Detection of triplet states in algae by zero-field resonance.

Triplet states have been detected and characterized by zero-field splitting parameters in $\text{Anacystis nidulans}$, $\text{Euglena gracilis}$, $\text{Porphyridium cruentum}$, and $\text{Chlorella vulgaris}$, using fluorescence-detected magnetic resonance in zero-field at 4.2 K. Monitoring the 720 nm photosystem I emission, transitions between triplet spin levels have been assigned to antenna chlorophyll of one of both photosystems; photochemical reactions of chlorophyll are observed in the presence of an inhibitor and strong light, probably resulting in photoreduction and pheophytinization.     

Triplet states in photosynthesis

A comparison of zero field splitting (ZFS) and spin polarization of triplet spectra of bacteriochlorophyll $\text{a in vitro}$ and $\text{in vivo}$ provides support for the special pair model of photoreactive chlorophyll in photosynthetic bacteria. Spin polarization of the triplet spectra is a new and unique probe of primary events in the light conversion act in photosynthesis.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides: A photoselection study

The relative orientation of the pigments of reaction centers from Rhodopseudomonas sphaeroides has been studied by the photoselection technique.A high value (+0.45) of $\text{p =}\text{(ΔA}{}_{\mathrm{<mtext>V</mtext>}}\text{? ΔA}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{(ΔA}{}_{\mathrm{<mtext>V</mtext>}}\text{+ ΔA}{}_{\mathrm{<mtext>H</mtext>}}$) is obtained when exciting and observing within the 870 nm band which is contradictory to the results of Mar and Gingras (Mar, T. and Gingras, G. (1976) Biochim. Biophys. Acta 440, 609–621) and Shuvalov et al. (Shuvalov, V.A., Asadov, A.A. and Krakhmaleva, I.N. (1977) FEBS Lett. 76, 240–245). It is shown that the low values of p obtained by both groups were erroneous due to excitation conditions.Analysis of the polarization of light-induced changes when exciting with polarized light in single transitions (spheroiden band and bacteriopheophytin Q_x bands) enable us to propose a possible arrangement of the pigments within the reaction center. It is concluded that the 870 nm band corresponds to a single transition and is one of the two bands of the primary electron donor (P-870). The second band of the bacteriochlorophyll dimer is centred at 805 nm. The Q_y transitions of the molecules constituting the bacteriochlorophyll dimer are nearly parallel (angle less than 25°).The two bacteriopheophytin molecules present slightly different absorption spectra in the near infra-red. Both bacteriopheophytin absorption bands are subject to a small shift under illumination. The angle between the Q_y bacteriopheophytin transitions is 55° or 125°. Both Q_y transitions are nearly perpendicular to the 870 nm absorption band. Finally, the carotenoid molecules makes an angle greater than 70° with the 870 nm band and the other bacteriochlorophyll molecules.     

Triplet states of bacteriochlorophyll and carotenoids in chromatophores of photosynthetic bacteria

Chromatophores from photosynthetic bacteria were excited with flashes lasting approx. 15 ns. Transient optical absorbance changes not associated with the photochemical electron-transfer reactions were interpreted as reflecting the conversion of bacteriochlorophyll or carotenoids into triplet states. Triplet states of various carotenoids were detected in five strains of bacteria; triplet states of bacteriochlorophyll, in two strains that lack carotenoids. Triplet states of antenna pigments could be distinguished from those of pigments specifically associated with the photochemical reaction centers. Antenna pigments were converted into their triplet states if the photochemical apparatus was oversaturated with light, if the primary photochemical reaction was blocked by prior chemical oxidation of P-870 or reduction of the primary electron acceptor, or if the bacteria were genetically devoid of reaction centers. Only the reduction of the electron acceptor appeared to lead to the formation of triplet states in the reaction centers.In the antenna bacteriochlorophyll, triplet states probably arise from excited singlet states by intersystem crossing. The antenna carotenoid triplets probably are formed by energy transfer from triplet antenna bacteriochlorophyll. The energy transfer process has a half time of approx. 20 ns, and is about 1 × 10³ times more rapid than the reaction of the bacteriochlorophyll triplet states with O₂. This is consistent with a role of carotenoids in preventing the formation of singlet O₂ in vivo. In the absence of carotenoids and O₂, the decay half times of the triplet states are 70 μs for the antenna bacteriochlorophyll and 6–10 μs for the reaction center bacteriochlorophyll. The carotenoid triplets decay with half times of 2–8 μs.With weak flashes, the quantum yields of the antenna triplet states are in the order of 0.02. The quantum yields decline severely after approximately one triplet state is formed per photosynthetic unit, so that even extremely strong flashes convert only a very small fraction of the antenna pigments into triplet states. The yield of fluorescence from the antenna bacteriochlorophyll declines similarly. These observations can be explained by the proposal that singlet-triplet fusion causes rapid quenching of excited singlet states in the antenna bacteriochlorophyll.     

