Linda B. Giorgi

Characterisation of triplet states in isolated Photosystem II reaction centres: Oxygen quenching as a mechanism for photodamage

Abstract Transient absorption spectroscopy has been used to study the isolated D1/D2/cytochrome b-559 reaction centre complex at 4°C. The D1/D2 reaction centre is observed to have an increased susceptibility to photodamage under aerobic conditions. This is attributed to oxygen quenching of a P680 triplet state, which results in the formation of highly oxidising singlet oxygen. This P680 triplet state is observed to have a lifetime of (1.0±0.1) ms under anaerobic conditions, shortening to (33±3) μs in the presence of oxygen. This state, which has a quantum yield of approx. 30%, is identified as residing upon the primary electron donor P680 by the transient bleaching of its reddest absorption band, which peaks at (680.5±0.5) nm. The shape of the P680 triplet-minus-singlet absorption difference spectrum, and particularly the (12±1) nm bandwidth of the red absorption band bleach, indicate that P680 is probably a pair of excitonically coupled chlorophyll molecules, with the P680 triplet state being localised upon one of these chlorophyll molecules. The red absorption band bleached by P680 triplet formation has a peak extinction coefficient of 133000 M−1 · cm−1 and an oscillator strength 1.1-times larger than that of the Qy-band of a chlorophyll a monomer in ether. It is shown that this P680 triplet state is formed primarily by charge recombination from the primary radical pair state at 4°C. A 3% quantum yield of a carotenoid triple state characterised by an absorption peak at 526 nm is also observed. The observed P680 triplet does not appear to be quenched by this carotenoid.

The state of detergent solubilised light-harvesting chlorophyll-a/b protein complex as monitored by picosecond time-resolved fluorescence and circular dichroism

Abstract Steady-state and picosecond time-resolved fluorescence techniques in conjunction with circular dichroism have been used to study the light-harvesting chlorophyll-a/b protein complex (LHC) isolated from pea chloroplasts. In particular, the effect of changing the detergent / chlorophyll ratio on the state of the LHC has been investigated. Our results have been interpreted in light of the known protein geometry of the LHC in 2-dimensional crystals (Kuhlbrandt, W. (1984) Nature 307, 478–479). The fluorescence lifetime data reveals 1 / e-lifetimes of 3.53 (±0.04) ns and 1.10 (±0.01) ns for a stable, efficiently energy-transferring state of the LHC. Subnanosecond lifetimes are observed under conditions leading to aggregation, while a long component of 5.50 (±0.16) ns corresponding to free Chl a is found when the detergent / chlorophyll ratio is high. The circular dichroism shows a major Chl-b exciton, a Chl-a / b exciton and a further ‘quenching’ Chl-b exciton. These have been attributed to: a C3 symmetric Chl-b interaction for which the intact C3 protein trimer geometry is a prerequisite; a dimeric Chl-a / b interaction, the presence of which is critically dependent on the detergent type; and a further Chl-b interaction which arises from the presence of aggregated trimers, respectively. We have found that the degree of heterogeneity with respect to the oligomeric state of the pigment-protein trimers is dependent upon the detergent / chlorophyll ratio used. Low detergent / chlorophyll ratios result in extensive aggregation of the trimers with a geometry similar to that found in 2-dimensional crystals of the LHC. Moderate detergent conditions yield predominantly non-aggregated trimers. Excess detergent conditions result in considerable chromophore heterogeneity and loss of the main Chl-b exciton consistent with protein denaturation through an initial break up of the trimer geometry. From these results we believe that in vitro the minimum stable functional unit corresponds to a C3 symmetric protein trimer.

