Femtosecond optical studies of the primary charge separation reactions in far-red photosystem II from Synechococcus sp. PCC 7335.

Primary processes of light energy conversion by Photosystem II (PSII) were studied using femtosecond broadband pump-probe absorption difference spectroscopy. Transient absorption changes of core complexes isolated from the cyanobacterium Synechococcus sp. PCC 7335 grown under far-red light (FRL-PSII) were compared with the canonical Chl a containing spinach PSII core complexes upon excitation into the red edge of the Qy band. Absorption changes of FRL-PSII were monitored at 278 K in the 400-800 nm spectral range on a timescale of 0.1-500 ps upon selective excitation at 740 nm of four chlorophyll (Chl) f molecules in the light harvesting antenna, or of one Chl d molecule at the ChlD1 position in the reaction center (RC) upon pumping at 710 nm. Numerical analysis of absorption changes and assessment of the energy levels of the presumed ion-radical states made it possible to identify PD1+ChlD1- as the predominant primary charge-separated radical pair, the formation of which upon selective excitation of Chl d has an apparent time of ∼1.6 ps. Electron transfer to the secondary acceptor pheophytin PheoD1 has an apparent time of ∼7 ps with a variety of excitation wavelengths. The energy redistribution between Chl a and Chl f in the antenna occurs within 1 ps, whereas the energy migration from Chl f to the RC occurs mostly with lifetimes of 60 and 400 ps. Potentiometric analysis suggests that in canonical PSII, PD1+ChlD1- can be partially formed from the excited (PD1ChlD1)* state.

Primary charge separation in native and plant pheophytin a-modified reaction centers of Chloroflexus aurantiacus: Ultrafast transient absorption measurements at low temperature.

Ultrafast transient absorption (TA) spectroscopy was used to study electron transfer (ET) at 100 K in native (as isolated) reaction centers (RCs) of the green filamentous photosynthetic bacterium Chloroflexus (Cfl.) aurantiacus. The rise and decay of the 1028 nm anion absorption band of the monomeric bacteriochlorophyll a molecule at the BA binding site were monitored as indicators of the formation and decay of the P+BA- state, respectively (P is the primary electron donor, a dimer of bacteriochlorophyll a molecules). Global analysis of the TA data indicated the presence of at least two populations of the P⁎ excited state, which decay by distinct means, forming the state P+HA- (HA is a photochemically active bacteriopheophytin a molecule). In one population (~65 %), P⁎ decays in ~2 ps with the formation of P+HA- via a short-lived P+BA- intermediate in a two-step ET process P⁎ â†’ P+BA-→ P+HA-. In another population (~35 %), P⁎ decays in ~20 ps to form P+HA- via a superexchange mechanism without producing measurable amounts of P+BA-. Similar TA measurements performed on chemically modified RCs of Cfl. aurantiacus containing plant pheophytin a at the HA binding site also showed the presence of two P⁎ populations (~2 and ~20 ps), with P⁎ decaying through P+BA- only in the ~2 ps population. At 100 K, the quantum yield of primary charge separation in native RCs is determined to be close to unity. The results are discussed in terms of involving a one-step P⁎ â†’ P+HA- superexchange process as an alternative highly efficient ET pathway in Cfl. aurantiacus RCs.

On the Mechanism of Selective Chemical Exchange of Bacteriopheophytins in the Reaction Centers of Rhodobacter sphaeroides R-26.

To elucidate the mechanism of site-selective chemical replacement of chromophores in the reaction centers (RCs) of photosynthetic bacteria by external pigments, we investigated how the efficiency of incorporation of plant pheophytin a (Pheo) into the binding sites for bacteriopheophytin a molecules (BPheo) in the isolated Rhodobacter sphaeroides R-26 RCs depended on the incubation medium temperature, Pheo aggregation state, and the presence of organic solvent (acetone). When Pheo was in a form of monomers in free detergent micelles in a water-detergent incubation medium, the degree of selective replacement of photochemically inactive BPheo HB molecules upon incubation of the RC/Pheo mixture at 5°C was ~15%. The exchange efficiency increased to 40% upon incubation at 25°C and reached 100% at the same temperature when 10% acetone was added to the incubation medium. At both 5 and 25°C, the degree of pigment exchange increased approximately twice, when a mixture of Pheo monomers and dimers in the presence of 10% acetone was used as the incubation medium. The removal of acetone from this medium with the preservation of pigment forms led to a significant decrease in the efficiency of Pheo incorporation. The effect of acetone on the pigment exchange was also observed at an elevated incubation temperature (43.5°C), when functionally active BPheo HA molecules were partially replaced. The results are discussed in terms of the mechanism according to which (i) the temperature-dependent internal movements of the RC protein facilitate the release of the BPheo molecule from the binding site with simultaneous insertion of the Pheo molecule into the same site in a coupled process, (ii) the role of temperature largely depends on the steric accessibility of binding pockets in the RC protein, (iii) the incorporation of Pheo occurs from a pool of monomeric molecules included in the RC-detergent micelles, and (iv) the presence of acetone in the incubation medium facilitates the exchange of Pheo monomers between micelles in the solution and the detergent belt of the RC complex.

