Rhodopseudomonas Sphaeroides Reaction Centers Research Articles

Magnitude and direction of the change in dipole moment associated with excitation of the primary electron donor in Rhodopseudomonas sphaeroides reaction centers

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMagnitude and direction of the change in dipole moment associated with excitation of the primary electron donor in Rhodopseudomonas sphaeroides reaction centersDavid J. Lockhart and Steven G. BoxerCite this: Biochemistry 1987, 26, 3, 664–668Publication Date (Print):February 1, 1987Publication History Published online1 May 2002Published inissue 1 February 1987https://doi.org/10.1021/bi00377a001RIGHTS & PERMISSIONSArticle Views213Altmetric-Citations126LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (552 KB) Get e-Alerts Get e-Alerts

Triplet-minus-singlet absorbance difference spectra of reaction centers of Rhodopseudomonas sphaeroides R-26 in the temperature range 24?290 K measured by Magneto-Optical Difference Spectroscopy (MODS)

The recently developed technique of Magneto-Optical Difference Spectroscopy (MODS) [10] has been applied to reaction centers (RC) of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. Absorbance changes induced by a magnetic field are measured as a function of wavelength yielding the triplet-minus-singlet (T-S) absorbance difference spectrum. (T-S) spectra thus obtained have been measured from 24-290 K. Going from low to high temperature the (T-S) spectra show the following features: (a) A rapid decrease of positive absorption bands at 809 and 819 nm. (b) A slow appearance of a band shift at 798 nm. (c) A shift of the peak wavelength of the Qy absorbance band of the primary donor P-860 from 992 to 861 nm, and of its Qx band from 603 to 600 nm. The spectra at 24, 66, 116, and 290 K have been analyzed by Gaussian deconvolution. The 800 nm region of the spectrum at 24 K can be decomposed in a combination of two band shifts and an appearing band. The temperature dependence of the spectra in this region is well explained by spectral broadening of the two shifting bands combined with a decrease in intensity of the appearing band when the temperature increases.The two shifting bands in the 800 nm region are identified as the two bands at 803 and 813 nm which together make up the 800 nm band in the absorption spectrum and are assigned to the two accessory RC bacteriochlorophylls (BChls). The band shift of the 813 nm pigment is appreciably larger than that of the 803 nm pigment. The appearing band at 808 nm is attributed to monomeric absorption of (3)P-860, the triplet state being localized on one BChl.We find no evidence for admixture of a charge transfer (CT) state of (3)P-860 with one of the accessory BChls at higher temperature.

Estimation of the rate of photochemical charge separation in Rhodopseudomonas sphaeroides reaction centers by fluorescence and absorption picosecond spectroscopy

Time-resolved fluorometry of reaction center (RC) preparations from Rhodopseudomonas sphaeroides, wild strain 1760-1, shows that the lifetime of the excited state of bacteriochlorophyll P870 ∗ is τ = 6± 1.5 ps and independent of temperature within the range 293 – 77 K. This value was found to coincide well with the time (7 ± 3 ps) of the RC porphyrin pigment transition into the ion-radical pair state P F, as measured by picosecond absorption spectroscopy of the same preparations.

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.

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. II. Free energy and kinetic relations between the acceptor states Q A−Q B− and Q AQ 2−B

