Vladimir A. Shuvalov

Reduction of pheophytin in the primary light reaction of photosystem II

The photochemical reduction of pheophytin and bacteriopheophytin has been shown in vitro [l-3] . In reaction centers of photosynthetic bacteria bacteriopheophytin a [4-91 and bacteriopheophytin b [ 10, 1 l] act as an intermediary electron carrier between bacteriochlorophyll dimer and the ‘primary’ electron acceptor, a complex of ubiquinone and Fe. When the ubiquinone is in the reduced form the photoaccumulation of reduced bacteriopheophytin can be observed [5-111. In various species of green plants 1.5-2.3 molecules of pheophytin have been found per 100 molecules of chlorophyll [ 121 . In photosystem II of green plants the photoreduction of the primary electron acceptor, Q (plastoquinone), is accompanied by a blue shift of absorption bands at 54.5 nm and 685 nm [13,14] which can belong to a bound or aggregated form of pheophytin in reaction centers of photosystem II [ 141. The photoreduction of pheophytin may be observed in photosystem II preparations from pea chloroplasts at 20°C [ 151. In this work a reversible reduction of pheophytin in the primary light reaction of photosystem II in pea subchloroplast particles at redox potentials (Eh) from -50 mV to -550 mV (when Q is in the reduced form) is demonstrated. This photoreaction is observed at -170°C as well as at 20°C and is accompanied by a 2-3 fold decrease in the chlorophyll fluorescence yield.

Picosecond absorbance difference spectroscopy on the primary reactions and the antenna-excited states in Photosystem I particles

Abstract Absorbance difference spectra at various delay times, and kinetics of absorbance changes induced by a 35 ps excitation pulse at 532 nm, were measured of relatively intact Photosystem I particles from spinach containing about 70 chlorophyll a molecules per photoactive primary electron donor P-700. The excitation pulse produced absorbance changes due to the formation of singlet- and triplet-excited antenna chlorophyll a , and, in the case of active reaction centers, also those due to the oxidation of P-700. The formation of excited chlorophyll a was accompanied by the bleaching of the Q y ground state absorption band and by the appearance of a rather flat absorption increase in the region 550–900 nm. The lifetime of singlet-excited chlorophyll a was found to be 40 ± 5 ps. When the iron-sulfur centers were prereduced (photo)chemically, the formation of a radical pair consisting of P-700 + and a chlorophyllous anion was observed. The absorbance-difference spectrum calculated for the reduction of the acceptor was similar to that measured earlier (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Kryukov, P.G. and Ke, B. (1979) FEBS Lett. 107, 313–316), and indicated that the acceptor is a chlorophyll a species absorbing around 693 nm. The lifetime of the radical pair was at least 25 ns. If, however, the acceptor complex was in the oxidized state before the flash, only the oxidation of P-700 was observed. No direct evidence was obtained for the reduction of the chlorophyllous acceptor, implying that if such an anion is formed, it must be reoxidized within 50 ps.

EFFECT OF EXTRACTION AND RE-ADDITION OF MANGANESE ON LIGHT REACTIONS OF PHOTOSYSTEM-II PREPARATIONS

Manganese plays an important role in photosynthetic oxidation of H20 (reviews [l-3]). Reaction centers (RC) of photosystem II (PS II) carry out successive 4-step oxidation of a special (Mncontaining) enzymatic system which in turn oxidizes Hz0 [l-3]. The minimal quantity of Mn necessary for 02 evolution is 5-6 atoms/400 chlorophyll (chl) molecules or /l RC of PS II [l-4]. The greater part (-2/3rds) of this Mn is ‘loosely bound’ and can be easily extracted by alkaline Tris, NH20H, Triton X-100 or by heating, and the extraction leads to inhibition of 02 evolution and associated light reactions of PS II [l-lo]. ‘Firmly bound’ Mn ( 1/3rd of the pool) which remains in PS II after the extraction procedures seems not to be required for electron transport in PS II 141. However, up to 70% of Mn can be removed from chloroplasts without essential loss of their ability to evolve 02 [ll]. Reported characteristics of EPR spectra of Mn in chloroplasts [ 12151 may indicate participation of either 4 or 2 atoms of Mn in PS II reactions. of Mg2+ or any other divalent cation of metals, M2+). New results from a thorough investigation of these effects reported here show that activity of the Mn-containing system in the donor side of PS II requires 4 Mn atoms, 2 of which can be replaced by either Mg2+ or some other divalent metal ions (M2+).

