Ultrafast transient absorption studies on Photosystem I reaction centers from Chlamydomonas reinhardtii. 1. A new interpretation of the energy trapping and early electron transfer steps in Photosystem I

The energy transfer and charge separation kinetics in core Photosystem I (PSI) particles of Chlamydomonas reinhardtii has been studied using ultrafast transient absorption in the femtosecond-to-nanosecond time range. Although the energy transfer processes in the antenna are found to be generally in good agreement with previous interpretations, we present evidence that the interpretation of the energy trapping and electron transfer processes in terms of both kinetics and mechanisms has to be revised substantially as compared to current interpretations in the literature. We resolved for the first time i), the transient difference spectrum for the excited reaction center state, and ii), the formation and decay of the primary radical pair and its intermediate spectrum directly from measurements on open PSI reaction centers. It is shown that the dominant energy trapping lifetime due to charge separation is only 6-9 ps, i.e., by a factor of 3 shorter than assumed so far. The spectrum of the first radical pair shows the expected strong bleaching band at 680 nm which decays again in the next electron transfer step. We show furthermore that the early electron transfer processes up to approximately 100 ps are more complex than assumed so far. Several possibilities are discussed for the intermediate redox states and their sequence which involve oxidation of P700 in the first electron transfer step, as assumed so far, or only in the second electron transfer step, which would represent a fundamental change from the presently assumed mechanism. To explain the data we favor the inclusion of an additional redox state in the electron transfer scheme. Thus we distinguish three different redox intermediates on the timescale up to 100 ps. At this level no final conclusion as to the exact mechanism and the nature of the intermediates can be drawn, however. From comparison of our data with fluorescence kinetics in the literature we also propose a reversible first charge separation step which has been excluded so far for open PSI reaction centers. For the first time an ultrafast 150-fs equilibration process, occurring among exciton states in the reaction center proper, upon direct excitation of the reaction center at 700 nm, has been resolved. Taken together the data call for a fundamental revision of the present understanding of the energy trapping and early electron transfer kinetics in the PSI reaction center. Due to the fact that it shows the fastest trapping time observed so far of any intact PSI particle, the PSI core of C. reinhardtii seems to be best suited to further characterize the electron transfer steps and mechanisms in the reaction center of PSI.     

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     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides II. Analysis of the band shifts of carotenoid and bacteriochlorophyll

An analysis was made of the changes of pigment absorption upon illumination of chromatophores of Rhodopseudomonas sphaeroides at ?35 °C, described in the preceding paper (de Grooth, B. G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 237–246). Comparison of the light-induced difference spectra in the carotenoid region obtained without additions, and in the presence of N-methylphenazonium methosulphate and ascorbate as donor-acceptor system showed that the latter spectrum was not only about 10 times larger in amplitude, but also red-shifted with respect to the first one. Together with the shape of the difference spectrum, this indicated that the spectrum obtained in the presence of a donor-acceptor system is due to an electrochromic shift of the absorption spectrum of a carotenoid by a few nm towards longer wavelength, caused by a delocalized potential across the chromatophore membrane. The results of an analysis of the kinetics of the absorbance changes near the zero points of the spectrum were in quantitative agreement with the extent of the red shift and indicated a shift of 0.25 nm for a single electron transfer per reaction center, and shifts of up to 4 nm when the electron transport is stimulated by a donor-acceptor system. For bacteriochlorophyll B-850 the shift is three times smaller.Analysis of the overall absorption spectrum showed that there are at least two pools of carotenoid. The carotenoid that shows electrochromism has absorption bands at 452, 481 and 515 nm, and comprises about one-third of the total carotenoid present; the remaining pool absorbs at about 7 nm shorter wavelength and does not show an electrochromic response to illumination. Both pools presumably consist of spheroidene; the differences in band location may be explained by the assumption that only the first pool is subjected to a local electric field which induces an electric dipole even at zero membrane potential. Similar results were obtained at room temperature and with a mutant of Rps. sphaeroides (G1C)-containing neurosporene.     

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.     

