Energy transfer and charge separation in photosystem I: P700 oxidation upon selective excitation of the long-wavelength antenna chlorophylls of Synechococcus elongatus.

Photosystem I of the cyanobacterium Synechococcus elongatus contains two spectral pools of chlorophylls called C-708 and C-719 that absorb at longer wavelengths than the primary electron donor P700. We investigated the relative quantum yields of photochemical charge separation and fluorescence as a function of excitation wavelength and temperature in trimeric and monomeric photosystem I complexes of this cyanobacterium. The monomeric complexes are characterized by a reduced content of the C-719 spectral form. At room temperature, an analysis of the wavelength dependence of P700 oxidation indicated that all absorbed light, even of wavelengths of up to 750 nm, has the same probability of resulting in a stable P700 photooxidation. Upon cooling from 295 K to 5 K, the nonselectively excited steady-state emission increased by 11- and 16-fold in the trimeric and monomeric complexes, respectively, whereas the quantum yield of P700 oxidation decreased 2.2- and 1.7-fold. Fluorescence excitation spectra at 5 K indicate that the fluorescence quantum yield further increases upon scanning of the excitation wavelength from 690 nm to 710 nm, whereas the quantum yield of P700 oxidation decreases significantly upon excitation at wavelengths longer than 700 nm. Based on these findings, we conclude that at 5 K the excited state is not equilibrated over the antenna before charge separation occurs, and that approximately 50% of the excitations reach P700 before they become irreversibly trapped on one of the long-wavelength antenna pigments. Possible spatial organizations of the long-wavelength antenna pigments in the three-dimensional structure of photosystem I are discussed.     

Red antenna states of photosystem I from Synechococcus sp. PCC 7002

Absorption, fluorescence and single-molecule spectroscopy at low temperatures were used to elucidate spectral properties, heterogeneities and dynamics of the red-shifted chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacterium Synechoccocus sp. PCC 7002. The 77 K absorption spectrum indicates the presence of 2–3 red-shifted Chla’s absorbing at about 708 nm. The fluorescence emission spectrum is dominated by a broad band at 714 nm. The emission spectra of single PSI complexes show zero-phonon lines (ZPLs) as well as a broad intensity distribution without ZPLs. The spectral region below 710 nm often shows ZPLs, they form a spectral band with a maximum at 698 nm (F698). The region above 710 nm is dominated by broad intensity distributions and the observation of ZPLs is less frequent. The broad distributions are due to the emission of the C708 Chla’s and the emission from F698 stems from a Chla species absorbing at the blue side of P700. The properties of these two emissions show a close relation to those of the C708 and C719 pools observed in T. elongatus. Therefore an assignment of F698 and C708 to Chla-species with similarities to C708 and C719 in T. elongatus is proposed.     

Excitation wavelength dependence of the fluorescence kinetics in Photosystem I particles from Synechocystis PCC 6803 and Synechococcus elongatus

The excitation-wavelength dependence of the excited-state dynamics of monomeric and trimeric Photosystem I (PSI) particles from Synechocystis PCC 6803 as well as trimeric PSI particles from Synechococcus elongatus has been studied at room temperature using time-resolved fluorescence spectroscopy. For aselective (400 nm), carotenoid (505 nm), and bulk chlorophyll (approximately 650 nm) excitation in all species, a downhill energy-transfer component is observed, corresponding to a lifetime of 3.4-5.5 ps. For selective red excitation (702-719 nm) in all species, a significantly faster, an approximately 1-ps, uphill transfer component was recorded. In Synechococcus PSI, an additional approximately 10-ps downhill energy-transfer component is found for all wavelengths of excitation, except 719 nm. Each of the species exhibits its own characteristic trap spectrum, the shape of which is independent of the wavelength of excitation. This trap spectrum decays in approximately 23 ps in both monomeric and trimeric Synechocystis PSI and in approximately 35 ps in trimeric Synechococcus PSI. The data were simulated based on the 2.5 A structural model of PSI of Synechococcus elongatus using the F?rster equation for energy transfer, and using the 0.6-1-ps charge-separation time and the value of 1.2-1.3 for the index of refraction that were obtained from the dynamics of a hypothetical PSI particle without red chls. The experimentally obtained lifetimes and spectra were reproduced well by assigning three of the chlorophyll-a (chla) dimers observed in the structure to the C708/C702RT pool of red chls present in PSI from both species. Essential for the simulation of the dynamics of Synechococcus PSI is the assignment of the single chla trimer in the structure to the C719/C708RT pool present in this species.     

