Enhanced hydrogen bonding induced by optical excitation: unexpected subnanosecond photoinduced dynamics in a peptide-based [2]rotaxane.
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Ethers, Cyclic↗
Biomedical subjects
I H van Stokkum
Publications and source records attributed to I H van Stokkum.
Oligomerization of light-harvesting I antenna peptides of Rhodospirillum rubrum.
We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum rubrum from the separated alpha beta BChl(2) subunits (B820) and the oligomerization of the B820 subunit from its monomeric peptides. The full LH1 complex was reversibly associated from B820 subunits by either varying the temperature in the range 277-300 K or by varying the detergent concentration in the buffer from 0.36 to 0.52% n-octyl-beta-D-glucopyranoside. Temperature-induced transition measurements showed hysteresis: raising the temperature induced dissociation of B873 directly into B820 subunits whereas upon recooling an intermediate spectral form was observed with an absorption maximum located around 850 nm. This intermediate form was also observed in detergent-induced transitions. It is speculated that the B850 form is a small aggregate of B820, for instance a dimer. Additionally, during a temperature-mediated transition at low detergent concentration, a set of spectral forms with maxima slightly blue-shifted from 873 nm were observed, possibly due to opened rings with one or only a few alpha beta BChl(2) units missing. The temperature-induced transition of LH1 is discussed in terms of a simple assembly model. It is concluded that a moderately cooperative assembly explains the formation of small aggregates of B820 as well as of incomplete rings. Furthermore, the B820 subunits were reversibly dissociated into the monomeric B777 form by increasing either the temperature or the detergent concentration. Estimations of the enthalpy and entropy changes for the dimeric association reaction of B777 into B820 yielded an enthalpy change of -216 kJ mol(-1) and an entropy change of -0.59 kJ mol(-1)K(-1), at a detergent concentration of 0.8% n-octyl-beta-D-glucopyranoside.
Bacterial Proteins↗
The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation.
It is shown that the N-terminal domain of photoactive yellow protein (PYP), which appears relatively independently folded in the ground state of the protein, plays a key role in the transient unfolding during signalling state formation: genetic truncation of the N-terminal domain of PYP significantly decreases the extent of cooperativity of the titration curve that describes chromophore protonation in the ground state of PYP, which is in agreement with the notion that the N-terminal domain is linked through a hydrogen-bonding network with the chromophore-containing domain of the protein. Furthermore, deletion of the N-terminal domain completely abolishes the non-linearity of the Arrhenius plot of the rate of ground state recovery.
Bacterial Proteins↗
An unusual pathway of excitation energy deactivation in carotenoids: singlet-to-triplet conversion on an ultrafast timescale in a photosynthetic antenna.
Carotenoids are important biomolecules that are ubiquitous in nature and find widespread application in medicine. In photosynthesis, they have a large role in light harvesting (LH) and photoprotection. They exert their LH function by donating their excited singlet state to nearby (bacterio)chlorophyll molecules. In photosynthetic bacteria, the efficiency of this energy transfer process can be as low as 30%. Here, we present evidence that an unusual pathway of excited state relaxation in carotenoids underlies this poor LH function, by which carotenoid triplet states are generated directly from carotenoid singlet states. This pathway, operative on a femtosecond and picosecond timescale, involves an intermediate state, which we identify as a new, hitherto uncharacterized carotenoid singlet excited state. In LH complex-bound carotenoids, this state is the precursor on the reaction pathway to the triplet state, whereas in extracted carotenoids in solution, this state returns to the singlet ground state without forming any triplets. We discuss the possible identity of this excited state and argue that fission of the singlet state into a pair of triplet states on individual carotenoid molecules constitutes the mechanism by which the triplets are generated. This is, to our knowledge, the first ever direct observation of a singlet-to-triplet conversion process on an ultrafast timescale in a photosynthetic antenna.
Carotenoids↗
Time-resolved fluorescence emission measurements of photosystem I particles of various cyanobacteria: a unified compartmental model.
