Long-lived charge-separated states in bacterial reaction centers isolated from Rhodobacter sphaeroides

AbstractWe studied the accumulation of long-lived charge-separated states in reaction centers isolated from Rhodobacter sphaeroides, using continuous illumination, or trains of single-turnover flashes. We found that under both conditions a long-lived state was produced with a quantum yield of about 1%. This long-lived species resembles the normal P+Q− state in all respects, but has a lifetime of several minutes. Under continuous illumination the long-lived state can be accumulated, leading to close to full conversion of the reaction centers into this state. The lifetime of this accumulated state varies from a few minutes up to more than 20 min, and depends on the illumination history. Surprisingly, the lifetime and quantum yield do not depend on the presence of the secondary quinone, QB. Under oxygen-free conditions the accumulation was reversible, no changes in the normal recombination times were observed due to the intense illumination. The long-lived state is responsible for most of the dark adaptation and hysteresis effects observed in room temperature experiments. A simple method for quinone extraction and reconstitution was developed

On the analysis of non-photochemical chlorophyll fluorescence quenching curves I. Theoretical considerations

AbstractNon-photochemical quenching (NPQ) protects photosynthetic organisms against photodamage by high light. One of the key measuring parameters for characterizing NPQ is the high-light induced decrease in chlorophyll fluorescence. The originally measured data are maximal fluorescence (Fm′) signals as a function of actinic illumination time (Fm′(t)). Usually these original data are converted into the so-called Stern–Volmer quenching function, NPQSV(t), which is then analyzed and interpreted in terms of various NPQ mechanisms and kinetics. However, the interpretation of this analysis essentially depends on the assumption that NPQ follows indeed a Stern–Volmer relationship. Here, we question this commonly assumed relationship, which surprisingly has never been proven. We demonstrate by simulation of quenching data that particularly the conversion of time-dependent quenching curves like Fm′(t) into NPQSV(t) is (mathematically) not “innocent” in terms of its effects. It distorts the kinetic quenching information contained in the originally measured function Fm′(t), leading to a severe (often sigmoidal) distortion of the time-dependence of quenching and has negative impact on the ability to uncover the underlying quenching mechanisms and their contribution to the quenching kinetics. We conclude that the commonly applied analysis of time-dependent NPQ in NPQSV(t) space should be reconsidered. First, there exists no sound theoretical basis for this common practice. Second, there occurs no loss of information whatsoever when analyzing and interpreting the originally measured Fm′(t) data directly. Consequently, the analysis of Fm′(t) data has a much higher potential to provide correct mechanistic answers when trying to correlate quenching data with other biochemical information related to quenching

Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise

AbstractExperiments were carried out to identify a process co-determining with QA the fluorescence rise between F0 and FM. With 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), the fluorescence rise is sigmoidal, in its absence it is not. Lowering the temperature to −10°C the sigmoidicity is lost. It is shown that the sigmoidicity is due to the kinetic overlap between the reduction kinetics of QA and a second process; an overlap that disappears at low temperature because the temperature dependences of the two processes differ. This second process can still relax at −60°C where recombination between QA− and the donor side of photosystem (PS) II is blocked. This suggests that it is not a redox reaction but a conformational change can explain the data. Without DCMU, a reduced photosynthetic electron transport chain (ETC) is a pre-condition for reaching the FM. About 40% of the variable fluorescence relaxes in 100ms. Re-induction while the ETC is still reduced takes a few ms and this is a photochemical process. The fact that the process can relax and be re-induced in the absence of changes in the redox state of the plastoquinone (PQ) pool implies that it is unrelated to the QB-occupancy state and PQ-pool quenching. In both +/−DCMU the process studied represents ~30% of the fluorescence rise. The presented observations are best described within a conformational protein relaxation concept. In untreated leaves we assume that conformational changes are only induced when QA is reduced and relax rapidly on re-oxidation. This would explain the relationship between the fluorescence rise and the ETC-reduction

Far-red fluorescence:A direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching

AbstractTime-resolved fluorescence on oligomers of the main light-harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter-trimer chlorophyll–chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far-red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400ps fluorescence component appearing in intact leaves of Arabidopsis when non-photochemical quenching (NPQ) is induced. The fluorescence components with high far-red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far-red emitting state is shown to be an emissive Chl–Chl charge transfer state which plays a crucial part in the quenching

Primary reactions - From isolated complexes to intact plants

It is shown that in all photosystems of oxygenic photosynthesis, with the exception of the largest PS II units, the energy trapping for the excited states is limited by charge separation in the reaction centers, and not by energy transfer from the antenna to the trap. Recent ultrafast spectroscopic studies show that in the reaction centers of oxygenic photosynthesis the primary electron donors are the monomeric accessory chorophylls, and not the “special pairs” P680 or P700. These findings change dramatically our understanding of the ultrafast electron transfer processes in reaction centers. The insight gained from the ultrafast studies on isolated antenna/reaction center units has been applied to unravel the quenching sites in the so-called “non-photochemical quenching” processes that protect in particular photosystem II against photodamage in high light

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F(0) level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λ(max)(em) ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ(max)(em) = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ(max)(em) = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ(max)(em) = 685 nm) derives from dead chlorophyll. (b) At the F(max) level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F(0) level, from closed PS II α-centers (τ ≈ 2.2 ns, λ(max)(em) = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ(max)(em) = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202)