Red Chlorophylls in the Exciton Model of Photosystem I

Structural arrangement of pigment molecules of Photosystem I of photosynthetic cyanobacterium Synechococcus elongatus is used for theoretical modeling of the excitation energy spectrum. It is demonstrated that a straightforward application of the exciton theory with the assumption of the same molecular transition energy does not describe the red side of the absorption spectrum. Since the inhomogeneity in the molecular transition energies caused by a dispersive interaction with the molecular surrounding cannot be identified directly from the structural model, the evolutionary search procedure is used for fitting the low temperature absorption and circular dichroism spectra. As a result, one dimer, three trimers and one tetramer of chlorophyll molecules responsible for the red side of the absorption spectrum with their assignment to the spectroscopically established three bands at 708, 714 and 719 nm are determined. All of them are found to be situated not in the very close vicinity of the reaction center but are encircling it almost at the same distance. In order to explain the unusual broadening on the red side of the spectrum the exciton state mixing with the charge transfer (CT) states is considered. It is shown that two effects can be distinguished as caused by mixing of those states: (i) the oscillator strength borrowing by the CT state from the exciton transition and (ii) the borrowing of the high density of the CT state by the exciton state. The intermolecular vibrations between two counter-charged molecules determine the high density in the CT state. From the broad red absorption wing it is concluded that the CT state should be the lowest state in the complexes under consideration. Such mixing effect enables resolving the diversity in the molecular transition energies as determined by different theoretical approaches.     

The far red induced slow component of delayed light from chloroplasts is emitted from Photosystem II

In spinach chloroplasts illuminated with far red light, the relative intensity maximum during the decay of delayed light is emitted at 680–690 nm. This finding supports previous models predicting emission from Photosystem II, and contradicts earlier attributions to Photosystem I.Due to self absorption, the emission spectrum of the relative maximum is shifted to longer wavelengths and displays apparent Photosystem I characteristics in chloroplast samples of higher concentration or in leaves. This may have caused earlier investigators to ascribe the emission to Photosystem I.A differences between the spectral width of the emission spectra of delayed fluorescence and the relative maximum indicates that these two phenomena represent emission from different sub-populations of Photosystem II centers.Abbreviations PS I Photosystem I - PS II Photosystem II - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Steady-state polarized light spectroscopy of isolated Photosystem I complexes

Monomeric and trimeric Photosystem I core complexes from the cyanobacterium Synechocystis PCC 6803 and LHC-I containing Photosystem I (PS I-200) complexes from spinach have been characterized by steady-state, polarized light spectroscopy at 77 K. The absorption spectra of the monomeric and trimeric core complexes from Synechocystis were remarkably similar, except for the amplitude of a spectral component at long wavelength, which was about twice as large in the trimeric complexes. This spectral component did not contribute significantly to the CD-spectrum. The (77 K) steady-state emission spectra showed prominent peaks at 724 nm (for the Synechocystis core complexes) and at 735 nm (for PS I-200). A comparison of the excitation spectra of the main emission band and the absorption spectra suggested that a significant part of the excitations do not pass the red pigments before being trapped by P-700. Polarized fluorescence excitation spectra of the monomeric and trimeric core complexes revealed a remarkably high anisotropy (0.3) above 705 nm. This suggested one or more of the following possibilities: 1) there is one red-most pigment to which all excitations are directed, 2) there are more red-most pigments but with (almost) parallel orientations, 3) there are more red-most pigments, but they are not connected by energy transfer. The high anisotropy above 705 nm of the trimeric complexes indicated that the long-wavelength pigments on different monomers are not connected by energy transfer. In contrary to the Synechocystis core complexes, the anisotropy spectrum of the LHC I containing complexes from spinach was not constant in the region of the long-wavelength pigments, and decreased significantly below 720 nm, the wavelength where the long-wavelength pigments on the core complexes start to absorb. These results suggested that in spinach the long-wavelength pigments on core and LHC-I are connected by energy transfer and have a non-parallel average Q_y(0-0) transitions.Abbreviations PS Photosystem - P Primary donor - Chl chlorophyll - LHC light-harvesting complex - CD circular dichroism - LD linear dichroism - BisTris 2-[bis(2-hydroxyethyl)amino]-2-hydroxy-methylpropane-1,3-diol - RC reaction center     

