Primary charge separation within P870* in wild type and heterodimer mutants in femtosecond time domain

Primary charge separation dynamics in the reaction center (RC) of purple bacterium Rhodobacter sphaeroides and its P870 heterodimer mutants have been studied using femtosecond time-resolved spectroscopy with 20 and 40fs excitation at 870nm at 293K. Absorbance increase in the 1060-1130nm region that is presumably attributed to P(A)(δ+) cation radical molecule as a part of mixed state with a charge transfer character P*(P(A)(δ+)P(B)(δ-)) was found. This state appears at 120-180fs time delay in the wild type RC and even faster in H(L173)L and H(M202)L heterodimer mutants and precedes electron transfer (ET) to B(A) bacteriochlorophyll with absorption band at 1020nm in WT. The formation of the P(A)(δ+)B(A)(δ-) state is a result of the electron transfer from P*(P(A)(δ+)P(B)(δ-)) to the primary electron acceptor B(A) (still mixed with P*) with the apparent time delay of ~1.1ps. Next step of ET is accompanied by the 3-ps appearance of bacteriopheophytin a(-) (H(A)(-)) band at 960nm. The study of the wave packet formation upon 20-fs illumination has shown that the vibration energy of the wave packet promotes reversible overcoming of an energy barrier between two potential energy surfaces P* and P*(P(A)(δ+)B(A)(δ-)) at ~500fs. For longer excitation pulses (40fs) this promotion is absent and tunneling through an energy barrier takes about 3ps. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Evidence for delocalized anticooperative flash induced proton binding as revealed by mutants at the M266His iron ligand in bacterial reaction centers

Bacterial reaction centers (RCs) convert light energy into chemical free energy via the double reduction and protonation of the secondary quinone electron acceptor, QB, to the dihydroquinone QBH2. Two RC mutants (M266His --> Leu and M266His --> Ala) with a modified ligand of the non-heme iron have been studied by flash-induced absorbance change spectroscopy. No important changes were observed for the rate constants of the first and second electron transfers between the first quinone electron acceptor, QA, and QB. However, in the M266HL mutant a destabilization of approximately 40 meV of the free energy level of QA- was observed, at variance with the M266HA mutant. The superposition of the three-dimensional X-ray structures of the three proteins in the QA region provides no obvious explanation for the energy modification in the M266HL mutant. The shift of the midpoint redox potential of QA/QA- in M266HL caused accelerated recombination of the charges in the P+ QA- state of the RCs where the native QA was replaced by a low potential anthraquinone (AQA). As previously reported for the native RCs, in the M266HL we observed a biphasicity of the P+ AQA- --> P AQA charge recombination. Interestingly, both phases present a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependencies of the proton uptake upon QA- and QB- formations are superimposable in both mutants but very different from those of native RCs. The data measured in mutants are similar to those that we previously obtained on strains modified at various sites of the cytoplasmic region. The similarity of the response to these different mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidic residues resulting in strongly anticooperative proton binding. The unspecific disappearance of the high pH band of proton uptake observed in all these mutants appears as the natural consequence of removing any member of an interactive proton cluster. This long range interaction also accounts for the similar responses to mutations of the proton uptake pattern induced by either QA- or QB-. We surmise that the presence of an extended protonated water H-bond network providing protons to QB is responsible for these effects.     

Excitation pressure regulates the activation energy for recombination events in the photosystem II reaction centres of Chlamydomonas reinhardtii.

