Absorption and fluorescence spectra and molecular organization of bacteriochlorophyll in reaction centers of Rhodopseudomonas spheroides

Second derivative spectroscopy, computer curve analysis and Stepanov's equation show that the absorbance and fluorescence spectra of primary electron donor in reaction center of Rhodopseudomonas sphaeroides are splitting each into two asymmetric Gaussian components. Their absorption maxima at -196 degrees are 880 and 896 nm and emission maxima-906 and 923 nm, respectively. The absorption spectrum of Bchl-800 splits in the near infrared region into two bands with maxima at 790 and 803 nm. These components are ascribed to an exciton coupling in the two dimers of bacteriochlorophyll in the reaction center. The Qy transition moments of the two bacteriochlorophyll molecules of primary electron donor make an angle of 110 degrees and the angle between two Qy transitions of the pigment in Bchl-800 dimer is 150 degrees. The distance between the centers of chromophores in the dimers is estimated to be 8-11 A.     

Temperature and detection-wavelength dependence of the picosecond electron-transfer kinetics measured in Rhodopseudomonas sphaeroides reaction centers. Resolution of new spectral and kinetic components in the primary charge-separation process

We have examined the temperature dependence of the rate of electron transfer to ubiquinone from the bacteriopheophytin (BPh) that serves as an initial electron acceptor (I) in reaction centers of Rhodopseudomonas sphaeroides. The kinetics were measured from the decay of the 665-nm absorption band of the reduced BPh (BPh⁻ or I⁻) and from the recovery of the BPh band at 545 nm, following excitation of reaction centers in polyvinyl alcohol films with 30-ps flashes. The measured time constant decreases from 229 ± 25 ps at 295 K to 97 ± 8 ps near 100 K and then remains constant down to 5 K. The temperature dependence of the kinetics can be rationalized on the assumption that the reaction results in changes in the frequencies of numerous low-energy nuclear (vibrational) modes of the electron carriers and/or the protein. The kinetics measured in the absorption bands near 765 and 795 nm show essentially the same temperature dependence as those measured at 545 or 665 nm, but the time constants vary with detection wavelength. The time constant measured in the 795-nm region (70 ± 10 ps at 5 and 76 K) is shorter than that seen in the absorption bands of the BPh; the time constant measured at 758 nm is longer. Time constants measured with reaction centers in solution at 288 K also vary with the detection wavelength. These results can be explained on the assumption that the absorption changes measured at some wavelengths reflect nuclear relaxations rather than electron transfer. The absorption changes at 795 nm probably reflect a relaxation of the bacteriochlorophyll molecules that are near neighbors of the BPh and the primary electron donor (P). Those near 530 and 755 nm probably are due to the second BPh molecule, which does not appear to undergo oxidation or reduction.     

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 B(A) [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 B(A) 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 B(A)-. These results are discussed in terms of a reversible electron transfer between P* and B(A) induced by a motion of the wavepacket near the intersection of potential energy surfaces of P* and P+B(A)-, when a maximal value of the Franck-Condon factor is created.     

Differentiation of the intracytoplasmic membrane of Rhodopseudomonas palustris induced by variations of oxygen partial pressure or light intensity

The photosynthetic apparatus of Rhodopseudomonas palustris contains, in addition to reaction center bacteriochlorophyll (Bchl) two spectral forms of light harvesting (LH) Bchl, i.e. LH Bchl I, characterized by an infrared absorption maximum at 880 nm (890 nm at 77°K) and LH Bchl II absorbing at 805 and 855 nm (805 and 870 nm at 77°K). LH Bchl I seems to be associated with a single protein species of an apparent mol. wt. of 13000 whereas LH Bchl II is apparently associated with two proteins of mol. wts. of 9000 and 11000.Cells in anaerobic cultures adapt to changes of light intensity 1. by variation of the size of the photosynthetic unit, i.e. the molar ratio of LH Bchl II to reaction center Bchl, 2. by variation of the number of photosynthetic units per unit of membrane area, 3. by regulation of the size of the intracytoplasmic membrane system.During adaptation of changes of oxygen partial pressure cells are able to synthesize reaction center Bchl, LH Bchl and intracytoplasmic membranes at different rates. The synthesis of reaction center Bchl and LH Bchl I are, however, coordinated with each other, while the syntheses of LH Bchl II and reaction center Bchl proceed independently.List of Non-Standard Abbreviations Bchl bacteriochlorophyll - ICM mitracytoplasmic membrane - LDAO lauryldimethyl aminoxide - R Rhodopseudomonas - RC reaction center - SDS sodium dodecylsulfate     

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275–283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P⁺H^– charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200–300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P^* and P⁺H^–.Abbreviations PSU photosynthetic unit - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - P reaction center primary electron donor - RC reaction center - Rb. Rhodobacter - Rs. Rhodospirillum - EDTA (ethylenediamine)tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Author for correspondence     

Nanosecond fluorescence from isolated photosynthetic reaction centers of Rhodopseudomonas sphaeroides

The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, PF. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to PF, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates PF from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and PF of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of PF with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.     