The type,amount, location,and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7′,8′-tetrahydro-ψ,ψ-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1,2,7′,8′-tetrahydro-ψ,ψ-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm ($\text{Q}{}_{\mathrm{<mtext>x</mtext>}}$) transition of the bacteriochlorophyll complex.     

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2°K

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2°K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are $\text{D = 0.02794 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, $\text{E = 0.00382 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, i.e. equal to those of monomeric chlorophyll $\text{a}$ to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperature 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Magnetophotoselection of the triplet state of reaction centers from Rhodopseudomonas sphaeroides R-26

Reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, give rise to large triplet state EPR signals upon illumination at low temperature (11 K). Utilizing monochromatic polarized light to generate the EPR spectra (magnetophotoselection) we have shown that the intensities of the observed triplet signals are strongly dependent upon the wavelength and polarization direction of the excitation. These data can be used to calculate the orientations of the excited transition moments with respect to each other and with respect to the triplet state principal magnetic axes system. Our quantitative approach is to follow the procedure outlined in a previous publication (Frank, H.A., Friesner, R., Nairn, J.A., Dismukes, G.C. and Sauer, K. (1979) Biochim. Biophys. Acta 547, 484–501) where computer simulations of the observed triplet state spectra were employed.The results presented in the present work indicate that the transition moment at 870 nm which is associated with the bacteriochlorophyll ‘special pair’ lies almost entirely along one of the principal magnetic axes of the triplet state. Also, the 870 nm transition moment makes an angle of approx. 60° with the 546 nm transition moment which is associated with a bacteriopheophytin. This latter result is in agreement with previous photoselection studies on the same bacterial species (Vermeglio, A., Breton, J., Paillotin, G. and Cogdell, R. (1978) Biochim. Biophys. Acta 501, 514–530).     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.     

Orientation of reaction center and antenna chromophores in the photosynthetic membrane of rhodopseudomonas viridis

Whole cells of Rhodopseudomonas viridis were oriented in a magnetic field. The degree of orientation of the cells was determined by using a photoselection technique. In order to deduce the orientation of the antennae and chromophores of the reaction centers with respect to the membrane plane, we performed linear dichroism measurements of absolute spectra and light induced difference spectra linked to states P⁺I and PI^? on oriented cells. These measurements lead to the following conclusions:The antennae bacteriochlorophyll molecular plane is nearly perpendicular to the membrane. The Q_y and Q_x transitions moments of these molecules make respectively angles of 20 and 70°ith the membrane plane. The antenna carotenoid molecules make an angle of 45°ith the membrane.The primary electron donor possesses two transition moments centered respectively at 970 and 850 nm. The 970 nm transition moment is parallel to the membrane plane, the 850 nm transition is tilted out of the plane. Upon photooxidation of this primary electron donor, a monomer-like absorption band appears at 805 nm. Its transition makes an angle smaller than 25° with the membrane. The photooxidation of the dimer also induces an absorption band shift for the two other bacteriochlorophyll molecules of the reaction center. The absorption band shifts of the two bacteriochlorophyll molecules occur in opposite direction.One bacteriopheophytin molecule is photoreduced in state PI^?. This photoreduction induces an absorption band shift for only one bacteriochlorophyll molecule. Finally, the geometry of the dimeric primary donor seems to be affected by the presence of a negative charge in the reaction center.     