Thermodynamic equilibria and electron transfer kinetics involving the quinone acceptor complex in reaction centers from Rhodopseudomonas sphaeroides were investigated. We focussed on reactions involving the two-electron states Q A -Q B - and Q AQ 2− B, described by the scheme ▪ The equilibrium partitioning between Q A -Q B - and Q AQ 2− B was determined spectroscopically from either the concentration of oxidized cytochrome c or the concentration of semiquinone after successive flashes of light. At pH < 9.5, Q AQ 2− B is stabilized relative to Q A -Q B -, while for pH > 9.5, Q A -Q B - is energetically favored. The reduction of Q A, to form Q A -Q B -, is not associated with a protonation step (p K < 8). However, the reduction of Q B -, to form the final state Q AQ 2− B, is accompanied by an uptake of a proton (p K ≥ 10.7). The preferential interaction of a proton with Q AQ 2− B provides the driving force for the forward electron transfer. The shift toward the photochemically inactive state Q A -Q B - with increasing pH may serve as a feedback mechanism in photosynthetic organisms to limit the rise in intracellular pH. The electron-transfer rate constants were determined from the observed kinetics and the equilibria between the states Q AQ 2− B and Q A -Q B -. The forward rate constant k (2) AB was approximately proportional to the proton concentration, whereas k (2) BA depended only weakly on pH. The recombination kinetics of D +Q AQ 2− B was biphasic. The slow rate agreed with the predicted charge recombination via the intermediate state D +Q A -Q B -; the fast rate may be due to the recombination from a separate (conformational) state. The results of this work were combined with those of a previous study on reactions involving the one-electron precursor states Q A -Q B and Q AQ B - (Kleinfeld, D., Okamura, M.Y., and Feher, G. (1984) Biochim. Biophys. Acta 766, 126–140). The overall sequence for the protonation of the reaction center in response to successive reductions of the acceptor complex involves the uptake of one proton for each electron transferred to Q B. This sequential uptake initiates the formation of a proton gradient across the cell membrane.

Picosecond-photodichroism studies of the transient states in Rhodopseudomonas sphaeroides reaction centers at 5 K. Effects of electron transfer on the six bacteriochlorin pigments

We have examined the dichroism of the visible and near-infrared absorption changes due to the early transient states in Rhodopseudomonas sphaeroides reaction centers imbedded in polyvinyl alcohol films at 5 K. The transient-state, ground-state and derivative spectra acquired under these conditions are highly resolved. Spectral features have been assigned to the bacteriochlorophyll (BChl) dimer (P) that serves as the primary electron donor, to each of the two additional BChls, and to the two bacteriopheophytin (BPh) molecules. The dichroism of the absorption changes, taken together with earlier results including our observation of a detection-wavelength dependence of the kinetics, argues that only one of the BPhs is a clearly resolved electron carrier prior to ubiquinone. The second BPh and the two BChls not constituting P display electrochromic effects and/or nuclear relaxations, possibly involving the protein, in response to the charge-separation process.

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides

Replacement of Fe 2+ by Zn 2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors Q − A and Q − B. Differences between the Q A and Q B sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.

Electron spin echo envelope modulation (ESEEM) spectroscopy of the triplet state of the primary donor of 14N and 15N bacterial photosynthetic reaction centers and of 14N and 15N bacteriochlorophyll a

An electron spin echo envelope modulation frequency analysis is performed on the triplet state of the primary electron donor (P-860) in 14N reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 and 15N-enriched reaction centers of Rhodopseudomonas sphaeroides 2.4.1, and of the triplet state of 14N or 15N bacteriochlorophyll a in vitro. The hyperfine coupling constants for 15N 3P-860 are 1.42, 1.74 and 2.04 MHz. The triplet state of the primary donor in bacterial photosynthesis is, on a timescale of a few MHz, delocalized over the two bacteriochlorophyll a molecules making up P-860.

15N electron nuclear double resonance of the primary donor cation radical P+.865 in reaction centers of Rhodopseudomonas sphaeroides: additional evidence for the dimer model.

Four 15N hyperfine coupling constants, including signs, have been measured by electron nuclear double resonance (ENDOR) and electron nuclear nuclear triple resonance (TRIPLE) for the bacteriochlorophyll a radical cation, BChla+., in vitro and for the light-induced primary donor radical cation, P+.865, in reaction centers of Rhodopseudomonas sphaeroides R-26. A comparison of the data shows that the hyperfine coupling constants have the same sign in both radicals and are, on the average, smaller by a factor of 2 in P+.865. These results provide additional evidence that P+.865 is a bacteriochlorophyll dimer and are in contradiction with the monomer structure of P+.865 recently proposed by O'Malley and Babcock. The reduction factors of the individual 15N couplings, together with the evidence from proton ENDOR data and molecular orbital calculations, indicate a dimer structure in which only two rings (either I and I or III and III) of the bacteriochlorophyll macrocycles overlap.