Photoreactions of bacteriopheophytins and bacteriochlorophylls in reaction centers of Rhodopseudomonas sphaeroides and Chloroflexus aurantiacus

Abstract A comparison of spectral properties of reaction centers from Chloroflexus aurantiacus and Rhodopseudomonas sphaeroides (R-26) is reported. Treatment of reaction centers from Rps. sphaeroides with NaBH4 leads to a decrease of the dipole strength of the 800-nm band by factor of approx. 1.75-1.95 and to the formation of new bacteriopheophytin, BPh-715, which is almost completely removed during the purification of reaction centers. The modification of the reaction centers does not change the quantum yield of P photooxidation and the spectrum of BPh-545 (H1) photoreduction which includes the changing of the 800-nm band. This implies the preservation of the photoactive chain P-B1-H1-QA (where B1 is the bacteriochlorophyll (BChl)-800 molecule situated between P and H1) and the modification of the second BChl-800 (B2). The preparation of modified reaction centers is a mixture of at least three types of reaction centers with different contents of B2 and of the second BPh (H2). Some of the reaction centers (5-25%) contain the original B2 and H2 molecules (type I). In the CD spectrum of modified reaction centers a decrease of the 800-nm band and the appearance of a positive band at 765 nm is observed. This spectrum is similar to the CD spectrum of Chloroflexus reaction centers containing 3 BPhs and 3 BChls. This implies that in some (approx. 40%) of the modified Rps. sphaeroides reaction centers (type II) B2 has been replaced by BPh a which interacts with H2. Probably some of the modified reaction centers (approx. 40%) have lost both B2 and H2 (type III). The modification of reaction centers leads to a considerable decrease of the CD bands at 800 (+) nm and 810 (−) nm and to a decrease of the absorbance changes near 800 nm in the difference absorption spectrum of the oxidation of P. The data are interpreted in terms of the interaction between P and B1 molecules which gives two transitions at 790-800 and 810 nm with different orientations in modified Rps. sphaeroides as well as in Chloroflexus reaction centers. Similar transitions are observed for the interaction between P and B2. The spectral analysis shows the existence of two chains P-B1-H1, and P-B2-H2 in which the distances between the centers of molecules are 1.3 nm or less.

Energy dissipation in photosynthesis: Does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?

Abstract. Dissipation of light energy was studied in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst., and in leaves of Spinacia oleracea L. and Arabidopsis thaliana (L.) Heynh., using chlorophyll fluorescence as an indicator reaction. Maximum chlorophyll fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated spinach leaves, as produced by saturating light and studied between +5 and −20 °C, revealed an activation energy ΔE of 0.11 eV. As this suggested recombination fluorescence produced by charge recombination between the oxidized primary donor of photosystem II and reduced pheophytin, a mathematical model explaining fluorescence, and based in part on known characteristics of primary electron-transport reactions, was developed. The model permitted analysis of different modes of fluorescence quenching, two localized in the reaction center of photosystem II and one in the light-harvesting system of the antenna complexes. It predicted differences in the relationship between quenching of variable fluorescence Fv and quenching of basal, so-called F0 fluorescence depending on whether quenching originated from antenna complexes or from reaction centers. Such differences were found experimentally, suggesting antenna quenching as the predominant mechanism of dissipation of light energy in the moss Rhytidiadelphus, whereas reaction-center quenching appeared to be important in spinach and Arabidopsis. Both reaction-center and antenna quenching required activation by thylakoid protonation but only antenna quenching depended on or was strongly enhanced by zeaxanthin. De-protonation permitted relaxation of this quenching with half-times below 1 min. More slowly reversible quenching, tentatively identified as so-called qI or photoinhibitory quenching, required protonation but persisted for prolonged times after de-protonation. It appeared to originate in reaction centers.

Femtosecond spectroscopy of primary charge separation in modified reaction centers of Rhodobacter sphaeroides (R-26)

Femtosecond measurements of kinetics and spectra of absorbance changes (ΔA) were carried out with modified reaction centers (RCs) from Rhodobacter sphaeroides (R‐26) from which nonactive bacteriochlorophyll BM (located in the M protein subunit) was removed. The band of BM at 800 nm in native RCs is shifted in femtosecond measurements and obscures the ΔA of active bacteriochlorophyll BL (L subunit). The spectrum of ΔA in modified RCs at 6 ps delay includes the bleachings of the bands of P (primary electron donor) at 870 nm, of BL at 805 nm and of HL (bacteriopheophytin located in the L subunit) at 755 nm showing the reduction of 0̃.5 mol BL and 0̃.5 mol HL per mol P+. These data confirm an earlier suggestion that BL participates as an electron acceptor in the light‐induced primary charge separation and agree with recent X‐ray analysis of Rhodopseudomonas viridis and R. sphaeroides RCs which shows a location of BL between P and HL.