Using in vivo thermoluminescence, we examined the effects of growth irradiance and growth temperature on charge recombination events in photosystem II reaction centres of the model green alga Chlamydomonas reinhardtii. We report that growth at increasing irradiance at either 29 or 15 degrees C resulted in comparable downward shifts in the temperature peak maxima (T(M)) for S2QB- charge pair recombination events, with minimal changes in S2QA- recombination events. This indicates that such growth conditions decrease the activation energy required for S2QB- charge pair recombination events with no concomitant change in the activation energy for S2QA- recombination events. This resulted in a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events, which was reversible when shifting cells from low to high irradiance and back to low irradiance at 29 degrees C. We interpret these results to indicate that the redox potential of QB was modulated independently of QA, which consequently narrowed the redox potential gap between QA and QB in photosystem II reaction centres. Since a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events correlated with growth at increasing excitation pressure, we conclude that acclimation to growth under high excitation pressure narrows the redox potential gap between QA and QB in photosystem II reaction centres, enhancing the probability for reaction center quenching in C. reinhardtii. We discuss the molecular basis for the modulation of the redox state of QB, and suggest that the potential for reaction center quenching complements antenna quenching via the xanthophyll cycle in the photoprotection of C. reinhardtii from excess light.     

P+QA- and P+QB- charge recombinations in Rhodopseudomonas viridis chromatophores and in reaction centers reconstituted in phosphatidylcholine liposomes. Existence of two conformational states of the reaction centers and effects of pH and o-phenanthroline

The P+QA- and P+QB- charge recombination decay kinetics were studied in reaction centers from Rhodopseudomonas viridis reconstituted in phosphatidylcholine bilayer vesicles (proteoliposomes) and in chromatophores. P represents the primary electron donor, a dimer of bacteriochlorophyll; QA and QB are the primary and secondary stable quinone electron acceptors, respectively. In agreement with recent findings for reaction centers isolated in detergent [Sebban, P., & Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54-65] the P+QA- decay kinetics were biphasic (kfast and kslow). Arrhenius plots of the kinetics were linear, in agreement with the hypothesis of a thermally activated process (probably via P+I-; I is the first electron acceptor, a bacteriopheophytin) for the P+QA- charge recombination. Similar activation free energies (delta G) for this process were found in chromatophores and in proteoliposomes. Significant pH dependences of kfast and kslow were observed in chromtophores and in proteoliposomes. In the pH range 5.5-11, the pH titration curves of kfast and kslow were interpreted in terms of the existence of three protonable groups, situated between I- and QA-, which modulate the free energy difference between P+I- and P+QA-. In proteoliposomes, a marked effect of o-phenanthroline was observed on two of the three pKs, shifting one of them by more than 2 pH units. On the basis of recent structural data, we suggest a possible interpretation for this effect, which is much smaller in Rhodobacter sphaeroides. The decay kinetics of P+QB- were also biphasic. Marked pH dependences of the rate constants and of the relative proportions of both phases were also detected for these decays. The major conclusion of this work comes from the biphasicity of the P+QB- decay kinetics. We had suggested previously that biphasicity of the P+QA- charge recombination in Rps. viridis comes from nonequilibrium between protonation states of the reaction centers due to comparable rates of the protonation events and charge recombination. This hypothesis does not hold since the P+QB- decays occur on a time scale (tau approximately 300 ms at pH 8) much longer than protonation events. This leads to the conclusion that kfast and kslow (for both P+QA- and P+QB-) are related to conformational states of the reaction centers, existing before the flash. In addition, the fast and slow decays of P+QB- are related to those measured for P+QA-, via the calculations of the QA-QB in equilibrium QAQB- apparent equilibrium constants, K2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.     

P+HA- charge recombination reaction rate constant in Rhodobacter sphaeroides reaction centers is independent of the P/P+ midpoint potential.

The kinetics of the P+HA- (oxidized donor, reduced bacteriopheophytin acceptor) recombination reaction was measured in a series of reaction center mutants of Rhodobacter sphaeroides with altered P/P+ midpoint potentials between 410 and 765 mV. The time constant for P+HA- recombination was found to range between 14 and 26 ns and was essentially independent of P/P+ midpoint potential. Previous work has shown that the time constant for initial electron transfer in these mutants at room temperature is also only weakly dependent on the P/P+ midpoint potential, ranging from about 2.5 ps to about 50 ps. These results, taken together, imply that heterogeneity in the P/P+ midpoint potential within the reaction center population is not likely the dominant cause of the substantial kinetic complexity observed in the decay of the excited singlet state of P on the picosecond to nanosecond time scale. In addition, the pathway of P+HA- decay appears to be direct or via P+BA- rather than proceeding back through P, even in the highest-potential mutant, as is evident from the fact that the rate of P+HA- recombination is unaltered by pushing P+HA- much closer to P in energy. Finally, the midpoint potential independence of the P+HA- recombination rate constant suggests that the slow rate of P+HA- recombination arises from an inherent limitation in the maximum rate of this process rather than because it occurs in the inverted region of a classical Marcus rate vs free energy curve.     

Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization

Site-directed mutations were introduced to replace D1-His198 and D2-His197 of the D1 and D2 polypeptides, respectively, of the photosystem II (PSII) reaction center of Synechocystis PCC 6803. These residues coordinate chlorophylls P(A) and P(B) which are homologous to the special pair Bchlorophylls of the bacterial reaction centers that are coordinated respectively by histidines L-173 and M-200 (202). P(A) and P(B) together serve as the primary electron donor, P, in purple bacterial reaction centers. In PS II, the site-directed mutations at D1 His198 affect the P(+)--P-absorbance difference spectrum. The bleaching maximum in the Soret region (in WT at 433 nm) is blue-shifted by as much as 3 nm. In the D1 His198Gln mutant, a similar displacement to the blue is observed for the bleaching maximum in the Q(y) region (672.5 nm in WT at 80 K), whereas features attributed to a band shift centered at 681 nm are not altered. In the Y(Z*)--Y(Z)-difference spectrum, the band shift of a reaction center chlorophyll centered in WT at 433--434 nm is shifted by 2--3 nm to the blue in the D1-His198Gln mutant. The D1-His198Gln mutation has little effect on the optical difference spectrum, (3)P--(1)P, of the reaction center triplet formed by P(+)Pheo(-) charge recombination (bleaching at 681--684 nm), measured at 5--80 K, but becomes visible as a pronounced shoulder at 669 nm at temperatures > or =150 K. Measurements of the kinetics of oxidized donor--Q(A)(-) charge recombination and of the reduction of P(+) by redox active tyrosine, Y(Z), indicate that the reduction potential of the redox couple P(+)/P can be appreciably modulated both positively and negatively by ligand replacement at D1-198 but somewhat less so at D2-197. On the basis of these observations and others in the literature, we propose that the monomeric accessory chlorophyll, B(A), is a long-wavelength trap located at 684 nm at 5 K. B(A)* initiates primary charge separation at low temperature, a function that is increasingly shared with P(A)* in an activated process as the temperature rises. Charge separation from B(A)* would be potentially very fast and form P(A)(+)B(A)(-) and/or B(A)(+)Pheo(-) as observed in bacterial reaction centers upon direct excitation of B(A) (van Brederode, M. E., et al. (1999) Proc. Natl. Acad Sci. 96, 2054--2059). The cation, generated upon primary charge separation in PSII, is stabilized at all temperatures primarily on P(A), the absorbance spectrum of which is displaced to the blue by the mutations. In WT, the cation is proposed to be shared to a minor extent (approximately 20%) with P(B), the contribution of which can be modulated up or down by mutation. The band shift at 681 nm, observed in the P(+)-P difference spectrum, is attributed to an electrochromic effect of P(A)(+) on neighboring B(A). Because of its low-energy singlet and therefore triplet state, the reaction center triplet state is stabilized on B(A) at < or =80 K but can be shared with P(A) at >80 K in a thermally activated process.     

Temperature dependence of charge recombination from the P+QA- and P+QB- states in photosynthetic reaction centers isolated from the thermophilic bacterium Chloroflexus aurantiacus.

