Characterization of the primary photochemistry of proteorhodopsin with femtosecond spectroscopy

Proteorhodopsin is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization, leading to transmembrane translocation of a proton toward the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of proteorhodopsin, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the midinfrared spectral regions. Experiments were performed both at pH 9.5 (a physiologically relevant pH value in which the primary proton acceptor of the protonated Schiff base, Asp⁹⁷, is deprotonated) and at pH 6.5 (with Asp⁹⁷ protonated). Simultaneous analysis of the data sets recorded in the two spectral regions and at both pH values reveals a multiexponential excited state decay, with time constants of ∼0.2 ps, ∼2 ps, and ∼20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerizaton pathways that lead to the K-state: one with an effective rate of ∼(2 ps)⁻¹ and the other with a rate of ∼(20 ps)⁻¹. At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of ∼0.7. At pH 6.5, the slower pathway is less productive, which results in an isomerization quantum yield of 0.5. We further observe an ultrafast response of residue Asp²²⁷, which forms part of the counterion complex, corresponding to a strengthening of its hydrogen bond with the Schiff base on K-state formation; and a feature that develops on the 0.2 ps and 2 ps timescale and probably reflects a response of an amide II band in reaction to the isomerization process.     

Magnetic field-stimulated luminescence and a matrix model for energy transfer. A new method for determining the redox state of the first quinone acceptor in the reaction center of whole cells of Rhodospirillum rubrum

In whole cells of Rhodospirillum rubrum the light-induced absorbance difference spectrum of the reduction of the first quinone electron acceptor Q₁ was determined in order to relate the emission yield ф and the magnetic field-induced emission increase Δф to the redox state of Q₁. It was found that Δф/ф² is a linear function of the number of reaction centers, in which Q₁ is reduced, independent of the fraction of reaction centers in the oxidized state. The emission yield is a hyperbolic function of the fraction of reaction centers closed, either by reduction of the acceptor Q₁ or by oxidation of the primary electron donor P. Apparently, in whole cells of R. rubrum a matrix model for energy transfer between various photosynthetic units can be applied. A model is presented, which is a generalization of theoretical considerations reported before (Duysens, L.N.M. (1978) in Chlorophyll Organization and Energy Transfer in Photosynthesis, Ciba Found. Symp. 61 (New Series), pp. 323–340, Elsevier/North-Holland, Amsterdam) and which is in excellent agreement with the experiments. From simultaneous measurements of Δф and ф the redox state of the reaction center can relatively easily be determined. So far, this is the only method for simultaneously measuring the fractions P⁺ and Q⁻₁ in intact cells under steady-state conditions.     

The Specificity of Controlled Protein Disorder in the Photoprotection of?Plants

Light-harvesting pigment-protein complexes of photosystem II of plants have a dual function: they efficiently use absorbed energy for photosynthesis at limiting sunlight intensity and dissipate the excess energy at saturating intensity for photoprotection. Recent single-molecule spectroscopy studies on the trimeric LHCII complex showed that environmental control of the intrinsic protein disorder could in principle explain the switch between their light-harvesting and photoprotective conformations in vivo. However, the validity of this proposal depends strongly on the specificity of the protein dynamics. Here, a similar study has been performed on the minor monomeric antenna complexes of photosystem II (CP29, CP26, and CP24). Despite their high structural homology, similar pigment content and organization compared to LHCII trimers, the environmental response of these proteins was found to be rather distinct. A much larger proportion of the minor antenna complexes were present in permanently weakly fluorescent states under most conditions used; however, unlike LHCII trimers the distribution of the single-molecule population between the strongly and weakly fluorescent states showed no significant sensitivity to low pH, zeaxanthin, or low detergent conditions. The results support a unique role for LHCII trimers in the regulation of light harvesting by controlled fluorescence blinking and suggest that any contribution of the minor antenna complexes to photoprotection would probably involve a distinct mechanism.     

