Reaction Centers Of Chloroflexus Aurantiacus Research Articles

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⁎ → 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⁎ → P+HA− superexchange process as an alternative highly efficient ET pathway in Cfl. aurantiacus RCs.

Femtosecond phase of charge separation in reaction centers of Chloroflexus aurantiacus

Difference absorption spectroscopy with temporal resolution of approximately 20 fsec was used to study the primary phase of charge separation in isolated reaction centers (RCs) of Chloroflexus aurantiacus at 90 K. An ensemble of difference (light-minus-dark) absorption spectra in the 730-795 nm region measured at -0.1 to 4 psec delays relative to the excitation pulse was analyzed. Comparison with analogous data for RCs of HM182L mutant of Rhodobacter sphaeroides having the same pigment composition identified the 785 nm absorption band as the band of bacteriopheophytin Phi(B) in the B-branch. By study the bleaching of this absorption band due to formation of Phi(B)(-), it was found that a coherent electron transfer from P* to the B-branch occurs with a very small delay of 10-20 fsec after excitation of dimer bacteriochlorophyll P. Only at 120 fsec delay electron transfer from P* to the A-branch occurs with the formation of bacteriochlorophyll anion B(A)(-) absorption band at 1028 nm and the appearance of P* stimulated emission at 940 nm, as also occurs in native RCs of Rb. sphaeroides. It is concluded that a nuclear wave packet motion on the potential energy surface of P* after a 20-fsec light pulse excitation leads to the coherent formation of the P(+)Phi(B)(-) and P(+)B(A)(-) states.

Energetics and Mechanism of Primary Charge Separation in Bacterial Photosynthesis. A Comparative Study on Reaction Centers of Rhodobacter sphaeroides and Chloroflexus aurantiacus

The high efficiency of charge separation in photosynthetic reaction centers arises from the interplay of energetics, electronic couplings, and reorganization energies relevant for the fast charge separation and slow recombination processes. All these parameters can be determined unambiguously only from magnetic-field-dependent measurements of the recombination dynamics of the intermediate radical pair P+HA- and the lifetime of the recombination product 3P*. Results obtained on QA-depleted reaction centers of Chloroflexus aurantiacus are compared with those for the well-characterized reaction centers of Rhodobacter sphaeroides. In contrast to Rb. sphaeroides, the magnetic field dependence of the triplet yield in Cf. aurantiacus has a pronounced resonance structure, allowing the direct determination of the exchange interaction of P+HA-, J = 21 G. The recombination rate kT is slightly larger for Cf. aurantiacus and shows a different temperature dependence. All these differences can be explained by the free energy of P+HA-, found to be larger by 0.04 eV in Cf. aurantiacus compared to Rb. sphaeroides. We propose that this different energy arises largely from the different amino acid at position L104, which is glutamic acid in the case of Rb. sphaeroides and glutamine in the case of Cf. aurantiacus. The electronic couplings and the reorganization energies, on the other hand, are very similar in both reaction centers. Implications for the mechanism of primary charge separation are discussed. The pronounced nonexponential kinetics of charge separation in Cf. aurantiacus is explained by the energetic inhomogeneity of the primary radical pair P+BA-.

Charge separation kinetics in isolated photosynthetic reaction centers of Chloroflexus aurantiacus (with Q A reduced) at low temperatures

The picosecond fluorescence kinetics of closed (quinone acceptor Q A reduced) reaction centers isolated from the phototrophic bacterium Chloroflexus aurantiacus shows time constants of ≈ 20 and ≈ 300 ps (amplitude ratio ≈ 1:1), which are nearly independent of temperature (7 to 80 K). Assuming a three-state kinetic model, we tested various assignments of the kinetics to the electron transfer processes. Only two of these assignments seem to be physically reasonable. One of these includes a fast reversible electron transfer between P* and H M, the pheophytin(s) in the M branch of the reaction center which up to now has been considered virtually inactive in electron transfer. The other possible model involves the formation of a monomeric bacteriochlorophyll anion.

High quantum yield of charge separation in reaction centers of Chloroflexus aurantiacus

The relative quantum yield of charge separation in photosynthetic reaction centers of Chloroflexus aurantiacus and Rhodobacter sphaeroides was measured on the nanosecond timescale. Thereby the quantum yield becomes independent of a potential loss of both quinones during reaction center isolation. Based on the quantum yield of 1.02±0.04 for reaction centers of Rhodobacter sphaeroides at room temperature (Wraight, C.A. and Clayton, R.K. (1973) Biochim. Biophys. Acta 333, 246–260), the quantum yield of initial charge separation in reaction centers of Chloroflexus aurantiacus at 280 K was determined to be 1.06 ± 0.11.

