Study of the acceptor portion of photosystem I according to the temperature relationships of P700 photoconversions

The functioning of the acceptor part of photosystem I was studied by temperature dependence of time course of light induced absorbtion changes at 700 nm of digitonin chloroplast fragments, enriched by photosystem I. Partial irreversibility of P700 photooxidation at low temperatures and appearance of two components (rapid and slow) in the time course of P700+ dark reduction reflect the contribution of different acceptors in electron transport. Thermoinactivation of fragments incubation at acid pH or treatment by glutaraldehyde cause complete inhibition of irreversible P700 photooxidation and slow dark reduction of P700+ at -170 degrees. The slow component of P700+ reduction and irreversible photooxidation of P700 are ascribed to contribution of secondary ferredoxin acceptors. The accurence of rapid component of P700+ dark reduction in light induced signal of treated fragments indicate that this component is due to recombination of reduced primary acceptor and P700+. Because only one electron transport takes at -170 degrees, the occurence of rapid and slow components in dark decay kinetics of P700+ suggests, that secondary acceptors of some reaction centers are incapable to reduction at -170 degrees. The shape of temperature dependence curve of the slow P700+ reduction component is interpreted as an indication of the tunneling electron transport.     

Characterization of a photosystem I core containing P700 and intermediate electron acceptor A1

A new photosystem I core has been isolated that is devoid of the bound iron-sulfur clusters but preserves electron flow from P700 to the intermediate electron acceptor A1. The particle is prepared by incubation of a Synechococcus sp. PCC 6301 photosystem I core protein (which contains electron acceptors A0, A1, and FX) with 3 M urea and 5 mM K3Fe(CN)6 to oxidatively denature the FX iron-sulfur cluster to the level of zero-valence sulfur. In this apo-FX preparation, over 90% of the flash-induced absorption change at 820 nm decays with a 10-microseconds half-time characteristic of the decay of the P700 triplet state formed from the backreaction of P700+ with an acceptor earlier than FX. Chemical reduction at high pH values with aminoiminomethanesulfinic acid results in kinetics identical with those seen in the P700 chlorophyll a protein prepared with sodium dodecyl sulfate (SDS-CP1, which contains only electron acceptor A0); the flash-induced absorption change decays primarily with a 25-ns half-time characteristic of the backreaction between P700+ and A0-, and the magnitude of the total absorption change is larger than can be accounted for by the P700 content alone. Addition of oxygen results in a reversion to the 10-microseconds kinetic decay component attributed to the decay of the P700 triplet state. At 77 K, the optical transient in the apo-FX preparation decays with a 200-microseconds half-time characteristic of the backreaction between P700+ and A1-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the generation of a photo-induced electric potential in chromatophores of photosynthetizing bacteria

Flash-induced formation of an electric potential difference (delta psi) was monitored by a direct method in chromatophores associated with the collodion phospholipid membrane. In Rhodospirillum rubrum and Rhodopseudomonas sphaeriodes chromatophores, the kinetics of delta psi generation exhibit fast (tau less than or equal to 0.3 microseconds) and slow (tau congruent to 200 microseconds) phases, the latter observed in the presence of exogenous quinones. Comparison of the kinetic and potentiometric characteristics of the process with those of electron transport reactions suggests that the fast phase of delta psi rise is due to charge separation between the primary electron donor, P870, and primary electron acceptor QIFe; the slow phase, which is inhibited by o-phenanthroline, is due to electron donation from QIFe to the secondary acceptor, quinone QII. The kinetics of delta psi decay include components arising form the recombination of primary separated charges (tau congruent to 30 ms) and from the passive discharge of the membrane (tau congruent to 400 ms; tau congruent to 1400 ms). From a redox titration of the photo-induced electric signal and the photo-induced absorption changes of P870 at different pH meanings, the value of pK for the primary acceptor FeQI was found to be 7.4 in Rps. sphaeroides chromatophores. In Chromatium minutissimum, a phase ( tau congruent to 20 microseconds) was observed in addition to those seen in Rps. sphaeroids and R. rubrum which was explained by the reduction of P890+ from the high potential cytochrome c555. Possible distribution of the electron transport components in the chromatophore membrane are discussed.     