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.

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D +Q AQ −B and free energy and kinetic relations between Q −AQ B and Q AQ −B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D +Q AQ − B proceeds via the intermediate state D +Q − AQ B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k AD-times the fraction of reaction centers in the Q − AQ B state) with the observed recombination rate, k obs D + →D . The kinetic measurements were used to obtain the pH dependence (6.1 ⩽ pH ⩽ 11.7) of the free energy difference between the states Q − AQ B and Q AQ − B. At low pH (less than 9) Q AQ − B is stabilized relative to Q − AQ B by 67 meV, whereas at high pH Q − AQ B is energetically favored. Both Q − A and Q − B associate with a proton, with p K values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q − B provides the driving force for the forward electron transfer.

Absorption and structure of an LM unit isolated with sodium dodecyl sulfate from reaction centers of Rhodopseudomonas sphaeroides

Low-temperature absorption, circular dichroism and resonance Raman spectra of the LM units isolated with sodium dodecyl sulfate from wild-type Rhodopseudomonas sphaeroides reaction centers (Agalidis, I. and Reiss-Husson, F. (1983) Biochim. Biophys. Acta 724, 340–351) are described in comparison with those of intact reaction centers. In LM unit, the Q y absorption band of P-870 at 77 K shifted from 890 nm (in reaction center) to 870 nm and was broadened by about 30%. In contrast, the 800 nm bacteriochlorophyll absorption band including the 810 species remained unmodified. It was concluded that the 810 nm transition is not the higher excitonic component of P-870. The Q x band of P-870 shifted from 602 nm (in reaction center) to 598 nm in LM, whereas the Q x band of the other bacteriochlorophylls was the same in reaction center and LM and had two components at about 605 and 598 nm. The Q xII band of bacteriopheophytin was upshifted to 538 nm and a slight blue shift of the Q y band of bacteriopheophytin was observed. Resonance Raman spectra of spheroidene in LM showed that its native cis-conformation was preserved. Resonance Raman spectroscopy also demonstrated that in LM the molecular interactions assumed by the conjugated carbonyls of bacteriochlorophyll molecules were altered, but not those assumed by the bacteriopheophytins carbonyls. In particular at least one Keto group of bacteriochlorophyll free in reaction center, becomes intermolecularly bounded in LM (possibly with extraneous water). This group may belong to the primary donor molecules.

Photoaffinity labeling of the azidoatrazine receptor site in reaction centers of Rhodopseudomonas sphaeroides

Photoaffinity labeling of photosynthetic reaction centers of Rhodopseudomonas sphaeroides by the herbicide inhibitor, azido[ 14C]atrazine, occurs principally on the L-subunit. The specificity of labeling is greater at 77 than at 295 K. Kinetic studies of charge recombination in reaction centers indicate competition between azidoatrazine and ubiquinone-1 (Q-1) for binding to the reaction center. This competition occurs through the L-subunit binding site, as increasing concentrations of Q-1 decrease azido[ 14C]atrazine labeling of this site. It is proposed that the inhibitor binding site, predominantly on the L-subunit, and the secondary quinone binding site on the M-subunit, are adjacent so that there is partial overlap by one molecule of the domain occupied by the other.