Spectral heterogeneity and time-resolved spectroscopy of excitation energy transfer in membranes of Heliobacillus mobilis at low temperatures.

Transient absorption difference spectra in the Qy absorption band from membranes of Heliobacillus mobilis were recorded at 140 and 20 K upon 200 fs laser pulse excitation at 590 nm. Excitation transfer from short wavelength absorbing forms of bacteriochlorophyll g to long wavelength bacteriochlorophyll g occurred within 1-2 ps at both long wavelength bacteriochlorophyll g occurred within 1-2 ps at both temperatures. In addition, a slower energy transfer process with a time constant of 15 ps was observed at 20 K within the pool of long wavelength-absorbing bacteriochlorophyll g. Energy transfer from long wavelength antenna pigments to the primary electron donor P798 was observed, yielding the primary charge-separated state P798+A0-. The time constant for this process was 30 ps at 140 K and about 70 ps at 20 K. A decay component with smaller amplitude and a lifetime of up to hundreds of picoseconds was observed that was centered around 814 nm at 20 K. Kinetic simulations using simple lattice models reproduce the observed decay kinetics at 295 and 140 K, but not at 20 K. The kinetics of energy redistribution within the spectrally heterogeneous antenna system at low temperature argue against a simple "funnel" model for the organization of the antenna of Heliobacillus mobilis and favor a more random spatial distribution of spectral forms. However, the relatively high rate of energy transfer from long wavelength antenna bacteriochlorophyll g to the primary electron donor P798 at low temperature is difficult to explain with either of these models.     

The effect of exchange of bacteriopheophytin a with plant pheophytin a on charge separation in Y(M210)W mutant reaction centers of Rhodobacter sphaeroides at low temperature

The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B(B) binding pocket.     

Nanosecond fluorescence from isolated photosynthetic reaction centers of Rhodopseudomonas sphaeroides

The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, P^F. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to P^F, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates P^F from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and P^F of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of P^F with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.     

Excited states of photosynthetic reaction centers at low redox potentials

In preparations of photochemical reaction centers from Rhodopseudomonas spheroides R-26, lowering the redox potential so as to reduce the primary electron acceptor prevents the photochemical transfer of an electron from bacteriochlorophyll to the acceptor. Measuring absorbance changes under these conditions, we found that a 20-ns actinic flash converts the reaction center to a new state, P^F, which then decays with a half-time that is between 1 and 10 ns at 295 °K. At 25 °K, the decay half-time is approx. 20 ns. The quantum yield of state P^F appears to be near 1.0, both at 295 and at 15 °K. State P^F could be an intermediate in the photochemical electron-transfer reaction which occurs when the acceptor is in the oxidized form.Following the decay of state P^F, we detected another state, P^R, with a decay half-time of 6 μs at 295 °K and 120 μs at 15 °K. The quantum yield of state P^R is approx. 0.1 at 295 °K, but rises to a value nearer 1.0 at 15 °K. The kinetics and quantum yields are consistent with the view that state P^R forms from P^F. State P^R seems likely to be a side-product, rather than an intermediate in the electron-transfer process.The decay kinetics indicate that state P^F cannot be identical with the lowest excited singlet state of the reaction center. One of the two states, P^F or P^R, probably is the lowest excited triplet state of the reaction center, but it remains unclear which one.     

Comparative study of reaction centers from purple photosynthetic bacteria: Isolation and optical spectroscopy