Fluorescence of bacteriochlorophyll as related to the photochemistry of chromatophores of photosynthetic bacteria

Time courses and the emission spectra of fluorescence and light-induced absorption changes of P890 in chromatophores of the photosynthetic bacteria Chromatium D, Rhodopseudomonas spheroides and Rhodospirillum rubrum were investigated.

The time course of fluorescence in chromatophores was separated into two phases, i.e. an initial rapid rise (ƒ_i) and a subsequent slow increase towards a steady level of emission (ƒ_v). The ƒ_i and the ƒ_v components showed different emission spectra having different peak position. The ƒ_v component was emitted from the longest wavelength-absorbing form of bulk bacteriochlorophyll (B890), the ƒ_i component from both B890 and B850.

The magnitude of the ƒ_v component depended on experimental conditions controlling the states of the cyclic electron transport in chromatophores, including changes in levels of redox potential of the medium, additions of electron donors and inhibitors. The magnitude of the ƒ_i component was not affected by these experimental conditions. It was, therefore, concluded that only the ƒ_v component is related to the cyclic electron transport, and that the magnitude of ƒ_v is controlled by the oxidation-reduction state of the primary electron acceptor for the photochemical reaction center in chromatophores.     

Temperature dependence of the kinetics of dark reduction of bacteriochlorophyll P870, oxidized by impulse laser and continuous light in photosynthetic reaction center preparations from Rhodosuedomonas spheroides strain 1760-1]

A comparative study was carried out of temperature dependence of kinetics of dark reduction of bacteriochlorophyll P870 oxidized both by pulsed laser and continuous actinic light in preparations of photosynthetic reaction centres-bacteriochlorophyll-protein complexes from Rhodopseudomonas spheroides, strain 1760-1. Half-time of the recombination of primary products--P870+ and reduced primary electron acceptor A1, which decreases with temperature lowering from 180-240 ms at 120K, is determined. Values of the rate constant of electron transfer from A1 to secondary acceptors at 29,K (2.7.10-1s-1) and the activation energy of this reaction in the range of 298-255K which is 11.8 kcal/mole are calculated.     

Coupling of nuclear wavepacket motion and charge separation in bacterial reaction centers

The mechanism of the charge separation and stabilization of separated charges was studied using the femtosecond absorption spectroscopy. It was found that nuclear wavepacket motions on potential energy surface of the excited state of the primary electron donor P* leads to a coherent formation of the charge separated states P⁺B_A⁻, P⁺H_A⁻ and P⁺H_B⁻ (where B_A, H_B and H_A are the primary and secondary electron acceptors, respectively) in native, pheophytin-modified and mutant reaction centers (RCs) of Rhodobacter sphaeroides R-26 and in Chloroflexus aurantiacus RCs. The processes were studied by measurements of coherent oscillations in kinetics at 890 and 935 nm (the stimulated emission bands of P*), at 800 nm (the absorption band of B_A) and at 1020 nm (the absorption band of B_A⁻) as well as at 760 nm (the absorption band of H_A) and at 750 nm (the absorption band of H_B). It was found that wavepacket motion on the 130–150 cm⁻¹ potential surface of P* is accompanied by approaches to the intercrossing region between P* and P⁺B_A⁻ surfaces at 120 and 380 fs delays emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A⁻). In the presence of Tyr M210 (Rb. sphaeroides) or M195 (C. aurantiacus) the stabilization of P⁺B_A⁻ is observed within a few picosseconds in contrast to YM210W. At even earlier delay (40 fs) the emission at 895 nm and bleaching at 748 nm are observed in C. aurantiacus RCs showing the wavepacket approach to the intercrossing between the P* and P⁺H_B⁻ surfaces at that time. The 32 cm⁻¹ rotation mode of HOH was found to modulate the electron transfer rate probably due to including of this molecule in polar chain connecting P_B and B_A and participating in the charge separation. The mechanism of the charge separation and stabilization of separated charges is discussed in terms of the role of nuclear motions, of polar groups connecting P and acceptors and of proton of OH group of TyrM210.     