Pigment-modified reaction centers of Chloroflexus aurantiacus: chemical exchange of bacteriopheophytins with plant-type pheophytins.

The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo- radical anion instead of the BPheo-. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.

Primary electron transfer in Rhodobacter sphaeroides R-26 reaction centers under dehydration conditions.

The photoinduced charge separation in QB-depleted reaction centers (RCs) from Rhodobacter sphaeroides R-26 in solid air-dried and vacuum-dried (~10-2 Torr) films, obtained in the presence of detergent n-dodecyl-ß-D-maltoside (DM), is characterized using ultrafast transient absorption spectroscopy. It is shown that drying of RC-DM complexes is accompanied by reversible blue shifts of the ground-state absorption bands of the pigment ensemble, which suggest that no dehydration-induced structural destruction of RCs occurs in both types of films. In air-dried films, electron transfer from the excited primary electron donor P⁎ to the photoactive bacteriopheophytin HA proceeds in 4.7 ps to form the P+HA- state with essentially 100% yield. P+HA- decays in 260 ps both by electron transfer to the primary quinone QA to give the state P+QA- (87% yield) and by charge recombination to the ground state (13% yield). In vacuum-dried films, P⁎ decay is characterized by two kinetic components with time constants of 4.1 and 46 ps in a proportion of ~55%/45%, and P+HA- decays about 2-fold slower (462 ps) than in air-dried films. Deactivation of both P⁎ and P+HA- to the ground state effectively competes with the corresponding forward electron-transfer reactions in vacuum-dried RCs, reducing the yield of P+QA- to 68%. The results are compared with the data obtained for fully hydrated RCs in solution and are discussed in terms of the presence in the RC complexes of different water molecules, the removal/displacement of which affects spectral properties of pigment cofactors and rates and yields of the electron-transfer reactions.

Mutation H(M202)L does not lead to the formation of a heterodimer of the primary electron donor in reaction centers of Rhodobacter sphaeroides when combined with mutation I(M206)H.

In photosynthetic reaction centers (RCs) of purple bacteria, conserved histidine residues [His L173 and His M202 in Rhodobacter (Rba.) sphaeroides] are known to serve as fifth axial ligands to the central Mg atom of the bacteriochlorophyll (BChl) molecules (PA and PB, respectively) that constitute the homodimer (BChl/BChl) primary electron donor P. In a number of previous studies, it has been found that replacing these residues with leucine, which cannot serve as a ligand to the Mg ion of BChl, leads to the assembly of heterodimer RCs with P represented by the BChl/BPheo pair. Here, we show that a homodimer P is assembled in Rba. sphaeroides RCs if the mutation H(M202)L is combined with the mutation of isoleucine to histidine at position M206 located in the immediate vicinity of PB. The resulting mutant H(M202)L/I(M206)H RCs are characterized using pigment analysis, redox titration, and a number of spectroscopic methods. It is shown that, compared to wild-type RCs, the double mutation causes significant changes in the absorption spectrum of the P homodimer and the electronic structure of the radical cation P+, but has only minor effect on the pigment composition, the P/P+ midpoint potential, and the initial electron-transfer reaction. The results are discussed in terms of the nature of the axial ligand to the Mg of PB in mutant H(M202)L/I(M206)H RCs and the possibility of His M202 participation in the previously proposed through-bond route for electron transfer from the excited state P* to the monomeric BChl BA in wild-type RCs.

Interaction of HydSL hydrogenase from Thiocapsa roseopersicina with cyanide leads to destruction of iron-sulfur clusters.

The effects of cyanide on enzymatic activity and absorption spectra in the visible and mid-IR (2150-1850cm-1) regions were characterized for purified HydSL hydrogenase from the purple sulfur bacterium Thiocapsa (T.) roseopersicina BBS. Prolonged incubation (over hours) of T. roseopersicina hydrogenase with exogenous cyanide was shown to result in an irreversible loss of activity of the enzyme in both the oxidized (as isolated) and H2-reduced states. The frequency position of the active site CO and CN- ligand stretching bands in the Fourier transform infrared (FTIR) spectrum of the oxidized form of hydrogenase was not influenced by cyanide treatment. The 410-nm absorption band characteristic of hydrogenase iron­sulfur clusters showed a bleaching concomitantly with cyanide inactivation. A new band at 2038cm-1 was present in the FTIR spectrum of the cyanide-inactivated preparation, which band is assignable to ferrocyanide as a possible product of a destructive interaction of hydrogenase with cyanide. The results are interpreted in terms of a slow destruction of iron­sulfur clusters of hydrogenase in the presence of cyanide accompanied by a release of iron ions in the form of ferrocyanide into the surrounding solution. Such a slow and irreversible cyanide-dependent inactivation seems to be complementary to a recently described rapid, reversible inhibitory reaction of cyanide with the active site of hydrogenases [S.V. Hexter, M.-W. Chung, K.A. Vincent, F.A. Armstrong, J. Am. Chem. Soc. 136 (2014) 10470-10477].