Reaction centers from two species of purple bacteria, Rhodospirillum rubrum and Rhodospirillum centenum, have been characterized and compared to reaction centers from Rhodobacter sphaeroides and Rhodobacter capsulatus. The reaction centers purified from these four species can be divided into two classes according to the spectral characteristics of the primary donor. Reaction centers from one class have a donor optical band at a longer wavelength, 865 nm compared to 850 nm, and an optical absorption band associated with the oxidized donor at 1250 nm that has a larger oscillator strength than reaction centers from the second class. Under normal buffering conditions, reaction centers isolated from Rb. sphaeroides and Rs. rubrum exhibit characteristics of the first class while those from Rb. capsulatus and Rs. centenum exhibit characteristics of the second class. However, the reaction centers can be converted between the two groups by the addition of charged detergents. Thus, the observed spectral differences are not due to intrinsic differences between reaction centers but represent changes in the electronic structure of the donor due to interactions with the detergents as has been confirmed by recent ENDOR measurements (Rautter J, Lendzian F, Lubitz W, Wang S and Allen JP (1994) Biochemistry 33: 12077–12084). The oxidation midpoint potential for the donor has values of 445 mV, 475 mV, 480 mV and 495 mV for Rs. rubrum, Rs. centenum, Rb. capsulatus, and Rb. sphaeroides, respectively. Despite this range of values for the midpoint potential, the decay rates of the stimulated emission are all fast with values of 4.1 ps, 4.5 ps. 5.5 ps and 6.1 ps for quinone-reduced RCs from Rs. rubrum, Rb. capsulatus, Rs. centenum, and Rb. sphaeroides, respectively. The general spectral features of the initial charge separated state are essentially the same for the four species, except for differences in the wavelengths of the absorption changes due to the different donor band positions. The pH dependence of the charge recombination rates from the primary and secondary quinones differ for reaction centers from the four species indicating different interactions between the quinones and ionizable residues. A different mechanism for charge recombination from the secondary quinone, that probably is direct recombination, is proposed for RCs from Rs. centenum.Abbreviations RC reaction center - P bacteriochlorophyll dimer - H bacteriopheophytin - Q quinone - Rb Rhodobacter - Rs Rhodospirillum - Rps Rhodopseudomonas - EDTA (ethylenediamine)tetraaceticacid - LDAO N,N-dimethyl-dodecylamine-N-oxide - CTAB cetyltrimethylammonium bromide - DOC deoxycholate - Tris Tris-(hydroxymethyl)aminomethane - ns nanosecond - ps picosecond - fs femtosecond     

Coherent phenomena of charge separation in reaction centers of LL131H and LL131H/LM160H/FM197H mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Primary stage of charge separation and transfer of charges was studied in reaction centers (RCs) of point mutants LL131H and LL131H/LM160H/FM197H of the purple bacterium Rhodobacter sphaeroides by differential absorption spectroscopy with temporal resolution of 18 fsec at 90 K. Difference absorption spectra measured at 0–4 psec delays after excitation of dimer P at 870 nm with 30 fsec step were obtained in the spectral range of 935–1060 nm. It was found that a decay of P* due to charge separation is considerably slower in the mutant RCs in comparison with native RCs of Rba. sphaeroides. Coherent oscillations were found in the kinetics of stimulated emission of the P* state at 940 nm. Fourier analysis of the oscillations revealed a set of characteristic bands in the frequency range of 20–500 cm⁻¹. The most intense band has the frequency of −30 cm⁻¹ in RCs of mutant LL131H and in native RCs and the frequency of ∼100 cm⁻¹ in RCs of the triple mutant. It was found that an absorption band of bacteriochlorophyll anion B_A⁻ which is registered in the difference absorption spectra of native RCs at 1020 nm is absent in the analogous spectra of the mutants. The results are analyzed in terms of the participation of the B_A molecule in the primary electron transfer in the presence of a nuclear wave packet moving along the inharmonic surface of P* potential energy.     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Nanosecond fluorescence from chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Single-photon counting techniques were used to measure the fluorescence decay from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum chromatophores after excitation with a 25-ps, 600-nm laser pulse. Electron transfer was blocked beyond the initial radical-pair state (PF) by chemical reduction of the quinone that serves as the next electron acceptor. Under these conditions, the fluorescence decays with multiphasic kinetics and at least three exponential decay components are required to describe the delayed fluorescence. Weak magnetic fields cause a small increase in the decay time of the longest component. The components of the delayed fluorescence are similar to those found previously with isolated reaction centers. We interpret the multi-exponential decay in terms of two small (0.01-0.02 eV) relaxations in the free energy of PF, as suggested previously for reaction centers. From the initial amplitudes of the delayed fluorescence, it is possible to calculate the standard free-energy difference between the earliest resolved form of PF and the excited singlet state of the antenna complexes in R. rubrum strains S1 and G9. The free-energy gap is found to be about 0.10 eV. It also is possible to calculate the standard free-energy difference between PF and the excited singlet state of the reaction center bacteriochlorophyll dimer (P). Values of 0.17 to 0.19 eV were found in both R. rubrum strains and also in Rps. sphaeroides strain 2.4.1. This free-energy gap agrees well with the standard free-energy difference between PF and P determined previously for reaction centers isolated from Rps. sphaeroides strain R26. The temperature dependence of the delayed fluorescence amplitudes between 180 K and 295 K is qualitatively different in isolated reaction centers and chromatophores. However, the temperature dependence of the calculated standard free-energy difference between P* and PF is similar in reaction centers and chromatophores of Rps. sphaeroides. The different temperature dependence of the fluorescence amplitudes in reaction centers and chromatophores arises because the free-energy difference between P* and the excited antenna is dominated by the entropy change associated with delocalization of the excitation in the antenna. We conclude that the state PF is similar in isolated reaction centers and in the intact photosynthetic membrane. Chromatophores from Rps. sphaeroides strain R-26 exhibit an anomalous fluorescence component that could reflect heterogeneity in their antenna.     