Triplet States in a Photosystem I Reaction Center Complex. Inhibition of Radical Pair Recombination by Bipyridinium Dyes and Naphthoquinones

Flash-induced absorption changes between 400 and 570 nm werestudied in a P700-chlorophyll a-protein complex from the thermophiliccyanobacterium Synechococcus sp. that lacked the bound secondaryelectron acceptors A₂ and P430. A positive peak at 520 nm, whichincreased linearly with the flash intensity and independentlyof the redox state of P700, is ascribed to a carotenoid triplet.Bleaching at 430 nm, which decayed with a half time of about10 µs, was abolished when P700 was oxidized with ferricyanideand saturated at a high flash intensity, indicative of its dependenceon the primary photochemistry of photosystem I. Several bipyridinium dyes and naphthoquinones suppressed the10 µs decay of the 430 nm signal in a way indicating thatthe 10 µs component represents the P700 triplet generatedby the back reaction between the reduced primary electron acceptorand oxidized P700 and that the added oxidants oxidize the reducedprimary acceptor so rapidly that back electron transfer to oxidizedP700 is prevented. Our results also show that the primary electronacceptor is located in a lypophilic environment in the chlorophyll-bindingsubunits of the photosystem I complexes. In a reaction centercomplex containing the secondary electron acceptors, the exogenousoxidants accept electrons only via P430. (Received February 23, 1984; Accepted May 1, 1984)     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Excitonic interactions in the reaction centre of photosystem II studied by using circular dichroism

Changes in excitonic interactions of photosystem II (PSII) reaction centre (RC) pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor (pheophytin (Pheo)) were analysed using circular dichroism (CD). The CD spectrum of PSII RC shows positive bands at 417, 435 and 681 and negative bands at 447 and 664 nm. Oxidation of the primary donor by illuminating the sample in the presence of silicomolybdate resulted in nearly symmetric decrease of CD amplitudes at 664 and 684 nm. In the Soret region, the maximum bleaching of CD signal was detected at 449 and 440 nm. Accumulation of reduced Pheo in the presence of dithionite brought about much lower changes in CD amplitudes than P680 oxidation. In this case, only a small asymmetric bleaching at 680 and 668 nm in the red region and a bleaching at 445, 435 and 416 nm in the Soret region has been detected. Therefore, we suppose that the contribution of the Pheo of the primary acceptor to the total CD signal of RC is negligible. In contrast to the oxidation of primary donor, the light-induced change in the CD spectrum upon primary acceptor reduction was strongly temperature-dependent. The reversible CD bleaching was completely inhibited below 200 K, although the reduced Pheo was accumulated even at a temperature of 77 K. Since the temperature does not influence the excitonic interaction, the temperature dependence of the CD changes upon Pheo reduction does not support the model of Pheo excitonically interacting with the other chlorophylls (Chl) of the RC. We propose that Pheo should not be considered as a part of a multimer model.     

Excitonic interactions in the reaction centre of photosystem II studied by using circular dichroism

Changes in excitonic interactions of photosystem II (PSII) reaction centre (RC) pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor (pheophytin (Pheo)) were analysed using circular dichroism (CD). The CD spectrum of PSII RC shows positive bands at 417, 435 and 681 and negative bands at 447 and 664 nm. Oxidation of the primary donor by illuminating the sample in the presence of silicomolybdate resulted in nearly symmetric decrease of CD amplitudes at 664 and 684 nm. In the Soret region, the maximum bleaching of CD signal was detected at 449 and 440 nm. Accumulation of reduced Pheo in the presence of dithionite brought about much lower changes in CD amplitudes than P680 oxidation. In this case, only a small asymmetric bleaching at 680 and 668 nm in the red region and a bleaching at 445, 435 and 416 nm in the Soret region has been detected. Therefore, we suppose that the contribution of the Pheo of the primary acceptor to the total CD signal of RC is negligible. In contrast to the oxidation of primary donor, the light-induced change in the CD spectrum upon primary acceptor reduction was strongly temperature-dependent. The reversible CD bleaching was completely inhibited below 200 K, although the reduced Pheo was accumulated even at a temperature of 77 K. Since the temperature does not influence the excitonic interaction, the temperature dependence of the CD changes upon Pheo reduction does not support the model of Pheo excitonically interacting with the other chlorophylls (Chl) of the RC. We propose that Pheo should not be considered as a part of a multimer model.     

