George Porter

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.

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.

Resolution of a long lived fluorescence component from D1/D2/cytochrome b-559 reaction centres

D1/D2/cytochrome b‐559 reaction centres rapidly degrade under continuous illumination. This is a problem in time‐resolved spectroscopy experiments. A long lived fluorescence component assigned to radical pair recombination has been reported previously, but contributing to less than 2% of the fluorescence. This letter reports that sample stability is significantly improved under anaerobic conditions. Fluorescence lifetime measurements described here assign 44% of the total fluorescence yield to a 36.5 ± 2.5 ns lifetime.

Primary processes in isolated Photosystem II reaction centres probed by magic angle transient absorption spectroscopy

Abstract There is currently some disagreement regarding the dominant time constant for formation of the radical pair state P680 + Ph − in isolated photosystem two reaction centres. It has recently been suggested that this disagreement may originate, at least in part, from different polarisations of the pump and probe beams used in optical experiments. In this paper, we present data collected using a magic angle configuration of the pump and probe polarisations. We find that these data support our previous interpretation of data collected using a parallel polarisation configuration. Moreover, we present further evidence to support our conclusion that formation of the P680 + Ph − state primarily occurs with a 21 ps time constant when P680 is directly excited. A 3 ps component is also observed; this component is not associated with a large proportion of the radical pair formation. We discuss our data and interpretation in comparison with those of other groups.

Determination of P680 singlet state lifetimes in photosystem two reaction centres

Abstract Photosystem two reaction centres have been studied using femtosecond transient absorption spectroscopy, employing sufficiently low excitation levels to avoid multiphoton processes. The absorption changes are shown to be strongly dependent on excitation wavelength. Transiently bleached P680 is found to have a higher oscillators strength than monomeric chlorophyll molecules, suggesting that the p680 excited singlet state is initially delocalised. When P680 is directly excited, its singlet excited state is observed to decay with lifetimes of 400±100 fs and 3.5± 1.5 ps.

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.

The influence of a solid/liquid interface on the fluorescence kinetics of the triphenylmethane dye malachite green

Abstract Time-resolved fluorescence measurements are reported of a triphenylmethane dye (malachite green), adsorbed at and aqueous/quartz interface, studied by evanescent wave excitation. The importance of internal conversion as the dominant deactivation pathway from the first excited singlet state of malachite green is observed to be greatly reduced for molecules at a solid/liquid interface compared to those in low viscosity solvents. Surface coverage dependent decay kinetics are observed, and at the lowest surface coverages studied, the excited singlet state lifetime of the dye is more than two orders of magnitude greater than the value observed for the dye in aqueous solution. Fluorescence decay kinetics are found to be non mono-exponential under all conditions, and the limitations of simple sum-of-exponentials fitting are discussed in light of other models currently available. It is possible to distinguish at least two populations of malachite green adsorbed at the surface.

Sub-picosecond oscillatory phenomena in silica glasses

Abstract We report the observation of sub-picosecond oscillatory signals during pump—probe studies of silica glasses. The behaviour of these oscillations is characteristic of impulsive stimulated Raman scattering. We demonstrate that these oscillations can be large enough to interfere with transient absorption experiments when silica cuvettes are used to retain the sample, and are potentially a cause of artifactual interference.