PICOSECOND FLUORESCENCE KINETICS OF THE D1-D2-CYT-B-559 PHOTOSYSTEM-II REACTION CENTER COMPLEX - ENERGY-TRANSFER AND PRIMARY CHARGE SEPARATION PROCESSES

The fluorescence kinetics of the D 1 -D 2 -cyt- b -559 Photosystem II reaction-center complex have been characterized in the picosecond and nanosecond time ranges. Measurements have been performed in the presence of β-lauryl maltoside under anaerobic conditions, both at 277 K and 77 K. Global analysis of decays recorded at different wavelengths and with different time resolutions yielded the decay-associated emission spectra of six exponential lifetime components, in the range of 1–6 ps up to 35 ns. We report here an ultrafast (τ≈1–6ps) fluorescence lifetime component with dominant amplitude (≥90%), which is thought to reflect the primary charge separation process. Furthermore, we have resolved a component with a 30–40 ps lifetime, its DAS indicating energy transfer. The lifetime of the primary radical pair P 680 + I ∼ , reflected by its recombination fluorescence, was found to be temperature-dependent. At 277 K this lifetime was 30 ns with a relative yield of 0.87, whereas at 77 K the radical-pair lifetime is increased to 35 ns with a relative yield of only 0.24 (both yields are corrected for the emission from uncoupled chlorophylls).

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (<100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A0 (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first approximately 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next approximately 40 fs the formation of a new broad band centered at approximately 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A1 (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700+A0A1) PS I reaction center (RC) from that of the open (state P700A0A1) RC reveals the pure spectrum of the P700+A0- ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An*-->k1[(PA0)*A1--><100 fs P+A0-A1]-->k2P+A0A1-, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (<100 fs) charge separation with the formation of the P700+A0-A1 state in approximately one half of the RCs, the approximately 5-ps energy transfer from antenna Chl* to P700A0A1 in the remaining RCs, and approximately 25-ps formation of the secondary radical pair P700+A0A1-.

Nuclear wavepacket motion producing a reversible charge separation in bacterial reaction centers

The excitation of bacterial reaction centers (RCs) at 870 nm by 30 fs pulses induces the nuclear wavepacket motions on the potential energy surface of the primary electron donor excited state P*, which lead to the fs oscillations in stimulated emission from P* [M.H. Vos, M.R. Jones, C.N. Hunter, J. Breton, J.‐C. Lambry and J.‐L. Martin (1994) Biochemistry 33, 6750–6757] and in QY absorption band of the primary electron acceptor, bacteriochlorophyll monomer BA [A.M. Streltsov, S.I.E. Vulto, A.Y. Shkuropatov, A.J. Hoff, T.J. Aartsma and V.A. Shuvalov (1998) J. Phys. Chem. B 102, 7293–7298] with a set of fundamental frequencies in the range of 10–300 cm−1. We have found that in pheophytin‐modified RCs, the fs oscillations with frequency around 130 cm−1 observed in the P*‐stimulated emission as well as in the BA absorption band at 800 nm are accompanied by remarkable and reversible formation of the 1020 nm absorption band which is characteristic of the radical anion band of bacteriochlorophyll monomer BA −. These results are discussed in terms of a reversible electron transfer between P* and BA induced by a motion of the wavepacket near the intersection of potential energy surfaces of P* and P+BA −, when a maximal value of the Franck–Condon factor is created.

Electron transfer in pheophytin a-modified reaction centers from Rhodobacter sphaeroides (R-26)

The major part (> 90%) of bacteriopheophytin a in reaction centers (RCs) of Rhodobacter sphaeroides was substituted by plant pheophytin a. In modified RCs the photochemical formation of P+Qa − occurs with with a quantum efficiency of 79%. The intermediary state P+I− displayed a recombination time constant of 1.5 ns, and the electron transfer from I− to Qa was characterized by a time constant of 540 ps. On the basis of spectral properties of P+I− for native and modified RCs, it was suggested that bacteriopheophytin, as well as bacteriochlorophyll monomers located in L protein branch, have a transition at 545 nm with approx. equal extinction coefficients. Accordingly, the state P+I− in modified RCs is proposed to consist of a thermodynamic mixture of P+BL− (∼ 80%) and P+Phe− (∼ 20%).