Light-induced absorbance changes due to Photosystems 1 and 2 in spinach chloroplasts at −50 °C

Absorbance changes in the region 500–565 nm and at 702 nm, brought about by excitation of Photosystems 1 and 2, respectively, were measured in spinach chloroplasts at ?50 °C. Either dark-adapted chloroplasts were used or chloroplasts preilluminated with a number of short saturating flashes just before cooling.Both photosystems were found to cause a light-induced increase of absorbance at 518 nm (due to “P518”). The System 1-induced change was not affected by preillumination. It decayed within 1 s in the dark and showed similar kinetics as P700. Experiments in the presence of external electron acceptors (methylviologen or Fe(CN)₆^3?) suggested that P518 was not affected by the redox state of the primary electron acceptor of System 1. The absorbance increase at 518 nm due to System 2 decayed in the dark with a half-time of several min. The kinetics were similar to those of C-550, the presumed indicator of the primary electron acceptor of System 2. After two flashes preillumination the changes due to P518 and C-550 were reduced by about 40%, and a relatively slow, System 2-induced oxidation of cytochrome b₅₅₉ occurred which proceeded at a similar rate as the increase in yield of chlorophyll a fluorescence. The results indicate that at ?50°C two different photoreactions of System 2 occur. One consists of a photoreduction of the primary electron acceptor associated with C-550, accompanied by the oxidation of an unknown electron donor; the other is less efficient and results in the photooxidation of cytochrome b₅₅₉.     

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.     

Light-induced absorption changes in Photosystem I at low temperatures

Light-induced absorption changes associated with the primary photochemical reaction and dark relaxation in Photosystem I were measured at various low temperatures. A possible temperature-dependent long-range electron tunneling process was suggested to account for the unique temperature dependence of the dark decay process. The kinetics of the light-induced absorption changes are in good agreement with the light-induced EPR changes reported earlier (Ke, B., Sugahara, K., Shaw, E.R., Hansen, R. E., Hamilton, W. D. and Beinert, H. (1974) Biochim. Biophys. Acta 368, 401–408) for the same Photosystem I subchloroplast fragments at comparable temperatures.All absorption changes between 400 and 725 nm at 86 °K have identical kinetics. The light-minus-dark difference spectrum is very similar to that of P-700 at room temperature, with an additional prominent positive change at 690 nm. Possible contributions by P-430 to the blue and red spectral changes were discussed.It was demonstrated that the intensity of the measuring beam has a drastic effect on the light-induced absorption changes of Photosystem I at low temperatures. Various pretreatments of the Photosystem I fragments such as those that photochemically (or chemically) oxidize the primary donor or photoreduce the primary acceptor abolish the subsequent photochemical reaction. Continuous illumination of the Photosystem I fragments before and during freezing has the same effect.In the temperature range of ?20 to ?60 °C, an unusual counter absorption change as well as a counter EPR change were observed.     

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.     

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.     

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii. After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a. With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q_y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q_y and Q_x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.     

Fast phases of the generation of the transmembrane electric potential in chromatophores of the photosynthetic bacterium Ectothiorhodospira shaposhnikovii

Ectothiorhodospira shaposhnikovii chromatophores were associated with a collodion film and kinetics of generation of the transmembrane electric potential (Δψ) were investigated in the ‘chromatophore-collodion film’ system, using an electrometric technique with a high resolution time (over 200 ns). A generation of Δψ (the chromatophore interior positive) following a laser flash was observed, the kinetics consisting of three components of the following half-times: less than 200 ns (phase 1); 2–7 μs (phase 2); and 120 μs (phase 3). A redox titration of the kinetic phases was performed. Computer analysis of the results has shown that the midpoint potentials (E_m) of the phase 1 at pH 7.5 are +400 mV and −75 mV, whereas those of the phase 2 are +310 mV and +35 mV. The comparison of the kinetic and potentiometric characteristics of the Δψ generation with analogous characteristics of the electron-transport processes, measured by optical spectroscopy, suggested that phases 1, 2 and 3 are associated with the electron transfer from P-890 to the primary quinone acceptor Q_a, from the high-potential cytochrome C_H to P-890, and with the reduction of secondary acceptor Q_b, respectively. From the amplitude characteristics of the Δψ components, a tentative scheme of the intramembrane localization of the electron carriers is presented.     