Several properties of the LM unit extracted with sodium dodecyl sulfate from Rhodopseudomonas sphaeroides purified reaction centers

We describe several characteristics of an LM unit extracted from purified wild-type Rhodopseudomonas sphaeroides reaction centers after treatment with SDS and lauryldimethylamine N-oxide. We also studied another preparation called RC-SDS in which after a similar detergent treatment the H-chain was not separated from the LM unit. The spectral properties of both preparations were similar to those of intact reaction centers; the main differences were a blue shift of the P-865 Q y band and a narrowing of the bacteriopheophytin Q x band. Studies of absorbance changes after steady light or flash illumination showed that LM was depleted of the primary electron acceptor Q I and of the transition metal (Mn), but was still able to react with quinones, with a partial restoration of the photochemistry. The primary acceptor Q 10 was still partly present in RC-SDS and formed in the light a very unstable semiquinone anion. In both preparations, in the presence of added quinones, the flash-induced P-865 + decayed in the dark with multiexponential kinetics, the apparent half-times ranging between 60 ms and 100 s. This recovery was accelerated either by the absence of O 2 or (in the case of LM only) by o-phenanthroline, which also decreased the amplitude of the P-865 + signal. From these experiments it was concluded that in these preparations the site of binding of the secondary quinone Q II was damaged or absent; the altered properties of this LM unit were considered to result from the denaturing action of the detergents, rather than from the absence of the H polypeptide.

Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: localization of the binding site by chemical cross-linking and immunochemical studies.

The location of the cytochrome binding site on the reaction center of Rhodopseudomonas sphaeroides was studied by two different approaches. In one, cross-linking agents, principally dithiobis(propionimidate) and dimethyl suberimidate, were used to link cytochrome c and cytochrome c2 to reaction centers; in the other, the inhibition of electron transfer by antibodies against the subunits was investigated. Cytochrome c (horse) cross-linked to the L and M subunits, whereas cytochrome c2 (R. sphaeroides) cross-linked only to the L subunit. The cross-linked reaction center-cytochrome complexes were isolated by affinity chromatography. The rate of electron transfer in the cross-linked cytochrome c2 complex was the same as that in the un-cross-linked complex. However, when cytochrome c was used, the rate in the cross-linked complex was about 15 times slower than that in the un-cross-linked complex. Fab fragments of antibodies specific against the L and M subunits blocked electron transfer from both cytochrome c (horse) and cytochrome c2 (R. sphaeroides). Antibodies specific for the H subunit did not block either reaction. We conclude that the cytochrome binding site on the reaction center is close (approximately 10 A) to both the L and M subunits, possibly in a cleft between them.

Localization of the secondary quinone-binding site in reaction centers from Rhodopseudomonas sphaeroides R-26 by antibody inhibition of electron transfer

Inhibition of the electron transfer from Q A to Q B was measured in the presence of Fab fragments of antibodies directed against the subunits of reaction centers of Rhodopseudomonas sphaeroides R-26. Anti-M Fab inhibited the electron transfer, whereas anti-L Fab and anti-H Fab did not. From these experiments, we conclude that the binding site for Q B is located on the M-subunit.

Resonance Raman scattering from Rhodopseudomonas sphaeroides reaction centers absorbed on a silver electrode

The reaction center complex from Rhodopseudomonas sphaeroides carotenoidless mutant R-26 has been well characterized as to its protein, pigment (4 bacteriochlorophylls (BChl) and 2 bacteriopheophytins (BPh)) and quinone content [ 11. The function of 3 or 4 of the pigment molecules as well as the quinones in the reaction center primary photochemistry is also becoming better understood [2,3]. Two of the BChls, the special pair, are believed to function as the primary electron donor on photoexcitation and transfer an electron to a ubiquinone molecule via one of the BPhs, which has been termed the intermediate electron acceptor. In addition, there is evidence that one of the remaining BChls is involved in the initial electron transfer from the special pair to BPh [3]. New spectroscopic methods are needed which can provide additional support to the picosecond optical absorption data, as well as structural information about the bonding interactions of BChl and BPh in the reaction center. finitive method for identifying the proposed transient intermediates in reaction center photochemistry. The vibrational spectrum of the BChl cation radical differs substantially from the neutral species [7,8]. We have undertaken a RR study of isolated Rps. sphaeroides reaction centers spontaneously adsorbed onto a Ag electrode: The advantages of studying the adsorbed complex include increased RR scattering intensity as a result of the surface enhanced effect on Ag [9], quenching of background fluorescence [lo], and the possibility of changing the redox state of the reaction center by varying the electrode potential.