Investigations by picosecond polarized fluorescence spectrochronography of structural aspects of energy transfer in living cells of the green bacterium Chlorobium limicola

The orientation of the long-wavelength (Q_y) transition moments of the antenna bacterioviridin (BVr) was examined in living cells of Chlorobium limicola. Previous linear dichroism studies [(1986) FEBS Lett. 199, 234–236] indicated that in each individual chromatophore of C. limicola the Q_y, transition moment vectors of the whole chlorosome BVr are essentially parallel to each other and are practically ideally oriented along the long axis of the chlorosome. We measured the picosecond polarized fluorescence decay kinetics for antenna bacteriochlorophyll (BChl) emissions upon selective excitation with polarized light of the Q_y, transition of BVr. The polarization (p) of the BVr fluorescence is measured to be constant during the BVr excited-state lifetime and to be equal to the limiting value of p achieved in monomeric BChl: P = + 0.42 ± 0.02. The results indicate convincingly that the excitation energy transfer within chlorosomes of C. limicola cells takes place between chromophores (or their coupled associates) with parallel transition moment vectors.     

Properties of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films.: Linear dichroism and low temperature spectra

Methods of preparing dried gelatin films containing purified reaction centers of Rhodopseudomonas sphaeroides are described. The spectral properties of reaction centers in solution are essentially maintained in dried gelatin films. These films are uniform and have excellent optical properties, showing little particulate scattering at temperatures down to about 4K. Film contraction on cooling to 90K is less than 1% in linear dimension. Linear dichroism spectra are reported for films at room and low temperature. Reaction centers show a moderate amount of linear dichroism in unstretched gelatin films; the magnitude of the linear dichroism becomes much greater when the films are stretched. In stretched films, linear dichroic ratios ($\text{A}{}_{\mathrm{\parallel }}\text{A}{}_{\mathrm{\perp }}$; absorbance measured with electric vector parallel and perpendicular to stretching direction) between 1.7 and 2.2 were obtained for the 860 nm absorption band of the bacteriochlorophyll component that undergoes primary photooxidation. The relative polarizations of light-induced absorption changes of reaction centers in stretched films are similar to those reported by Vermeglio and Clayton ((1976) Biochim. Biophys. Acta 449, 500–515) and support their hypothesis that absorbance decreases, maximal near 860 and 810 nm, and an increase near 790 nm are associated with the respective disappearance and appearance of discrete bands characteristic of the reduced and oxidized bacteriochlorophyll dimer. This interpretation is also supported by the polarization of the absolute absorption spectrum near 810 and 860 nm. An absorption band near 540 nm, ascribed to the Q_x transitions of two molecules of bacteriopheophytin in the reaction center, is split at low temperatures into two bands having similar polarizations. This splitting is probably not due to exciton coupling of the two molecules, since excition theory predicts different polarizations.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.     

The triplet exciton of chlorophyll a and carotenoid in solution and in photosynthetic antenna proteins

We have observed the development and decay of triplet excitons formed in the ‘antenna’ chlorophyll $\text{a}\text{b}$ protein complex by high-intensity laser excitation. The carotenoid triplet (³Car) appeared 5 ns after excitation in the protein isolation, commonly termed CP-II; the risetime in a larger antenna particle, called LHC (light-harvesting complex) was 12 ns. The quantum yield of ³Car in CP-II decreased 11-fold as intensity was increased from 10¹⁶ to 2 · 10¹⁷ photons/cm² per pulse. The effect is attributed to exciton annihilation during the initial period of triplet formation. Above 5 · 10¹⁶ photons/cm² per s, the ³Car lifetime decreases substantially from its low intensity value of 8.7 μs. A comparison of the transient absorption spectrum of CP-II with those of chlorophyll and carotenoid in vitro indicates that ‘trapped’ chlorophyll triplets formed at high intensities. We present a simple model of destructive interaction between ³Car and chlorophyll triplets which is compatible with the observed increased rate of ³Car decay. Indirect evidence suggests similar effects occur in LHC.     

Cytochrome c degrading activity in rat liver mitochondria

Benzophenone can be used as an extrinsic triplet state probe, as its phosphorescence, a broad band centered at 445 nm, is readily observable in aqueous solution at room temperature. When bound covalently as an acyl enzyme at the active site of chymotrypsin, the benzophenone probe produces phosphorescence which is unusually resistant to quenching by O₂, $\text{trans}$-cinnamic acid, and H₃O⁺. Sodium 2-naphthalenesulfonate quenches the phosphorescence, probably indirectly. The quenching data indicate that the local protein structure at the enzyme active site provides a rigid and protective substrate environment, which is not penetrated by even the smallest triplet quenchers.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.     