Excitation energy transfer to Photosystem I in filaments and heterocysts of Nostoc punctiforme

Cyanobacteria adapt to varying light conditions by controlling the amount of excitation energy to the photosystems. On the minute time scale this leads to redirection of the excitation energy, usually referred to as state transitions, which involves movement of the phycobilisomes. We have studied short-term light adaptation in isolated heterocysts and intact filaments from the cyanobacterium Nostoc punctiforme ATCC 29133. In N.punctiforme vegetative cells differentiate into heterocysts where nitrogen fixation takes place. Photosystem II is inactivated in the heterocysts, and the abundancy of Photosystem I is increased relative to the vegetative cells. To study light-induced changes in energy transfer to Photosystem I, pre-illumination was made to dark adapted isolated heterocysts. Illumination wavelengths were chosen to excite Photosystem I (708 nm) or phycobilisomes (560 nm) specifically. In heterocysts that were pre-illuminated at 708 nm, fluorescence from the phycobilisome terminal emitter was observed in the 77 K emission spectrum. However, illumination with 560 nm light caused quenching of the emission from the terminal emitter, with a simultaneous increase in the emission at 750 nm, indicating that the 560 nm pre-illumination caused trimerization of Photosystem I. Excitation spectra showed that 560 nm pre-illumination led to an increase in excitation transfer from the phycobilisomes to trimeric Photosystem I. Illumination at 708 nm did not lead to increased energy transfer from the phycobilisome to Photosystem I compared to dark adapted samples. The measurements were repeated using intact filaments containing vegetative cells, and found to give very similar results as the heterocysts. This demonstrates that molecular events leading to increased excitation energy transfer to Photosystem I, including trimerization, are independent of Photosystem II activity.     

Spectral and Kinetic Analysis of the Energy Coupling in the PS I–LHC I Supercomplex from the Green Alga Chlamydomonas reinhardtii at 77 K

Energy transfer processes in the chlorophyll antenna of the PS I–LHCI supercomplexes from the green alga Chlamydomonas reinhardtii have been studied at 77 K using transient absorption spectroscopy with multicolor excitation in the 640–670 nm region. Comparison of the kinetic data obtained at low and room temperatures indicates that the slow ∼ ∼100 ps excitation equilibration phase that is characteristic of energy coupling of the LHCI peripheral antenna to the PS I core at physiological temperatures (Melkozernov AN, Kargul J, Lin S, Barber J and Blankenship RE (2004) J Phys Chem B 108: 10547–10555) is not observed in the excitation dynamics of the PS I–LHCI supercomplex at 77 K. This suggests that at low temperatures the peripheral antenna is energetically uncoupled from the PS I core antenna. Under these conditions the observed kinetic phases on the time scales from subpicoseconds to tens of picoseconds represent the superposition of the processes occurring independently in the PS I core antenna and the Chl a/b containing LHCI antenna. In the PS I–LHCI supercomplex with two uncoupled antennas the excitation is channeled to the excitation sinks formed at low temperature by clusters of red pigments. A better spectral resolution of the transient absorption spectra at 77 K results in detection of two ΔA bands originating from the rise of photobleaching on the picosecond time scale of two clearly distinguished pools of low energy absorbing Chls in the PS I–LHCI supercomplex. The first pool of low energy pigments absorbing at 687 nm is likely to originate from the red pigments in the LHCI where the Lhca1 protein is most abundant. The second pool at 697 nm is suggested to result either from the structural interaction of the LHCI and the PS I core or from other Lhca proteins in the antenna. The kinetic data are discussed based on recent structural models of the PS I–LHCI. It is proposed that the uncoupling of pigment pools may be a control mechanism that regulates energy flow in Photosystem I.     

