003) was also retarded. Our results, thus, clearly demonstrated for the first time that co-solutes could minimize the alteration of photochemical activity and chlorophyll-protein complexes against photodamage of PS I submembranes particles.

Characterization of P700 as a photochemical quencher in isolated photosystem I particles using simultaneous measurements of absorbance changes at 830 nm and photoacoustic signal

The relationship between the redox state of P700, the primary donor of PS I, monitored using absorbance changes at 830 nm and photochemical energy storage in PS I reaction centers assayed with the photoacoustic method (PA) was studied in isolated PS I submembrane particles aspirated onto nitrocellulose filters. Several donors have been used to support the electron transport through PS I. NADPH and NADH demonstrated low rates of electron donation to PS I, while ascorbate and ascorbate plus 2,6-dichlorophenolindophenol (DCIP) couple have been found more effective in both P700+ reduction and stimulation of the variable component of the PA signal. A linear relationship was found in isolated PS I particles between the (A830,max – A830,steady)/A830,max and (PAmax – PAsteady)/PAmax ratios, which characterized the relative amount of P700 in the reduced state and the relative magnitude of the variable PA component, respectively. That linear relationship was obtained independently from the nature of electron donor used for the reduction of P700+. Such linear relationship was also obtained at various wavelengths of modulated light in the range of 660 to 720 nm, only the slope of the linear fits varied with wavelength. It is concluded that reduced P700 act as a photochemical quencher of absorbed energy. Variable thermal dissipation in PS I reaction centers of isolated submembrane particles linearly depends on the amount of reduced P700 and thus constitutes an appropriate indicator of the redox pressure applied to PS I.

Relationship between the redox state of P700 and variable thermal dissipation in isolated photosystem I particles

The relationship between the redox state of P700 monitored using absorbance changes at 830 nm and photochemical energy storage in PSI reaction centers assayed with the photoacoustic method (PA) was studied in isolated PSI submembrane particles aspirated onto nitrocellulose filters. Several donors have been used to support the electron transport through PSI. NADPH and NADH demonstrated low rates of electron donation to PSI, while ascorbate and ascorbate plus 2,6-dichlorophenolindophenol (DCIP) couple have been found more effective in both P700+ reduction and stimulation of the variable component of the PA signal. A linear relationship was found in isolated PSI particles between the (A830, max - A830, steady)/A830, max and (PAmax - PAsteady)/PAmax ratios, which characterized the relative amount of P700 in the reduced state and the relative magnitude of the variable PA component, respectively. That linear relationship was independent on the nature of electron donor used for the reduction of P700+. Such linear relationship were obtained at various wavelength of modulated light used for the measurements of the PA signal in the range of 660 to 720 nm. Only the slope of the linear fits varied with wavelength. It is concluded that in isolated submembrane particles the photochemical energy storage in PSI reaction centers linearly depends on the amount of reduced P700.