Resonance Raman scattering of bacteriochlorophyll, bacteriopheophytin and spheroidene in reaction centers of Rhodopseudomonas speroides.

We report and discuss Raman spectra of bacteriochlorophyll $\text{a}$ and of bacteriopheophytin $\text{a}$ obtained $\text{in vitro}$ by resonance effect in their Q_X and Soret electronic bands. Selective excitation of spectra of either of these molecules in reaction centers of $\text{Rhodopseudomonas spheroides}$, strains Y and R 26, was achieved by illumination in their respective Q_X bands. Preliminary interpretation of the spectra yields information about the interactions assumed by these molecules in the reaction centers. Spheroidene bound to reaction centers of strain Y probably affects a conformation different from that assumed by the bulk spheroidene of the chromatophore.     

Phase fluorimetric lifetime spectra. I. In algal cells at 77 K

A new method for decomposing fluorescence emission spectra into their elementary components, based on the simultaneous recording of fluorescence intensity and lifetime vs. the emission wavelength, has been applied to the spectra of algal cells at liquid nitrogen temperature. A model of Gaussian components fits both τ(λ) and F(λ) spectra with the same parameters. The fluorescence lifetimes have been measured by phase fluorimetry at two modulation frequencies: 29 and 139 MHz. The final Gaussian decomposition is able to describe both the 29 and 139 MHz spectra. The following conclusions concerning the fluorescence spectra of Chlorella cells at 77 K can be drawn. These conclusions are also valid with minor changes for the other examined species. (1) An overlapping of different emitting bands occurs in all the spectra; therefore, a direct lifetime reading from phase delay measurement necessitates measurements being made at several frequencies. (2) At the F_max fluorescence level, the lifetime values of the two emissions usually associated with variable fluorescence are 0.53 ns (for B′₁; λ peak 688 nm), and 1.46 ns (for B′₂; λ peak 698 nm); these lifetimes are shorter than those we have measured at room temperature (approx. 1.8 ns). (3) Superimposed on B′₁ and B′₂ and with approximatively the same peak location, two long-lifetime components (B″₁, 4.8 ns; B″₂, 5.6 ns) are present. Two hypotheses can be proposed to explain these emissions: (i) the long-lifetime components arise from subsets of chlorophyll a disconnected from the functional antenna by the cooling process; and (ii) charge recombination in reaction centers leads to delayed fluorescence. (4) In the λ > 710 nm region, two main bands are required to describe the so-called Photosystem I emission: B₃ (0.8 ns; λ peak 715 nm) and B₄ (3.3 ns; λ peak 724 nm). The former band, usually unresolved in the amplitude fluorescence spectra, is a specific finding from lifetime measurements and has been associated with the antenna core of Photosystem I. No additional information has been obtained for B₄. A supplementary small band (B₅, 0.40 ns; $\text{λ}\text{peak}\text{? 740}\text{nm}$) is necessary to take into account the frequency effect and the τ(λ) decrease in the λ > 740 nm spectral range.     

Magnetophotoselection of the triplet state of reaction centers from Rhodopseudomonas sphaeroides R-26.

Reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, give rise to large triplet state EPR signals upon illumination at low temperature (11 K). Utilizing monochromatic polarized light to generate the EPR spectra (magnetophotoselection) we have shown that the intensities of the observed triplet signals are strongly dependent upon the wavelength and polarization direction of the excitation. These data can be used to calculate the orientations of the excited transition moments with respect to each other and with respect to the triplet state principal magnetic axes system. Our quantitative approach is to follow the procedure outlined in a previous publication (Frank, H.A., Friesner, R., Nairn, J.A., Dismukes, G.C. and Sauer, K. (1979) Biochim. Biophys. Acta 547, 484-501) where computer simulations of the observed triplet state spectra were employed. The results presented in the present work indicate that the transition moment at 870 nm which is associated with the bacteriochlorophyll 'special pair' lies almost entirely along one of the principal magnetic axes of the triplet state. Aso, the 870 nm transition moment makes an angle of approx. 60 degrees with the 546 nm transition moment which is associated with a bacteriopheophytin. This latter result is in agreement with previous photoselection studies on the same bacterial species (Vermeglio, A., Breton, J., Paillotin, G. and Cogdell, R. (1978) Biochim. Biophys. Acta 501, 514-530).