Comparison of the Dl/D2/cytochrome b559 reaction centre complex of photosystem two isolated by two different methods

Photosystem 2 reaction centre complexes prepared either by solubilisation with Triton X‐100 and subsequent exchange into dodecyl maltoside or by a procedure involving a combination of dodecyl maltoside and LiC104, were characterised in terms of chlorophyll a, pheophytin a, β‐carotene and cytochrome b559 content. Time‐resolved chlorophyll fluorescence decay kinetics were measured using both types of complexes. Our data show that the isolated photosystem two reaction centre complex contain, for two pheophytin a molecules, close to six chlorophyll a, two β‐carotene and one cytochrome b559. No major differences were observed in the composition or the kinetic characteristics measured in the samples prepared by the different procedures. Time‐resolved fluorescence measurements indicate that more than 94% of the chlorophyll a in both preparations is coupled to the reaction centre complex.

Thermodynamic properties of D1/D2/cytochrome b-559 reaction centres investigated by time-resolved fluorescence measurements

Abstract Photosystem II reaction centres have been studied using time-correlated single photon counting to provide time-resolved fluorescence information. Comparative quantum yield measurements suggest that up to 94% of the chlorophyll in these preparations is coupled to the charge-separation pathway. Analysis of time-resolved fluorescence data suggests a free-energy gap between the excited chlorophyll special pair and the primary radical pair of ΔG( P 680 + Ph − - P 680∗) = −0.11 eV at 277 K. Measurements of this free-energy difference between 277 K and 77 K show that between 220 K and 77 K the contributions of entropy and enthalpy are ΔS( P 680 + Ph − - P 680∗) = +3.8 · 10 −4 eV · K −1 and ΔH( P 680 + Ph − - P 680∗) = −5.0 · 10 −3 eV , respectively. Thus the charge transfer reaction is dominated by entropy contributions between 77 K and 220 K, the ratio of entropy to enthalpy at 220 K being 17:1. The relationship between ΔG and temperature is non-linear over the range 220–270 K, which indicates that enthalpy and/or entropy are temperature dependent in this region.

Energy transfer to low energy chlorophyll species prior to trapping by P700 and subsequent electron transfer

It is found that the two singlet state lifetimes observed in medium sized isolated Photosystem One reaction centres belong to two distinct sets of particles. The nanosecond lifetime is due to PS1 particles in which P700 does not trap excitation energy, and the excitation energy is homogeneously distributed within the antennae of these particles. The spectral features of the picosecond component show that excitation energy in the antenna has become largely concentrated in one or more low energy (red) chlorophyll species within 3.5 ps. Antennae which have become decoupled from P700 also appear to be decoupled from these red “ancillary” chlorophylls, and this suggests that some substructure or level of organisation links them to P700.The rate of quenching of antenna singlet states appears to be independent of the redox state of P700 under the conditions used here, and oxidising P700 does not prevent excitation energy from reaching the red chlorophyll species in the antenna.We find no evidence in the data presented here of a chlorophyll molecule acting as a “metastable” primary acceptor (A0). The lower limit for the detection of such a species in these data is 20% of the optical density of the transient P700 bleach.

Redox potentials of cytochrome b-559 in the D1/D2/cytochrome b-559 reaction centre of Photosystem II

Abstract Redox titrations of a stable form of the Photosystem II (PS II) reaction centre, isolated from peas, have detected three redox forms of cytochrome b-559: a high-potential form (+ 430 mV), an intermediate-potential form (+ 180 mV) and a low-potential form (+ 25 mV) with relative amplitudes of 28%, 62% and 10%, respectively. These results contrast with the observations of Shuvalov et al. (Shuvalov, V.A., Heber, U. and Schreiber, U. (1989) FEBS Lett. 258, 27–31), who reported midpoint potentials much lower than these for cytochrome b-559 in isolated PS II reaction centres. Our data show that, if the reaction centre complex is handled appropriately, then cytochrome b-559 can have redox potentials quite close to those found in intact membrane systems.

Comparison of primary electron transfer in Photosystem II reaction centres isolated from the higher plant Pisum sativum and the green alga Chlamydomonas reinhardtii

Abstract We have used ultrafast spectroscopy to compare the photochemistry of Photosystem II (PS II) reaction centres isolated from the higher plant pea, Pisum sativum , and a green alga, Chlamydomonas reinhardtii . The apparatus now used for these experiments includes a highly sensitive multichannel detector and a magic angle configuration for the pump and probe beam polarisations. For both organisms, identical time constants were obtained for both the formation of the radical pair P680 + Ph − , and charge recombination from this state. We conclude that the use of the organism Chlamydomonas reinhardtii is well suited to future studies of the primary photochemistry of genetically mutated PS II reaction centres.