Study of the long-wavelength fluorescence band at 920 nm of isolated reaction centers of the photosynthetic bacterium Rhodopseudomonas spaeroides R-26 with fluorescence-detected magnetic resonance in zero field

The triplet state of isolated reaction centers of Rhodopseudomonas sphaeroides R-26 has been studied by fluorescence-detected electron spin resonance in zero magnetic field (FDMR) at 4.2 K. The sign of the FDMR resonance monitored at the long-wavelength fluorescence band is positive (fluorescence increase); this confirms the earlier interpretation (Hoff, A.J. and Gorter de Vries, H. (1978) Biochim. Biophys. Acta 503, 94–106) that the negative sign of the FDMR resonance of the reaction center triplet state in whole bacterial cells is caused by resonant transfer of the singlet excitations from the antenna pigments to the trap. By monitoring the FDMR response as a function of the wavelength of fluorescence, we have recorded microwave-induced fluorescence spectra. In addition to the positive microwave-induced fluorescence band peaking at 935 nm, at 905 nm a negative band was found. The resonant microwave frequencies for these two bands, i.e., the values of the zero-field splitting parameters | D| and | E| of the triplet state being monitored, were different, those of the 905 nm microwave-induced fluorescence band being identical to the resonant microwave frequencies measured with absorption-detected zero-field resonance (Den Blanken, H.J., Van der Zwet, G.P. and Hoff, A.J. (1982) Chem. Phys. Lett. 85, 335–338), a technique that monitors the bulk properties of the sample. From this result and its negative sign, we tentatively attribute the 905 nm microwave-induced fluorescence band to a small (possibly less than 1%) fraction of antenna bacteriochlorophylls that are in close contact with the trap. The positive 935 nm microwave-induced fluorescence band with resonant microwave frequencies deviating from the bulk material is ascribed to a minority of primary donor bacteriochlorophyll dimers, which have a higher than normal fluorescence yield because of a somewhat slower charge-separation reaction. Is it likely that practically all long-wavelength fluorescence of isolated reaction centers stems from such impaired reaction centers.

ESR in zero field of the photoinduced triplet state in isolated reaction centers of rhodopseudomonas sphaeroides R-26 detected by the singlet ground-state absorbance

We have measured zero-field resonance transitions of the triplet state of the primary donor monitoring the transmittance at 890 nm at 1.2 K in isolated reaction centers of Rhodopseudomonas sphaeroides R-26. The transitions correspond to a decrease in transmittance, confirming the energy transfer model for the transitions detected via the antenna fluorescence in whole cells.

Temperature dependence of electron transfer between bacteriopheophytin and ubiquinone in protonated and deuterated reaction centers of Rhodopseudomonas sphaeroides

The rate of the electron-transfer reaction between bacteriopheophytin and the first quinone in isolated reaction centers of Rhodopseudomonas sphaeroides has an unusual temperature dependence. The rate increases about threefold with decreasing temperature between 300 and 25 K, and decreases abruptly at temperatures below 25 K. Partial deuteration of the reaction centers alters the temperature dependence of the rate constant. Qualitative features of the temperature dependence can be understood in the context of a theory of nonadiabatic electron transfer (Sarai, 1980. Biochim. Biophys. Acta 589:71-83). We conclude that very low-energy (10-50 cm-1) processes, perhaps skeletal vibrations of the protein, are important to electron transfer. Higher-energy vibrations, possibly involving the pyrrolic N--H bonds of bacteriopheophytin, also are important in this process.