Temperature dependence of the initial electron-transfer kinetics in photosynthetic reaction centers of Chloroflexus aurantiacus

Absorbance changes in the near infrared were measured in photosynthetic reaction centers of the thermophilic, gliding, green bacterium Chloroflexus aurantiacus over the temperature range from 80 to 320 K, using 605-nm excitation pulses with an autocorrelation width of about 0.8 ps. Spectra collected during the first 1.5 ps after excitation show stimulated emission from the excited singlet state P*, absorbance increases at 762 and 802 nm, and absorbance decreases in the 865-nm band and at 783 and 815 nm. The absorbance changes associated with the formation of P* suggest that the electronic transitions in the ground-state spectrum are strongly mixed. Spectra collected up to 90 ps after the excitation pulse show the decay of the stimulated emission, and bleaching at 750 nm and 815 nm, reflecting the transfer of an electron from the bacteriochlorophyll dimer (P) to the initial electron acceptor(s). The electron-transfer reaction is slower in C. aurantiacus than in reaction centers of purple bacterial species; P* decays with a time-constant of 7.1±0.5 ps at 296 K and 9.0±0.1 ps at 320 K. At lower temperatures, the kinetics are non-exponential. The decay of the stimulated emission at 80 K can be fit either with a biexponential function with time constants of 3.0±0.1 and 32±12 ps, or with a stretched exponential function [exp — (t/τ0)α] with τ0 = 4.1 ± 1.2 ps and α = 0.45 ± 0.06. Several models that might account for the nonexponential decay kinetics of P* are discussed. A model based on rapid interconversion among conformational states in P* provides a simple interpretation of the temperature dependence of the kinetics.

Crystallization of photosynthetic reaction centres from Chloroflexus aurantiacus

The reaction center of Chloroflexus aurantiacus was crystallized in the presence of detergents. The crystals were obtained using polyethylene glycol as a precipitant. Three different crystal forms were obtained; one is supposed to be hexagonal, space group P6 322, the second rhombic, space group P2 122. Membrane protein crystallization; Reaction center; X-ray; ( Chloroflexus aurantiacus)

The photochemical reaction center of Chloroflexus aurantiacus is composed of two structurally similar polypeptides.

A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chloroflexus aurantiacus. The procedure consisted of three chromatography steps. The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl-N-methylglucamide (Mega-9). The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less. Under non-denaturing conditions, a pigmented protein band having a Mr of 52,000-55,000 was observed in sodium dodecyl sulfate gels. When the isolated complex was heat-dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar Mr (24,000 and 24,500) were observed. Two protein bands were also observed in two-dimensional isoelectric focusing/sodium-dodecyl-sulfate polyacrylamide gel electrophoresis; the PI values of the two polypeptides were 6.5 and 6.7. Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides. The N-terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria. The Chloroflexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins. Hence, it appears to be the smallest photochemically active reaction center isolated to date.

Picosecond spectroscopy of the charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation of the primary electron donor

Absorbance changes in the picosecond region were studied in isolated reaction centers of the green photosynthetic bacterium Chloroflexus aurantiacus upon selective excitation of the primary electron donor, P, at 870 nm. The results indicate that the first observed state is an excited state of P (P∗) which appears to transfer an electron to a bacteriochlorophyll a molecule absorbing at 812 nm (B 1) in 10 ± 2 ps as indicated by a bleaching at this wavelength. This reaction is followed by a rapid electron transfer (3 ± 1 ps) from B 1 − to bacteriopheophytin a, so that the fraction of reaction centers in the state P +B 1 − remains small during the experiment. An apparent bleaching at 925 nm is ascribed to stimulated emission from excited P, which emission disappears upon formation of P +. The difference between these time constants for electron transfer and those observed for the same reactions in reaction centers of the purple photosynthetic bacterium Rhodopseudomonas (Rhodobacter) sphaeroides is discussed in terms of the energy difference between P∗ and P +B 1 −, which appears to be larger for C. aurantiacus.

Primary photochemistry in the facultative green photosynthetic bacterium Chloroflexus aurantiacus.

The mechanism of primary photochemistry has been investigated in purified cytoplasmic membranes and isolated reaction centers of Chloroflexus aurantiacus. Redox titrations on the cytoplasmic membranes indicate that the midpoint redox potential of P870, the primary electron donor bacteriochlorophyll, is +362 mV. An early electron acceptor, presumably menaquinone has Em 8.1 = -50 mV, and a tightly bound photooxidizable cytochrome c554 has Em 8.1 = +245 mV. The isolated reaction center has a bacteriochlorophyll to bacteriopheophytin ratio of 0.94:1. A two-quinone acceptor system is present, and is inhibited by o-phenanthroline. Picosecond transient absorption and kinetic measurements indicate the bacteriopheophytin and bacteriochlorophyll form an earlier electron acceptor complex.