Spectral and photochemical properties of subchromatophore fractions derived from carotenoid-deficient Chromatium by Triton treatment

Triton treatment of chromatophores of carotenoid-deficient Chromatium followed by density-gradient centrifugation led to a separation into three subchromatophore fractions. Unlike the case with chromatophores of regular Chromatium, Triton releases about 1/3 of the total bulk bacteriochlorophyll into one fraction (designated G, for green) whose major absorption-band maximum is at 780 nm. One fraction (H, for heavy) absorbs at 805 and 885 nm, with an absorbance ratio A₈₈₅ nm/A₈₀₅ nm between 1.5 and 2; another fraction (L, for light) absorbs at 805 nm and has a shoulder at 825 nm. The absorption and fluorescence emission spectra of the three fractions at room temperature and 77°K indicate that the different bacteriochlorophyll forms are efficiently separated by Triton treatment.

The reaction center P890 is concentrated exclusively in the H-fraction, at a level of 5–7% of the bulk bacteriochlorophyll. The solubilized bacteriochlorophyll absorbing at 780 nm can be totally and irreversibly bleached by 5 mM ferricyanide. The other bacteriochlorophyll forms in the H- and L-fractions are also irreversibly bleached by ferricyanide to variable extents. P890 is the only component that can be re-reduced by ascorbate after ferricyanide oxidation. The P890 content estimated by reversible chemical bleaching agrees well with that obtained by reversible light bleaching. The different bacteriochlorophyll forms, with the exception of the 780-nm absorbing form, are relatively stable toward light bleaching. Again, only P890 is reversibly bleached by light.

Cytochromes-555 and -553 are distributed in both the H-and L-fractions, but not in the solubilized-bacteriochlorophyll G-fraction. However, only cytochromes in the H-fraction which contains all of the P890 can undergo coupled oxidation. Excitation with 20-nsec ruby-laser pulses shows that cytochrome-555 can be oxidized in 2–3 μsec by photooxidized P890, indicating that necessary conformation for rapid electron transport is retained in the subchromatophore particles.

The data on fractionation and redox reactions obtained here, together with direct kinetic measurements recently reported in the literature lend further support to the view that oxidation of these two cytochromes is mediated by the same reaction center, P890.     

Bacteriochlorophyll fluorescence changes related to the bacteriopheophytin photoreduction in the chromatophores of purple sulfur bacteria]

It is shown that illumination of chromatophores of sulfur bacterium Chromatium minutissimum at Eh of the medium --200 mV divided by --620 mV (when the photooxidation of pigment P890 is completely inhibited) induces a decrease in bacteriochlorophyll fluorescence yield, reversible in the dark. Under these conditions a reversible photoreduction of bacteriopheophytin is detected (bleaching of absorption bands at 543 and 760 nm and development of a band at 650 nm), which is accompanied by a blue shift of the absorption band at 8 nm. As a possible interpretation of these effects the suggestion is made on the function of bacteriopheophytin as a primary electron acceptor in reaction centers of bacteria. The bacteriopheophytin photoreduction, followed by a decrease in fluorescence yield, is also observed in other sulfur bacteria, Thiocapsa roseopersicina and Ectothiorodospira shaposhnikovii, but it is not detected in nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides. This is considered as an evidence for the difference in the functional organization of the reaction centers of these two groups of bacteria,     

Light- and chemically-induced oxidation-reduction reactions in chromatophore fractions of Rhodospirillum rubrum

Chromatophore fractions of Rhodospirillum rubrum were prepared by centrifugation of a ‘classical’ chromatophore preparation in a sucrose density gradient. Most of the experiments were carried out with a fraction separating out at about 23% sucrose. Although no light-induced reactions of cytochromes could be detected, the presence of two bound cytochromes, C422 and C428, in this fraction was indicated by oxidation-reduction reactions induced by KIO₄ and dithionite. Cytochrome C422 appeared to be the ‘high-potential’ cytochrome detected earlier in preparations from this organism. Titration of chemically-induced absorbance changes due to a reduction of oxidized cytochrome C428 has led to an estimated redox midpoint potential of E_m7.5 = −0.01 V for this component in a four-electron transport reaction.

Light-induced absorbance changes in the near-infrared spectral region measured in the light chromatophore fraction at different environmental redox potentials suggested a bacteriochlorophyll component, P', with spectral characteristics which were different from those of the high-potential bacteriochlorophyll component P890. The spectral shifts observed upon oxidation, either by light or by air were a bleaching in the 860–880-nm spectral region and an appearance of an absorption band at about 900 nm. The component could be oxidized and reduced reversibly and had an estimated midpoint potential of +0.06 V in a one-electron reaction.