Photosystem I (PS-I) contains a small fraction of chlorophylls (Chls) that absorb at wavelengths longer than the primary electron donor P700. The total number of these long wavelength Chls and their spectral distribution are strongly species dependent. In this contribution we present room temperature time-resolved fluorescence data of five PS-I core complexes that contain different amounts of these long wavelength Chls, i.e., monomeric and trimeric photosystem I particles of the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus elongatus, and Spirulina platensis, which were obtained using a synchroscan streak camera. Global analysis of the data reveals considerable differences between the equilibration components (3.4-15 ps) and trapping components (23-50 ps) of the various PS-I complexes. We show that a relatively simple compartmental model can be used to reproduce all of the observed kinetics and demonstrate that the large kinetic differences are purely the result of differences in the long wavelength Chl content. This procedure not only offers rate constants of energy transfer between and of trapping from the compartments, but also well-defined room temperature emission spectra of the individual Chl pools. A pool of red shifted Chls absorbing around 702 nm and emitting around 712 nm was found to be a common feature of all studied PS-I particles. These red shifted Chls were found to be located neither very close to P700 nor very remote from P700. In Synechococcus trimeric and Spirulina monomeric PS-I cores, a second pool of red Chls was present which absorbs around 708 nm, and emits around 721 nm. In Spirulina trimeric PS-I cores an even more red shifted second pool of red Chls was found, absorbing around 715 nm and emitting at 730 nm.
Chlorophyll↗
Probing the structure and dynamics of a DNA hairpin by ultrafast quenching and fluorescence depolarization.
DNA hairpins have been investigated in which individual adenines were replaced by their fluorescent analog 2-aminopurine (2AP). The temperature dependence of the time evolution of polarized emission spectra was monitored with picosecond time resolution. Four isotropic decay components for each oligonucleotide indicated the coexistence of at least four conformations. The fluorescence for three of these was significantly quenched, which is explained by hole transfer from 2AP to guanine(s). An approximately 8-ps component is ascribed to direct hole transfer, the approximately 50-ps and approximately 500-ps components are ascribed to structural reorganization, preceding hole transfer. At room temperature, a fraction remains unquenched on a 10-ns timescale, in contrast to higher temperatures, where the flexibility increases. Besides quenching due to base stacking, a second quenching process was needed to describe the data. Evidence for both intrastrand and interstrand hole transfer was found. The extracted probability for stacking between neighboring bases in double-stranded regions was estimated to be approximately 75% at room temperature and approximately 25% at 80 degrees C, demonstrating structural disorder of the DNA. Fluorescence depolarization revealed both local dynamics of the DNA and overall dynamics of the entire oligonucleotide. Upon raising the temperature, the C-N terminus of the hairpin appears to melt first; the rest of the hairpin denatures above the average melting temperature.
Base Sequence↗
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.
Absorption↗
Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.
The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.
Escherichia coli↗
Förster excitation energy transfer in peridinin-chlorophyll-a-protein.
Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the Förster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the Förster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex.
Carotenoids↗
Kinetics of and intermediates in a photocycle branching reaction of the photoactive yellow protein from Ectothiorhodospira halophila.
We have studied the kinetics of the blue light-induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy. As compared to the parallel dark recovery reaction of the presumed blue-shifted signaling state pB, the light-induced branching reaction showed a 1000-fold higher rate. In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.
Bacterial Proteins↗
Primary charge separation routes in the BChl:BPhe heterodimer reaction centers of Rhodobacter sphaeroides.
Energy transfer and the primary charge separation process are studied as a function of excitation wavelength in membrane-bound reaction centers of Rhodobacter sphaeroides in which the excitonically coupled bacteriochlorophyll homodimer is converted to a bacteriochlorophyll-bacteriopheophytin heterodimer, denoted D [Bylina, E. J., and Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 7226]. In the HM202L heterodimer reaction center, excitation of D using 880 nm excitation light results in a 43 ps decay of the excited heterodimer, D. The decay of D results for about 30% in the formation of the charge separated state D+QA- and for about 70% in a decay directly to the ground state. Upon excitation of the monomeric bacteriochlorophylls using 798 nm excitation light, approximately 60% of the excitation energy is transferred downhill to D, forming D. Clear evidence is obtained that the other 40% of the excitations results in the formation of D+QA- via the pathway BA --> BA+HA- --> D+HA- --> D+QA-. In the membrane-bound "reversed" heterodimer reaction center HL173L, the simplest interpretation of the transient absorption spectra following B excitation is that charge separation occurs solely via the slow D-driven route. However, since a bleach at 812 nm is associated with the spectrum of D in the HL173L reaction center, it cannot be excluded that a state including BB is involved in the charge separation process in this complex.
Bacteriochlorophylls↗
Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides.
A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA-. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA- was associated with similar time constants of 1.5 ps and 1. 7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA- and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.
Bacteriochlorophylls↗
Spectroscopic properties of the CP43 core antenna protein of photosystem II.