Redox-potentiometric titrations of the electrochromic absorption change in chloroplasts

Abstract Two phases of the electrochromic 515 nm absorption change in chloroplasts elicited by microsecond flashes can be resolved kinetically. Redox-potentiometric titrations indicate that the initial amplitude appearing within 0.5 ms, and designated as phase a, has three components in the low-potential region with E m7.5 values of +60 mV, −195 mV and less than −400 mV. From the insensitivity to DCMU, we propose that the species with E m7.5 values of −195 mV and less than −400 mV are both related to Photosystem I. This conclusion was supported by the loss of both components when the Photosystem I reaction centre (P-700) was chemically oxidised ( E m7.5 = +370 mV). The species having an E m7.5 less than −400 mV is presumed to be the Photosystem I primary acceptor, while the E m7.5 = −195 mV wave could be due to a secondary electron acceptor, such as cytochrome b -563 LP , whose photoreduction is possible owing to the long duration of the excitation flash. The DCMU-sensitive component with an E m7.5 of +60 mV is assumed to be the primary quinone acceptor (Q A ) of Photosystem II. Unlike the Photosystem I redox components, the midpoint potential of this species is sensitive to the background ionic level: the E m7.5 is shifted to −100 mV when the cation concentration is lowered to facilitate membrane unstacking. The slow phase of the electrochromic signal (phase b) has been estimated by measuring the 2,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone-sensitive amplitude of the absorption change at 20 ms. The signal appears with an estimated E m7.5 = +50 mV, becomes maximal at −50 mV and attenuates with an E m7.5 of about −180 mV. These results suggest that phase b occurs when the plastoquinone pool is reduced and cytochrome b -563 LP is oxidised.

The design of a picosecond flash spectroscope and its application to photosynthesis

The design and characteristics of the current picosecond flash-photolysis apparatus at the Royal Institution is described. This equipment is being applied to the investigation of various photosynthetic materials isolated from higher plants. Typical absorption spectra obtained with Photosystem 1 (PS1) reaction centres, isolated from pea chloroplast, are presented to show the quality of data now obtainable on a picosecond timescale.

A comparison of the photochemical activity of two forms of Photosystem II reaction centre isolated from sugar beet

Abstract Both time-resolved fluorescence and absorption measurements have been conducted on two different forms of Photosystem II reaction centre isolated from sugar beet. One form, called RC IIa, contained 6 chlorophylls and 2 β-carotenes per 2 pheophytins, while the other, called RC Iib, contained 4 chlorophylls and 1 β-carotene per 2 pheophytins. Single photon-counting fluorescence decay obtained from the two preparations showed similar charge recombination fluorescence lifetimes which could be resolved into two components of 46.1 and 14.2 ns. Analysis of the amplitude of the fluorescence of the fast component of 5.6 ns, which largely originates from non-functional chlorophyll, gave an estimate of the relative activity for RC IIb which was only a 5.5% lower compared to that of RC IIa. This small relative difference in photochemical activity was also confirmed by measuring the extent of primary charge separation activity using flash induced absorption spectroscopy. In this case the amplitude of the long-lived component, attributed to primary radical-pair formation and recombination, was 16% lower in RC IIb as compared with RC IIa. When the secondary electron transfer activity of the two forms of reaction centre were measured using MnCl 2 and silicomolibdate as electron donor and acceptor respectively, RC IIb was 16% less active than RC IIa. From the data we conclude that the removal of 2 chlorophylls and 1 β-carotene molecules from the isolated Photosystem II reaction centre only slightly impair its functional activity with respect to primary charge separation. This conclusion seems to suggest that photochemically active isolated reaction centres of Photosystem II and purple bacteria can have the same minimum pigment stoichiometry of 4 chlorophylls and 1 carotenoid per 2 pheophytins.