Aggregation of 8,12-diethyl farnesyl bacteriochlorophyll c at low temperature

The effect of temperature on the aggregation of 3^lR-8,12-diethyl farnesyl bacteriochlorophyll c in a mixture of n-pentane and methylcyclohexane (1/1, v/v) was studied by means of absorption, circular dichroism and fluorescence spectroscopy. At room temperature essentially only two aggregate species, absorbing at 702 nm (A-702) and 719 nm (A-719), were present. Upon cooling to 219 K, A-702 was quantitatively converted to A-719. Further lowering of the temperature led to the stepwise formation of larger aggregates by the conversion of A-719 to aggregate species absorbing at 743 nm (A-743) and 755 nm (A-755). All absorption changes were reversible. A-719 was highly fluorescent (maximum at 192 K: 744 nm), while A-743 and especially A-755 were weakly fluorescent. Below 130 K the mixture solidified, and no major changes in the absorption spectrum were observed upon further cooling. At 45 K, however, a relatively strong emission at 775 nm was observed. Below 200 K, the absorption, fluorescence and circular dichroism spectra resembled that of the chlorosome. These results open up the possibility to study higher aggregates of BChl c as models for the chlorosome by various methods at low temperature, thus avoiding interference by thermal processes.Abbreviations A-680, A-702, A-719, A-743 and A-755- BChl c aggregates absorbing at the wavelengths indicated - BChl- bacteriochlorophyll - R[E,E] BChl c _F- the 3¹ R isomer of 8,12-diethyl BChl c esterified with farnesol (F), analogously - M- methyl - Pr- propyl - S- stearol (see Smith 1994) - CD- circular dichroism     

Resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at 77 and 295 K through fluorescence excitation anisotropy

Fluorescence excitation spectra of highly anisotropic emission from Photosystem I (PS I) were measured at 295 and 77 K on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). When PS I was excited with light at wavelengths greater than 715 nm, fluorescence observed at 745 nm was highly polarized with anisotropies of 0.32 and 0.20 at 77 and 295 K, respectively. Upon excitation at shorter wavelengths, the 745-nm fluorescence had low anisotropy. The highly anisotropic emission observed at both 77 and 295 K is interpreted as evidence for low-energy chlorophylls (Chls) in cyanobacteria at room temperature. This indicates that low-energy Chls, defined as Chls with first excited singlet-state energy levels below or near that of the reaction center, P700, are not artifacts of low-temperature measurements.If the low-energy Chls are a distinct subset of Chls and a simple two-pool model describes the excitation transfer network adequately, one can take advantage of the low-energy Chls' high anisotropy to approximate their fluorescence excitation spectra. Maxima at 703 and 708 nm were calculated from 295 and 77 K data, respectively. Upper limits for the number of low-energy Chls per P700 in PS I from S. 6803 were calculated to be 8 (295 K) and 11 (77 K).Abbreviations Chl - chlorophyll - BChl - bacteriochlorophyll - LHC - light-harvesting chlorophyll - PS - Photosystem - RC - reaction center - S. 6803 - Synechocystis sp. PCC 6803     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Fluorescence of the various red antenna states in photosystem I complexes from cyanobacteria is affected differently by the redox state of P700

Photosystem I of cyanobacteria contains different spectral pools of chlorophylls called red or long-wavelength chlorophylls that absorb at longer wavelengths than the primary electron donor P700. We measured the fluorescence spectra at the ensemble and the single-molecule level at low temperatures in the presence of oxidized and reduced P700. In accordance with the literature, it was observed that the fluorescence is quenched by P700(+). However, the efficiency of the fluorescence quenching by oxidized P700(+) was found to be extremely different for the various red states in PS I from different cyanobacteria. The emission of the longest-wavelength absorbing antenna state in PS I trimers from Thermosynechococcus elongatus (absorption maximum at 5K: ≈ 719nm; emission maximum at 5K: ≈ 740nm) was found to be strongly quenched by P700(+) similar to the reddest state in PS I trimers from Arthrospira platensis emitting at 760nm at 5K. The fluorescence of these red states is diminished by more than a factor of 10 in the presence of oxidized P700. For the first time, the emission of the reddest states in A. platensis and T. elongatus has been monitored using single-molecule fluorescence techniques.     