CP43 is a chlorophyll-protein complex that funnels excitation energy from the main light-harvesting system of photosystem II to the photochemical reaction center. We purified CP43 from spinach photosystem II membranes in the presence of the nonionic detergent n-dodecyl-beta,D-maltoside and recorded its spectroscopic properties at various temperatures between 4 and 293 K by a number of polarized absorption and fluorescence techniques, fluorescence line narrowing, and Stark spectroscopy. The results indicate two "red" states in the Q(y) absorption region of the chlorophylls. The first peaks at 682.5 nm at 4 K, has an extremely narrow bandwidth with a full width at half-maximum of approximately 2.7 nm (58 cm(-1)) at 4 K, and has the oscillator strength of a single chlorophyll. The second peaks at approximately 679 nm, has a much broader bandshape, is caused by several excitonically interacting chlorophylls, and is responsible for all 4 K absorption at wavelengths longer than 685 nm. The Stark spectrum of CP43 resembles the first derivative of the absorption spectrum and has an exceptionally small overall size, which we attribute to opposing orientations of the monomer dipole moments of the excitonically coupled pigments.
Biophysical Phenomena↗
The flow of excitation energy in LHCII monomers: implications for the structural model of the major plant antenna.
Spectral and kinetic information on energy transfer within the light-harvesting complex II (LHCII) monomer was obtained from this subpicosecond transient absorption study, by using selective excitation (663, 669, 672, 678, and 682 nm) of various Chl a absorption bands and detecting the induced changes over the entire Qy region (650-700 nm). It is shown that transfer from the pigment(s) absorbing around 663 nm to the low energy ones occurs in 5 +/- 1 ps, whereas the 670-nm excitation is delivered to the same "destination" in two phases (0.30 +/- 0.05 ps, and 12 +/- 2 ps), and a fast equilibration (lifetime 0.45 +/- 0.05 ps) takes place within the main absorption band (675-680 nm). From comparison with results from similar time-resolved measurements on trimeric samples, it can be concluded that the intramonomeric energy transfer completely determines the spectral equilibration observed in native LHCII complexes. To correlate the measured lifetimes and their associated spectra with the pigment organization within the available structural model of LHCII (. Nature. 367:614-621), extensive but straightforward theoretical modeling was used. Thus it is demonstrated that the pigment assignment (Chl a or Chl b) given by Kuhlbrandt and co-workers cannot simultaneously describe the dichroic spectra and the transient absorption results for the rather homologous LHCII and CP29 proteins. A more recent assignment for CP29, in which a Chl b molecule ("Chl b5") is identified as a Chl a (Dr. R. Bassi, personal communication), leads to a much better description of both CP29 and LHCII. Furthermore, the orientations of the transition dipole moments, which have not been obtained in the crystal structure, are now assigned for most of the Chl's.
Biophysical Phenomena↗
Formation of a long-lived P+BA- state in plant pheophytin-exchanged reaction centers of Rhodobacter sphaeroides R26 at low temperature.
Femtosecond transient absorption spectroscopy in the range of 500-1040 nm was used to study electron transfer at 5 K in reaction centers of Rhodobacter sphaeroides R26 in which the bacteriopheophytins (BPhe) were replaced by plant pheophytin a (Phe). Primary charge separation took place with a time constant of 1.6 ps, similar to that found in native RCs. Spectral changes around 1020 nm indicated the formation of reduced bacteriochlorophyll (BChl) with the same time constant, and its subsequent decay in 620 ps. This observation identifies the accessory BChl as the primary electron acceptor. No evidence was found for electron transfer to Phe, indicating that electron transfer from BA- occurs directly to the quinone (QA) through superexchange. The results are explained by a model in which the free energy level of P+Phe- lies above that of P+BA-, which itself is below P*. Assuming that the pigment exchange does not affect the energy levels of P* and P+BA-, our results strongly support a two-step model for primary electron transfer in the native bacterial RC, with no, or very little, admixture of superexchange.
Bacteriochlorophylls↗
Energy transfer in LHCII monomers at 77K studied by sub-picosecond transient absorption spectroscopy.