The temperature dependence of charge recombination from the P+QA- and from the P+QB- states produced by a flash was studied in reaction centers isolated from the photosynthetic thermophilic bacterium Chloroflexus aurantiacus. P designates the primary electron donor; QA and QB the primary and secondary quinone electron acceptors respectively. In QB-depleted reaction centers the rate constant (kAP) for P+QA- recombination was temperature independent between 0-50 degrees C (17.6 +/- 0.7 s-1 at pH 8 and pH 10). The same value was obtained in intact membranes in the presence of o-phenanthroline. Upon lowering the temperature from 250 K to 160 K, kAP increased by a factor of two and remained constant down to 80 K. The overall temperature dependence of kAP was consistent with an activationless process. Ubiquinone (UQ-3) and different types of menaquinone were used for QB reconstitution. In UQ-3 reconstituted reaction centers charge recombination was monoexponential (rate constant k = 0.18 +/- 0.03 s-1) and temperature independent between 5-40 degrees C. In contrast, in menaquinone-3- and menaquinone-4-reconstituted reaction centers P+ rereduction following a flash was markedly biphasic and temperature dependent. In menaquinone-6-reconstituted reaction centers a minor contribution from a third kinetic phase corresponding to P+QA- charge recombination was detected. Analysis of these kinetics and of the effects of the inhibitor o-phenanthroline at high temperature suggest that in detergent suspensions of menaquinone-reconstituted reaction centers a redox reaction removing electrons from the quinone acceptor complex competes with charge recombination. Instability of the semiquinone anions is more pronounced when QB is a short-chain menaquinone. From the temperature dependence of P+ decay the activation parameters for the P+QB- recombination and for the competing side oxidation of the reduced menaquinone acceptor have been derived. For both reactions the activation enthalpies and entropies change markedly with menaquinone chain length but counterbalance each other, resulting in activation free energies at ambient temperature independent of the menaquinone tail. When reaction centers are incorporated into phospholipid vesicles containing menaquinone-8 a temperature-dependent, monophasic, o-phenanthroline-sensitive recombination from the P+QB- state is observed, which is consistent with the formation of stable semiquinone anions. This result seems to indicate a proper QB functioning in the two-subunit reaction center isolated from Chlorflexus aurantiacus when the complex is inserted into a lipid bilayer.     

Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.     

Excitation relaxation in the chemically modified photosynthetic reaction center from <Emphasis Type="Italic">Rhodobacter</Emphasis><Emphasis Type="Italic">sphaeroides</Emphasis> 601

The ultrafast excitation relaxation in the sodium borohydride-treated reaction center of Rhodobacter sphaeroides 601 was investigated with selective excitation. From the femtosecond pump-probe measurement at 790 nm, the excitation relaxation demonstrates a biexponential decay with time constants of about 200 fs and 1.4 ps. By comparison with the result from sodium ascorbate-pretreated modified RS601, it could be concluded that the dynamical trace at 790 nm mainly originates from the contribution of accessory bacteriochlorophyll in the active side, and the electrochromic shift arising from the induced positive charge on the special pair primarily affects the absorption band in the red region of the accessory bacteriochlorophyll in RS601. With direct excitation of the special pair, the charge separation and subsequent electron transfer were observed in borohydride-modified RS601. The 2.8 ps component was ascribed to the charge separation and electron transfer from P* to H(A). From the dynamical traces at 790, 800 and 818 nm, the ultrafast energy relaxation from the excited accessory bacteriochlorophyll in the active side is consistent with a two-step energy transfer mechanism. This dynamical observation in modified RS601 is of significance in understanding the physical mechanism of excitation relaxation and energy transfer in the photosynthetic primary process.     

Determination of the excitation migration time in Photosystem II consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes approximately 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. R?gner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

Conformational heterogeneity of the bacteriopheophytin electron acceptor HA in reaction centers from Rhodopseudomonas viridis revealed by Fourier transform infrared spectroscopy and site-directed mutagenesis.

The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.     