Electronic spectra of PS I mutants: the peripheral subunits do not bind red chlorophylls in Synechocystis sp. PCC 6803

Steady-state fluorescence and absorption spectra have been obtained in the Qy spectral region (690-780 nm and 600-750 nm, respectively) for several subunit-deficient photosystem I mutants from the cyanobacterium Synechocystis sp. PCC 6803. The 77 K fluorescence spectra of the wild-type and subunit-deficient mutant photosystem I particles are all very similar, peaking at approximately 720 nm with essentially the same excitation spectrum. Because emission from far-red chlorophylls absorbing near 708 nm dominates low-temperature fluorescence in Synechocystis sp., these pigments are not coordinated to any the subunits PsaF, Psa I, PsaJ, PsaK, PsaL, or psaM. The room temperature (wild-type-mutant) absorption difference spectra for trimeric mutants lacking the PsaF/J, PsaK, and PsaM subunits suggest that these mutants are deficient in core antenna chlorophylls (Chls) absorbing near 685, 670, 675, and 700 nm, respectively. The absorption difference spectrum for the PsaF/J/I/L-deficient photosystem I complexes at 5 K reveals considerably more structure than the room-temperature spectrum. The integrated absorbance difference spectra (when normalized to the total PS I Qy spectral area) are comparable to the fractions of Chls bound by the respective (groups of) subunits, according to the 4-A density map of PS I from Synechococcus elongatus. The spectrum of the monomeric PsaL-deficient mutant suggests that this subunit may bind pigments absorbing near 700 nm.     

Assignment of red antenna states in photosystem I from Thermosynechococcus elongatus by single-molecule spectroscopy

Single-molecule spectroscopy at cryogenic temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacteria Thermosynechococcus elongatus. Absorption and hole burning data suggest the presence of three pools absorbing at wavelengths greater than 700 nm with their absorption maxima at 708, 715, and 719 nm. The responsible Chla molecules are termed C708, C715, and C719. In the emission spectra of single PSI complexes, zero-phonon lines (ZPLs) were observed over the whole red emission range of PSI. The spectral region of the C708 pool is dominated by intense ZPLs; on the other hand, the broad emission of C715/C719 is unstructured and ZPLs are seen in this region much less frequently. Spectral jumps of ZPLs were observed. The dynamics as well as the spectral range covered by such jumps differ for C708 and C715/C719. This heterogeneity is likely caused by differences in the close environment of the chromophores. A tentative assignment of C708 and C715/C719 to Chla dimers and a Chla trimer is discussed, which is based on the remarkable structural differences in the environment of the most probable candidates for the red-most fluorescence.     

Excitation spectra for Photosystem I and Photosystem II in chloroplasts and the spectral characteristics of the distribution of quanta between the two photosystems

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis.

The fluorescence of variable yield at 750 nm at −196 °C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at −196 °C at the minimum, F_O, level and the maximum, F_M, level of the emission at 750 nm. The difference spectrum, F_M–F_O, which represents the excitation spectrum for F_V is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex.

Fluorescence at the F_O level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, , which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of F_O/F_V measured at 692 nm and the extent of F_V at 750 nm. According to this procedure the excitation spectrum of Photosystem I at −196 °C was determined by subtracting 1/3 of the excitation spectrum of F_V at 750 nm from the excitation spectrum of F_O at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex.

The wavelength dependence of was determined from fluorescence measurements at 692 and 750 nm at −196 °C. is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Spectral inhomogeneity of photosystem I and its influence on excitation equilibration and trapping in the cyanobacterium Synechocystis sp. PCC6803 at 77 K.

Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trapping at 77 K in the photosystem I (PSI) core antenna from the cyanobacterium Synechocystis sp. PCC 6803. Excitation of the bulk antenna at 670 and 680 nm induces a subpicosecond energy transfer process that populates the Chl a spectral form at 685--687 nm within few transfer steps (300--400 fs). On a picosecond time scale equilibration with the longest-wavelength absorbing pigments occurs within 4-6 ps, slightly slower than at room temperature. At low temperatures in the absence of uphill energy transfer the energy equilibration processes involve low-energy shifted chlorophyll spectral forms of the bulk antenna participating in a 30--50-ps process of photochemical trapping of the excitation by P(700). These spectral forms might originate from clustered pigments in the core antenna and coupled chlorophylls of the reaction center. Part of the excitation is trapped on a pool of the longest-wavelength absorbing pigments serving as deep traps at 77 K. Transient hole burning of the ground-state absorption of the PSI with excitation at 710 and 720 nm indicates heterogeneity of the red pigment absorption band with two broad homogeneous transitions at 708 nm and 714 nm (full-width at half-maximum (fwhm) approximately 200--300 cm(-1)). The origin of these two bands is attributed to the presence of two chlorophyll dimers, while the appearance of the early time bleaching bands at 683 nm and 678 nm under excitation into the red side of the absorption spectrum (>690 nm) can be explained by borrowing of the dipole strength by the ground-state absorption of the chlorophyll a monomers from the excited-state absorption of the dimeric red pigments.     

Excitation spectra for photosystem I and photosystem II in chloroplasts and the spectral characteristics of the distributions of quanta between the two photosystems.

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis. The fluorescence of variable yield at 750 nm at -196 degrees C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at -196 degrees C at the minimum, FO, level and the maximum, FM, level of the emission at 750 nm. The difference spectrum, FM-FO, which represents the excitation spectrum for FV is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex. Fluoresence at the FO level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, alpha, which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of FO/FV measured at 692 nm and the extent of FV at 750 nm. According to this procedure the excitation spectrum of Photosystem I at -196 degrees C was determined by subtracting 1/3 of the excitation spectrum of FV at 750 nm from the excitation spectrum of FO at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex. The wavelength dependence of alpha was determined from fluorescence measurements at 692 and 750 nm at -196 degrees C. Alpha is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where alpha shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, alpha increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Fluorescence detected magnetic resonance of the primary donor and inner core antenna chlorophyll in Photosystem I reaction centre protein: Sign inversion and energy transfer

The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state ³P700, created via radical pair formation and recombination. ³P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at _f > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10^-4 cm^-1 and E=38.5×10^-4 cm^-1, were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the ³P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < _f < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < _f < 700 nm (inner core antennae, F690), and _f > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of ³P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.Abbreviations Chl chlorophyll - CP1 core chlorophyll protein of Photosystem I - EPR electron paramagnetic resonance - F690, F720 chlorophyll forms having fluorescence maximum at 690–695 and 720 nm, respectively - F(A)(O)DMR fluorescence (absorption) (optical) detected magnetic resonance - FF fluorescence fading - ISC intramolecular intersystem crossing - _f fluorescence emission wave-length - LHC I light harvesting chlorophyll a/b protein of Photosystem I - P700 primary donor of Photosystem I - PS I Photosystem I - RC reaction centre - RP radical pair - SDS sodium dodecyl sulphate - ZFS zero field splitting     

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.     

Red region excitation spectra of protochlorophyllide in dark-grown leaves from plant species with different proportions of its spectral forms

Etiolated leaves of three different species, maize, wheat, and pea, as well as a pea mutant (lip1) were used to compare the excitation spectra of protochlorophyllide (Pchlide) in the red region. The species used have different composition of short-wavelength and long-wavelength Pchlide forms. The relation between different forms was furthermore changed through incubating the leaves in 5-aminolevulinic acid (ALA), which caused an accumulation of short-wavelength Pchlide forms, as shown by changes in absorption and fluorescence spectra. This is the first time a comprehensive comparison is made between excitation spectra from different species covering an emission wavelength range of 675–750 nm using fluorescence equipment with electronic compensation for the variations in excitation irradiance. The different forms of Pchlide having excitations peaks at 628, 632, 637, 650, and 672 nm could be best measured at 675, 700, 710, 725, and 750 nm, respectively. Measuring emission at wavelengths between 675– 710 nm gave an exaggeration of the short-wavelength forms and measuring at longer wavelengths gave for the pea leaves an exaggeration of the 672 nm peak. In general, an energy transfer from short-wavelength Pchlide forms to long-wavelength Pchlide forms occurred, but such an energy transfer sometimes seemed to be limited as a result of a discrete location of the Pchlide spectral forms. The excitation spectra resembling the absorption spectrum most were measured at an emission wavelength of 740 nm. Measuring the excitation at 710 nm gave higher intensity of the spectra but the short-wavelength forms were accentuated.