Energy transfer in the peridinin chlorophyll-a protein of Amphidinium carterae studied by polarized transient absorption and target analysis

The peridinin chlorophyll-a protein (PCP) of dinoflagellates differs from the well-studied light-harvesting complexes of purple bacteria and green plants in its large (4:1) carotenoid to chlorophyll ratio and the unusual properties of its primary pigment, the carotenoid peridinin. We utilized ultrafast polarized transient absorption spectroscopy to examine the flow of energy in PCP after initial excitation into the strongly allowed peridinin S2 state. Global and target analysis of the isotropic and anisotropic decays reveals that significant excitation (25-50%) is transferred to chlorophyll-a directly from the peridinin S2 state. Because of overlapping positive and negative features, this pathway was unseen in earlier single-wavelength experiments. In addition, the anisotropy remains constant and high in the peridinin population, indicating that energy transfer from peridinin to peridinin represents a minor or negligible pathway. The carotenoids are also coupled directly to chlorophyll-a via a low-lying singlet state S1 or the recently identified SCT. We model this energy transfer time scale as 2.3 +/- 0.2 ps, driven by a coupling of approximately 47 cm(-1). This coupling strength allows us to estimate that the peridinin S1/SCT donor state transition moment is approximately 3 D.     

Identifying the lowest electronic states of the chlorophylls in the CP47 core antenna protein of photosystem II

CP47 is a pigment-protein complex in the core of photosystem II that tranfers excitation energy to the reaction center. Here we report on a spectroscopic investigation of the isolated CP47 complex. By deconvoluting the 77 K absorption and linear dichroism, red-most states at 683 and 690 nm have been identified with oscillator strengths corresponding to approximately 3 and approximately 1 chlorophyll, respectively. Both states contribute to the 4 K emission, and the Stark spectrum shows that they have a large value for the difference polarizability between their ground and excited states. From site-selective polarized triplet-minus-singlet spectra, an excitonic origin for the 683 nm state was found. The red shift of the 690 nm state is most probably due to strong hydrogen bonding to a protein ligand, as follows from the position of the stretch frequency of the chlorophyll 13(1) keto group (1633 cm(-)(1)) in the fluorescence line narrowing spectrum at 4 K upon red-most excitation. We discuss how the 683 and 690 nm states may be linked to specific chlorophylls in the crystal structure [Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739-743].     

On the magnetic field dependence of the yield of the triplet state in reaction centers of photosynthetic bacteria

The yield of the triplet state in reaction centers of Rhodopseudomonas sphaeroides is dependent on the strength of an applied magnetic field. Reaction centers of the wild type that lack a functional iron complexed to the primary acceptor ubiquinone show a dependence similar to that of reaction centers of the mutant R-26 in which the iron-ubiquinone complex is intact. Apparently, the iron of the iron-ubiquinone complex is not essential to the effect, but it does exert an influence on its extent. In chromatophores, the effect is about 2-fold decreased; the value of the magnetic field at which half the effect is found is about 500 G, in contrast to this value for reaction centers, which is 50–100 G. The magnetodependence of the triplet yield is discussed in terms of the Chemically Induced Dynamic Electron Polarization mechanism (CIDEP).     

Fluorescence and absorption spectroscopy of the weakly fluorescent chlorophyll a in cytochrome b6f of Synechocystis PCC6803.

A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1.     

Pathways of energy flow through the light-harvesting antenna of the photosynthetic purple bacterium rhodobacter sphaeroides

Using low intensity picosecond absorption spectroscopy with independently tunable excitation and probing infrared pulses, we have studied the pathways of energy transport through the light-harvesting antenna pigments of the photosynthetic purple bacterium Rhodobacter sphaeroides. From the observed excited-state rise time of the red-most pigment B896 as a function of excitation wavelength it is concluded that the B850 pigment of LH2 is spectrally heterogeneous. For excitations originating in the B850 pigment this results in a fast channel (9 ps) that is mainly excited in the peak of the B850 absorption band, and a slow channel (35 ps) that is predominantly excited at ~840 nm. Upon excitation of B800, more than 90% of the excitations follow the fast path. From the observed kinetics it is concluded that the majority of the LH2 → LH1 energy transfer takes place within at most a few picoseconds. The rate-limiting step in the whole energy transfer sequence appears to be the B896 → reaction center transfer. The origin of the B850 heterogeneity and the slow 35-ps component is at the moment unclear. Possibly it represents a highly extended form of LH2 in which transfer to LH1 takes a relatively long time, due to a large number of transfer steps.     

The conformation of the complex of the helix destabilizing protein GP32 of bacteriophage T4 and single stranded DNA

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (less than or equal to -10 degrees) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.     

Aggregates of the chlorophyll-binding protein IsiA (CP43') dissipate energy in cyanobacteria

In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of approximately 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light.