The carotenoid band shift in reaction centers from Rhodopseudomonas sphaeroides

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the “special pair” of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Observation of pheophytin reduction in photosystem two reaction centers using femtosecond transient absorption spectroscopy.

Photosystem two reaction centers have been studied using a sensitive femtosecond transient absorption spectrometer. Measurements were performed at 295 K using different excitation wavelengths and excitation intensities which are shown to avoid multiphoton absorption by the reaction centers. Analyses of results collected over a range of time scales and probe wavelengths allowed the resolution of two exponential components in addition to those previously reported [Durrant, J. R., Hastings, G., Hong, Q., Barber, J., Porter, G., & Klug, D. R. (1992) Chem. Phys. Lett. 188, 54-60], plus the long-lived radical pair itself. A 21-ps component was observed. The process(es) responsible for this component was (were) found to produce bleaching of a pheophytin ground-state absorption band at 545 nm and the simultaneous appearance of a pheophytin anion absorption band at 460 nm resulting in a transient spectrum which was that of the radical pair P680+Ph-. This component is assigned to the production of reduced pheophytin. A lower limit of 60% of the final pheophytin reduction was found to occur at this rate. Despite subtle differences in transient spectra, the lifetime and yield of this pheophytin reduction are essentially independent of excitation wavelength within the signal to noise limitations of these experiments. A long-lived species was also observed. This species is produced by those processes which result in the 21-ps component, and it has a spectrum which is found to be independent of excitation wavelength. This spectrum is characteristic of the primary radical pair state P680+Ph-. In addition, a 200-ps component was found which is tentatively assigned to a slow energy-transfer/trapping process. This component was absent if P680 was excited directly and is therefore not integral to primary radical pair formation. Overall, it is concluded that the rate of pheophytin reduction is limited to (21 ps)-1, even when P680 is directly excited.     

Vibrational coherence in bacterial reaction centers with genetically modified B-branch pigment composition

Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.     

Formation of reaction centers and light-harvesting bacteriochlorophyll-protein complexes in Rhodopseudomonas capsulata

Formation of the photosynthetic apparatus was induced in aerobically grown dark cultures of Rhodopseudomonas capsulata by lowering of the oxygen tension. Besides the wild type strain the carotenoid-less mutant strain A1a+ was investigated. Both strains exhibited initially a decrease of the molar ratio of total bacteriochlorophyll (Bchl) to reaction center (RC) Bchl, followed by an increase. Synthesis of RC-Bchl preceded the synthesis of light-harvesting (LH) Bchl. Activities of photophosphorylation in membrane preparations, isolated from cultures after different periods of incubation at low aeration, decreased on the basis of total Bchl from about 9 to 2 mumole ATP/mumole total Bchl-min, whereas the rate on the basis of RC-Bchl remained constant (about 500 mumole ATP/mumole RC-Bchl-min). Under the same conditions the membrane proteins were labelled with U-14C-protein hydrolysate. Corresponding to RC-Bchl the synthesis of RC-proteins dominated during the first 30 min of incubation at PO2 below 3 mm Hg. After 45-60 min of membrane formation at low aeration the synthesis of LH-complex proteins exceeded the synthesis of RC proteins. The correlations between protein and Bchl synthesis in the sequential formation of RC- and LH-complexes are discussed.     

Photochemistry and electron transfer in borohydride-treated photosynthetic reaction centers

The photochemistry and electron-transfer activities of sodium-borohydride-treated reaction centers from the purple photosynthetic bacterium Rhodopseudomonas sphaeroides R26 have been investigated by both milliand picosecond absorption techniques. Separation from the treated reaction center of the reduction product, apparently a reduced form of one of the two molecules of bacteriochlorophyll contributing to the 800 nm ground-state absorption band, is also reported. In the near-infrared region, differences between treated and untreated reaction centers are observed in both milli- and picosecond light-induced difference spectra. However, borohydride-treated reaction centers exhibit photochemistry and electron transfer which are indistinguishable from those observed in untreated reaction centers. These results indicate that normal activity occurs in reaction centers that contain both molecules of bacteriopheophytin, but only three of the usual four molecules of bacteriochlorophyll.