Heme-copper relationship of cytochrome oxidase in rat brain in situ

The role of copper aa3 in relation to heme aa3 of cytochrome oxidase in electron transfer and oxygen utilization is poorly understood in vitro. In an attempt to study this in situ, we have simultaneously monitored the steady state redox changes of heme aa3 and copper aa3 in an isolated perfused rat head model (skull intact, muscle removed). By means of reflectance spectrophotometry the redox reactions of heme aa3 and copper aa3 were continuously monitored using 605-625 nm and 815-920 nm wavelength pairs, respectively. The reaction kinetics of these components in response to transient perfusion interruption in energized and de-energized preparations were then examined. We found that in response to perfusion interruption, soon after full reduction, the copper signal begins to change toward oxidation despite continuation of anoxic insult and progressive reduction of heme aa3. This phenomenon disappeared by pretreatment of the preparation with 2,4-dinitrophenol. A schematic sequence of electron transport in situ is proposed which emphasizes an active role for Cua in this sequence.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.     

Electrochromic detection of a coherent component in the formation of the charge pair P(+)H(L)(-) in bacterial reaction centers

We demonstrate coupling of an intraprotein electron transfer reaction to coherent vibrational motions. The kinetics of charge separation toward the radical pair state P(+)H(L)(-) were studied in reaction centers of Rhodobacter sphaeroides at 15 K. The electrochromic shift of the bacteriochlorophyll monomers is the most prominent spectral feature associated with this charge displacement. The newly reported absolute absorption spectrum of the P(+)H(L)(-) state is discussed in terms of this shift. In wild-type reaction centers, the rise kinetics of the electrochromic shift display a small but significant 30 cm(-)(1) periodic modulation (period of approximately 1 ps). This modulation is also present in FL181Y mutant reaction centers, where overall charge separation is somewhat more rapid than in the wild-type reaction center. In contrast, in YM210L mutant reaction centers, where the charge separation is much slower, the modulation is absent. The conclusion that the motion along the reaction coordinate has a 30 cm(-)(1) coherent component is discussed in light of possible mechanisms of electron transfer.     

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.     

Recruitment of a foreign quinone into the A1 site of photosystem I. Altered kinetics of electron transfer in phylloquinone biosynthetic pathway mutants studied by time-resolved optical, EPR, and electrometric techniques

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A(1) site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700(+) and an FeS(-) cluster; 2) a 750-microseconds lifetime that represents electron donation from an FeS(-) cluster to methyl viologen; and 3) an approximately 15-microseconds lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700(+) Q(-) during the lifetime of the spin polarization and give no evidence of a significant population of P700(+) FeS(-) for t 相似文献   

Electrometrical study of electron transfer from the terminal FA/FB iron-sulfur clusters to external acceptors in photosystem I

An electrometrical technique was used to investigate electron transfer between the terminal iron-sulfur centers F(A)/F(B) and external electron acceptors in photosystem I (PS I) complexes from the cyanobacterium Synechococcus sp. PCC 6301 and from spinach. The increase of the relative contribution of the slow components of the membrane potential decay kinetics in the presence of both native (ferredoxin, flavodoxin) and artificial (methyl viologen) electron acceptors indicate the effective interaction between the terminal 14Fe-4S] cluster and acceptors. The finding that FA fails to donate electrons to flavodoxin in F(B)-less (HgCl2-treated) PS I complexes suggests that F(B) is the direct electron donor to flavodoxin. The lack of additional electrogenicity under conditions of effective electron transfer from the F(B) redox center to soluble acceptors indicates that this reaction is electrically silent.     

Biochemical and biophysical characterization of photosystem I from phytoene desaturase and zeta-carotene desaturase deletion mutants of Synechocystis Sp. PCC 6803: evidence for PsaA- and PsaB-side electron transport in cyanobacteria