Energy transfer from chlorophyll b (Chl b) to chlorophyll a (Chl a) in monomeric preparations of light-harvesting complex II (LHCII) from spinach was studied at 77 K using pump-probe experiments. Sub-picosecond excitation pulses centered at 650 nm were used to excite preferentially Chl b and difference absorption spectra were detected from 630 to 700 nm. Two distinct Chl b to Chl a transfer times, approximately 200 fs and 3 ps, were found. A clearly distinguishable energy transfer process between Chl a molecules occurred with a time constant of 18 ps. The LHCII monomer data are compared to previously obtained LHCII trimer data, and both data sets are fitted simultaneously using a global analysis fitting routine. Both sets could be described with the following time constants: 140 fs, 600 fs, 8 ps, 20 ps, and 2.9 ns. In both monomers and trimers 50% of the Chl b to Chl a transfer is ultrafast (<200 fs). However, for monomers this transfer occurs to Chl a molecules that absorb significantly more toward shorter wavelengths than for trimers. Part of the transfer from Chl b to Chl a that occurs with a time constant of 600 fs in trimers is slowed down to several picoseconds in monomers. However, it is argued that observed differences between monomers and trimers should be ascribed to the loss of some Chl a upon monomerization or a shift of the absorption maximum of one or several Chl a molecules. It is concluded that Chl b to Chl a transfer occurs only within monomeric subunits of the trimers and not between different subunits.
Chlorophyll↗
Primary electron transfer kinetics in membrane-bound Rhodobacter sphaeroides reaction centers: a global and target analysis.
Absorbance difference kinetics were measured on quinone-reduced membrane-bound wild type Rhodobacter sphaeroides reaction centers in the wavelength region from 690 to 1060 nm using 800 nm excitation. Global analysis of the data revealed five lifetimes of 0.18, 1.9, 5.1, and 22 ps and a long-lived component for the processes that underlie the spectral evolution of the system. The 0.18 ps component was ascribed to energy transfer from the excited state of the accessory bacteriochlorophyll (B*) to the primary donor (P*). The 1.9 ps component was associated with a state involving a BChl anion absorbing in the 1020 nm region. This led to the conclusion that primary electron transfer is best described by a model in which the electron is passed from P* to the acceptor bacteriopheophytin (HL) via the monomeric bacteriochlorophyll (BL), with the formation of the radical pair state . An analysis assuming partial direct charge separation from B* [Van Brederode, M. E., Jones, M. R., and Van Grondelle, R. (1997) Chem. Phys. Lett. 268, 143-149] was also consistent with the data. Within the framework of a five component model, the 5.1 and 22 ps lifetimes were associated with charge separation and relaxation of the radical pair state respectively, providing a description which adequately accounted for the complex kinetics of decay of P*. Alternatively, by assuming that the 5.1 and 22 ps components originate from a single component with a multi-exponential decay, a simpler analysis with only four components could be employed, resulting in only a small increase (7%) in the weighted root mean square error of the fit. In both descriptions part of the decay of P* proceeds with a lifetime of about 2 ps. The relative merits of these alternative descriptions of the primary events in light-driven electron transfer are discussed. Similar measurements on YM210H mutant reaction centers revealed four lifetimes of 0.2, 3.1, and 12 ps and a long-lived component. The 3.1 and 12 ps lifetimes are ascribed to multi-exponential decay of the P* state. The differences with the WT data are discussed.
Electron Transport↗
Time-resolved and steady-state spectroscopic analysis of membrane-bound reaction centers from Rhodobacter sphaeroides: comparisons with detergent-solubilized complexes.
The spectroscopic analysis of the antenna-deficient Rhodobacter sphaeroides strain RCO1 has been extended to an investigation of the kinetics and spectroscopy of primary charge separation. Global analysis of time-resolved difference spectra demonstrated that the rate of charge separation in membrane-bound reaction centers is slightly slower than in detergent-solubilized reaction centers from the same strain. A kinetic analysis of the decay of the primary donor excited state at single wavelengths was carried out using a high repetition rate laser system, with the reaction centers being maintained in the open state using a combination of phenazine methosulfate and horse heart cytochrome c. The kinetics of primary charge separation in both membrane-bound and solubilized reaction centers were found to be non-monoexponential, with two exponential decay components required for a satisfactory description of the decay of the primary donor excited state. The overall rate of charge separation in membrane-bound reaction centers was slowed if the primary acceptor quinone was reduced using sodium ascorbate. This slowing was caused, in part, by an increase in the relative amplitude of the slower of the two exponential components. The acceleration in the rate of charge separation observed on removal of the reaction center from the membrane did not appear to be caused by a significant change in the electrochemical properties of the primary donor. The influence of the environment of the reaction center on primary charge separation is discussed together with the origins of the non-monoexponential decay of the primary donor excited state.
Ascorbic Acid↗