Fluorescence decay kinetics of chlorophyll in photosynthetic membranes

The absorption of light by the pigments of photosynthetic organisms results in electronic excitation that provides the energy to drive the energy-storing light reactions. A small fraction of this excitation gives rise to fluorescence emission, which serves as a sensitive probe of the energetics and kinetics of the excited states. The wavelength dependence of the excitation and emission spectra can be used to characterize the nature of the absorbing and fluorescing molecules and to monitor the process of sensitization of the excitation transfer from one pigment to another. This excitation transfer process can also be followed by the progressive depolarization of the emitted radiation. Using time-resolved fluorescence rise and decay kinetics, measurements of these processes can now be characterized to as short as a few picoseconds. Typically, excitation transfer among the antenna or light harvesting pigments occurs within 100 psec, whereupon the excitation has reached a photosynthetic reaction center capable of initiating electron transport. When this trap is functional and capable of charge separation, the fluorescence intensity is quenched and only rapidly decaying kinetic components resulting from the loss of excitation in transit in the antenna pigment bed are observed. When the reaction centers are blocked or saturated by high light intensities, the photochemical quenching is relieved, the fluorescence intensity rises severalfold, and an additional slower decay component appears and eventually dominates the decay kinetics. This slower (1-2 nsec) decay results from initial charge separation followed by recombination in the blocked reaction centers and repopulation of the excited electronic state, leading to a rapid delayed fluorescence component that is the origin of variable fluorescence. Recent growth in the literature in this area is reviewed here, with an emphasis on new information obtained on excitation transfer, trapping, and communication between different portions of the photosynthetic membranes.     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.     

High yield of M-side electron transfer in mutants of Rhodobacter capsulatus reaction centers lacking the L-side bacteriopheophytin

We present studies on a series of photosynthetic reaction center (RC) mutants created in the background of the Rhodobacter capsulatus D(LL) mutant, in which the D helix of the M subunit has been substituted with that from the L subunit. Previous work on the D(LL) mutant in chromatophore preparations showed that RCs assembled without the bacteriopheophytin H(L) electron acceptor and performed no charge separation following light absorption. We have successfully isolated poly-His-tagged D(LL) RCs by using the detergent Deriphat 160-C and shown that the RCs are devoid of H(L). The excited state of the primary electron donor, P*, is found to have a lifetime of 180 +/- 20 ps and to decay exclusively (>95%) via internal conversion to the ground state, with no evidence for formation of any charge-separated intermediates. By additional mutation in the D(LL) background of two residues that affect the P/P+ oxidation potential and one that facilitates M-side electron transfer, we achieve an unprecedented 70% yield of P+ H(M)-, more than doubling the highest yield of this state achieved previously. This result underscores the importance of the relative free energies of P* and the charge-separated states in governing the rates and yields of electron transfer in bacterial RCs and provides a basis for systematically investigating M-side electron transfer without any competition from the native L-side pathway.     

Directed alteration of the D1 polypeptide of photosystem II: evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680

In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R.J., Barry, B.A., Babcock, G.T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA- occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA- occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

EPR characterization of genetically modified reaction centers of Rhodobacter capsulatus

Electron paramagnetic resonance (EPR) has been used to investigate the cation and triplet states of Rhodobacter capsulatus reaction centers (RCs) containing amino acid substitutions affecting the primary donor, monomeric bacteriochlorophylls (Bchls), and the photoactive bacteriopheophytin (Bphe). The broadened line width of the cation radical in HisM200----Leu and HisM200----Phe reaction centers, whose primary donor consists of a Bchl-Bphe heterodimer, indicates a highly asymmetric distribution of the unpaired electron over the heterodimer. A T0 polarized triplet state with reduced yield is observed in heterodimer-containing RCs. The zero field splitting parameters indicate that this triplet essentially resides on the Bchl half of the heterodimer. The cation and triplet states of reaction centers containing HisM200----Gln, HisL173----Gln, GluL104----Gln, or GluL104----Leu substitutions are similar to those observed in wild type. Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that are positioned near the central Mg2+ ions of the reaction center monomeric bacteriochlorophylls. Reaction centers containing serine substitutions at M180 and L153 or a threonine substitution at L153 have unaltered pigment compositions and are photochemically active. The cation and triplet states of HisL153----Leu reaction centers are similar to those observed in wild type. Triplet energy transfer to carotenoid is not observed at 100 K in HisM180----Arg chromatophores. These results have important implications for the structural requirements of tetrapyrrole binding and for our understanding of the mechanisms of primary electron transfer in the reaction center.     

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