In photosystem I, oxidation of reduced acceptor A(1)(-) through iron-sulfur cluster F(X) is biphasic with half-times of approximately 5-30 ns ("fast" phase) and approximately 150-300 ns ("slow" phase). Whether these biphasic kinetics reflect unidirectional electron transfer, involving only the PsaA-side phylloquinone or bi-directional electron transfer, involving both the PsaA- and PsaB-side phylloquinones, has been the source of some controversy. Brettel (Brettel, K. (1988) FEBS Lett. 239, 93-98) and Joliot and Joliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130-11136) have attributed to nearby carotenoids electrochromic band shifts, accompanying A(1) reduction, centered at approximately 450 and 500-510 nm. As a test of these assignments, we separately deleted in Synechocystis sp. PCC 6803 the genes that encode phytoene desaturase (encoded by crtP (pds)) and zeta-carotene desaturase (encoded by crtQ (zds)). The pds(-) and zds(-) strains synthesize phytoene and zeta-carotene, respectively, both of which absorb to shorter wavelength than beta-carotene. Compared with wild type, the mutant A(1)(-) (FeS) - A(1)(FeS)(-) difference spectra, measured in cells and photosystem I complexes, retain the electrochromic band shift centered at 450 nm but show a complete loss of the electrochromic band shifts centered at 500-510 nm. Thus, the latter clearly arise from beta-carotene. In the wild type, the electrochromic band shift of the slow phase (centered at 500 nm) is shifted by 6 nm to the blue compared with the fast phase (centered at 506 nm). Thus, the carotenoid pigments acting as electrochromic markers during the fast and slow phases of A(1)(-) oxidation are different, indicating the involvement of both the PsaA- and the PsaB-side phylloquinones in photosystem I electron transport.     

Evidence for slow turnover in a fraction of Photosystem II complexes in thylakoid membranes

We used two different techniques to measure the recovery time of Photosystem II following the transfer of a single electron from P-680 to Q_A in thylakoid membranes isolated from spinach. Electron transfer in Photosystem II reaction centers was probed first by spectroscopic measurements of the electrochromic shift at 518 nm due to charge separation within the reaction centers. Using two short actinic flashes separated by a variable time interval we determined the time required after the first flash for the electrochromic shift at 518 nm to recover to the full extent on the second flash. In the second technique the redox state of Q_A at variable times after a saturating flash was monitored by measurement of the fluorescence induction in the absence of an inhibitor and in the presence of ferricyanide. The objective was to determine the time required after the actinic flash for the fluorescence induction to recover to the value observed after a 60 s dark period. Measurements were done under conditions in which (1) the electron donor for Photosystem II was water and the acceptor was the endogenous plastoquinone pool, and (2) Q₄₀₀, the Fe²⁺ near Q_A, remained reduced and therefore was not a participant in the flash-induced electron-transfer reactions. The electrochromic shift at 518 nm and the fluorescence induction revealed a prominent biphasic recovery time for Photosystem II reaction centers. The majority of the Photosystem II reaction centers recovered in less than 50 ms. However, approx. one-third of the Photosystem II reaction centers required a half-time of 2–3 s to recover. Our interpretation of these data is that Photosystem II reaction centers consist of at least two distinct populations. One population, typically 68% of the total amount of Photosystem II as determined by the electrochromic shift, has a steady-state turnover rate for the electron-transfer reaction from water to the plastoquinone pool of approx. 250 e⁻ / s, sufficiently rapid to account for measured rates of steady-state electron transport. The other population, typically 32%, has a turnover rate of approx. 0.2 e⁻ / s. Since this turnover rate is over 1000-times slower than normally active Photosystem II complexes, we conclude that the slowly turning over Photosystem II complexes are inconsequential in contributing to energy transduction. The slowly turning over Photosystem II complexes are able to transfer an electron from P-680 to Q_A rapidly, but the reoxidation of Q⁻_A is slow (t1/2 = 2 s). The fluorescence induction measurements lead us to conclude that there is significant overlap between the slowly turning over fraction of Photosystem II complexes and PS II_β reaction centers. One corollary of this conclusion is that electron transfer from P-680 to Q_A in PS II_β reaction centers results in charge separation across the membrane and gives rise to an electrochromic shift.     

Spectrophotometric and voltage clamp characterization of monolayers of bacterial photosynthetic reaction centers

Bacterial photosynthetic reaction centers from Rhodopseudomonas sphaeroides have been spread on an air/aqueous interface, compressed, and transferred quantitatively to either glass or transparent, tin oxide-coated slides. These assemblies permit the concomitant measurement of both optical and electrical activities to be made on protein films under voltage-clamp conditions. Optical spectra of the monolayer-coated slides reveal characteristic reaction center absorptions. Linear dichroism spectra of the monolayers indicate that the reaction center is aligned on the air/aqueous interface with an angle of inclination which is essentially the same as it is with respect to the membrane plane in vivo. The kinetics of the light-induced absorbance changes of the reaction center in the deposited films are essentially unaltered from those in solution; however, there is some loss in the extent of photochemical activity. Measurement of the light-induced electrical transients shows capacitative charging and discharging currents, which can be readily associated with the reaction center bacteriochlorophyll dimer to ubiquinone electron transfer. The extent of the photochemical activity detected by the voltage-clamp is at best only 10–12% of that measured by optical assay. This suggests that on the air/aqueous interface, the reaction centers must be predominately oriented with opposing directions of electron transfer, having only a slight, variable tendency to align with the ubiquinone directed toward the aqueous phase. In spite of the present shortcomings, these assemblies appear to be uniquely useful to study the effect of clamped potentials on the kinetics and mechanisms of electron transfer.     

Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex

We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc1 complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (bH heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b(H) heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the bH heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.     

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.     

The localized coupling of bacterial photophosphorylation: Effect of antimycin a and N,N-dicyclohexylcarbodiimide) in chromatophores from Rhodopseudomonas sphaeroides,Ga, studied by single turnover event analysis

1. The inhibition by antimycin A of the cyclic electron transfer has been studied in chromatophores from Rhodopseudomonas sphaeroides Ga following an approach based on the analysis of the relaxation kinetics of the reaction center optical changes in pulsed light. The recovery kinetics of the bacteriochlorophyll redox state have been found to be clearly biphasic. The half-times of the fast phase (13 ms) and slow phase (about 400 ms) were not modified by antimycin in a range of concentrations from 0.1 to 9 μM. On the other hand the percentage extent of the fast phase, which reflects the rate of the cyclic electron transfer, was monotonically decreased by increasing concentrations of the inhibitor. This indicates that antimycin decreases progressively the fraction of the photosynthetic units, active in cyclic electron transfer. 2. The ATP yield per flash observed under conditions of controlled inhibition of electron flow was strongly dependent upon the amount of active redox cycles. On the other hand, the amplitude of the carotenoid band shift, which has been demonstrated unequivocally to be correlated to the ATP yield per flash in uninhibited chromatophores, was not affected by antimycin up to a 40% inhibition of electron flow. 3. The effect of a progressive limitation by DCCD in the number of active ATP synthetase complexes on flash-induced phosphorylation has been examined. The decrease in ATP yield observed over a wide range of flash frequencies is related simply to the ATPase activity and to phosphorylation in continuous light, irrespective of the value of the membrane potential, which appears to be stabilized by this inhibitor. 4. As a whole, the results obtained at low concentrations of antimycin and under conditions of partial inhibition by DCCD evidence a localized coupling between the redox reactions and phosphorylation.     

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.     

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.     

Fluorescence induction of photosynthetic bacteria

The kinetics of bacteriochlorophyll fluorescence in intact cells of the purple nonsulfur bacterium Rhodobacter sphaeroides were measured under continuous and pulsed actinic laser diode (808 nm wavelength and maximum 2 W light power) illumination on the micro- and millisecond timescale. The fluorescence induction curve was interpreted in terms of a combination of photochemical and triplet fluorescence quenchers and was demonstrated to be a reflection of redox changes and electron carrier dynamics. By adjustment of the conditions of single and multiple turnovers of the reaction center, we obtained 11 ms^–1 and 120 μs^–1 for the rate constants of cytochrome c₂³⁺ detachment and cyclic electron flow, respectively. The effects of cytochrome c₂ deletion and chemical treatments of the bacteria and the advantages of the fluorescence induction study on the operation of the electron transport chain in vivo were discussed.     

Kinetics of the electrogenic step and cytochrome b6 and f redox changes in chloroplasts: Evidence for a Q cycle

The spectroscopic measurements of the slow phase of the electrochromic effect and the redox kinetics of cytochrome b₆ and f provide strong evidence that a Q cycle operates in chloroplasts under conditions of non-cyclic electron transport. The effect of HQNO and DBMIB on the extent and kinetics of these light-induced changes places several constraints on the mechanism of quinol oxidation by the cyt. b/f—FeS complex: for each electron removed from the cyt. b/f—FeS complex by P700 an additional charge is transferred across the membrane; the cyclic pathway of electrons involved in quinol oxidation by the cyt. b/f—FeS complex includes at least one of the two b₆ cytochromes; the electrogenic step associated with quinol oxidation is subsequent to the reduction of at least one cytochrome b₆ quinol oxidation may proceed in a stepwise manner, with the first electron going to cytochrome b₆ and the second electron going to the FeS center and cytochrome f.