Characterization of the low-temperature triplet state of chlorophyll in photosystem II core complexes: Application of phosphorescence measurements and Fourier transform infrared spectroscopy.

Phosphorescence measurements at 77 K and light-induced FTIR difference spectroscopy at 95 K were applied to study of the triplet state of chlorophyll a ((3)Chl) in photosystem II (PSII) core complexes isolated from spinach. Using both methods, (3)Chl was observed in the core preparations with doubly reduced primary quinone acceptor QA. The spectral parameters of Chl phosphorescence resemble those in the isolated PSII reaction centers (RCs). The main spectral maximum and the lifetime of the phosphorescence corresponded to 955±1 nm and of 1.65±0.05 ms respectively; in the excitation spectrum, the absorption maxima of all core complex pigments (Chl, pheophytin a (Pheo), and ß-carotene) were observed. The differential signal at 1667(-)/1628(+)cm(-1) reflecting a downshift of the stretching frequency of the 13(1)-keto C=O group of Chl was found to dominate in the triplet-minus-singlet FTIR difference spectrum of core complexes. Based on FTIR results and literature data, it is proposed that (3)Chl is mostly localized on the accessory chlorophyll that is in triplet equilibrium with P680. Analysis of the data suggests that the Chl triplet state responsible for the phosphorescence and the FTIR difference spectrum is mainly generated due to charge recombination in the reaction center radical pair P680(+)PheoD1(-), and the energy and temporal parameters of this triplet state as well as the molecular environment and interactions of the triplet-bearing Chl molecule are similar in the PSII core complexes and isolated PSII RCs.

Low-temperature (77 K) phosphorescence of triplet chlorophyll in isolated reaction centers of photosystem II.

Phosphorescence characterized by the main emission band at 952 ± 1 nm (1.30 eV), the lifetime of 1.5 ± 0.1 ms and the quantum yield nearly equal to that for monomeric chlorophyll a in aqueous detergent dispersions, has been detected in isolated reaction centers (RCs) of spinach photosystem II at 77 K. The excitation spectrum shows maxima corresponding to absorption bands of chlorophyll a, pheophytin a, and ß-carotene. The phosphorescence intensity strongly depends upon the redox state of RCs. The data suggest that the phosphorescence signal originates from the chlorophyll triplet state populated via charge recombination in the radical pair [Formula: see text].

Chemically modified reaction centers of photosystem II: Exchange of pheophytin a with 7-deformyl-7-hydroxymethyl-pheophytin b.

The native pheophytin a (Pheo a) in isolated reaction centers of photosystem II (PSII RCs) has been chemically exchanged with extraneous 7-deformyl-7-hydroxymethyl-Pheo b (7(1)-OH-Pheo b) which differs from Pheo a by the C-7 substituent (hydroxymethyl instead of methyl). The two pigments have similar reduction potentials in vitro [M. Meyer, Dissertation, Universität München, 1997], while their absorption spectra show small but distinct differences in the visible region. The resulting 7(1)-OH-Pheo b-modified reaction center preparations were characterized by high-performance liquid chromatography, electronic absorption and light-induced Fourier transform infra red absorption difference spectroscopies, together with photoaccumulation of the reduced pheophytin electron acceptor and NaBH4-treatment. About 70% of the total Pheo a molecules are found to be replaced by 7(1)-OH-Pheo b molecules in modified preparations, indicating that both the photochemically active (PheoD1) and inactive (PheoD2) binding sites were subjected to pigment exchange. The 7(1)-OH-Pheo b molecule located at the PheoD1 site is able to functionally replace the native Pheo a, participating in primary charge separation as an electron acceptor. The Qx absorption band of this modified pheophytin molecule is localized at ~546nm; its Qy band is blue-shifted with respect to the absorption of other reaction center core pigments, being located at ~665nm. The Qy and Qx optical transitions of the 7(1)-OH-Pheo b molecule exchanged into the PheoD2 site are identified at 677 and 543.5nm, respectively. The photochemically active double-modified PSII RCs additionally containing 7-deformyl-7-hydroxymethyl-13(1)-deoxo-13(1)-hydroxy-Pheo b at the PheoD2 site were obtained by treatment of the 7(1)-OH-Pheo b-modified RCs with NaBH4.