Spectroscopic characterization of a semi-stable, charge-separated state in Cu-substituted reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides exposed to continuous illumination in the presence of an inhibitor of the Q_A⁻ to Q_B electron transfer, a semi-stable, charge-separated state was formed with halftimes of formation and decay of several minutes. When the non-heme iron was replaced by Cu²⁺, the decay of the semi-stable, charge-separated state became much slower than in centers with bound Fe²⁺ with about the same rate constant for formation. In Cu²⁺-substituted reaction centers, the semi-stable state was associated with an EPR signal, significantly different from that observed after chemical reduction of the acceptor-side quinone or after illumination at low temperature, but similar to that of an isolated Cu²⁺ in the absence of magnetic interaction. The EPR results, obtained with Cu²⁺-substituted reaction centers, suggest that the slow kinetics of formation and decay of the charge-separated, semi-stable state is associated with a structural rearrangement of the acceptor side and the immediate environment of the metal-binding site.     

Characterization of a semi-stable,charge-separated state in reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides, subjected to continuous illumination in the presence of an inhibitor of the Q_A to Q_B electron transfer, the oxidation of P870 consisted of several kinetic phases with a fast initial reaction followed by very slow accumulation of P870⁺ with a halftime of several minutes. When the light was turned off, a phase of fast charge recombination was followed by an equally slow reduction of P870⁺. In reaction centers depleted of Q_B, where forward electron transfer from Q_A is also prevented, the slow reactions were also observed but with different kinetic properties. The kinetic traces of accumulation and decay of P870⁺ could be fitted to a simple three-state model where the initial, fast charge separation is followed by a slow reversible conversion to a long-lived, charge-stabilized state. Spectroscopic examination of the charge-separated, semi-stable state, using optical absorbance and EPR spectroscopy, suggests that the unpaired electron on the acceptor side is located in an environment significantly different from normal. The activation parameters and enthalpy and entropy changes, determined from the temperature dependence of the slow conversion reaction, suggest that this might be coupled to changes in the protein structure of the reaction centers, supporting the spectroscopic results. One model that is consistent with the present observations is that reaction centers, after the primary charge separation, undergo a slow, light-induced change in conformation affecting the acceptor side. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of copper and zinc ions on photosystem II studied by EPR spectroscopy.

The effect of Zn(2+) or Cu(2+) ions on Mn-depleted photosystem II (PS II) has been investigated using EPR spectroscopy. In Zn(2+)-treated and Cu(2+)-treated PS II, chemical reduction with sodium dithionite gives rise to a signal attributed to the plastosemiquinone, Q(A)(*)(-), the usual interaction with the non-heme iron being lost. The signal was identified by Q-band EPR spectroscopy which partially resolves the typical g-anisotropy of the semiquinone anion radical. Illumination at 200 K of the unreduced samples gives rise to a single organic free radical in Cu(2+)-treated PS II, and this is assigned to a monomeric chlorophyll cation radical, Chl a(*)(+), based on its (1)H-ENDOR spectrum. The Zn(2+)-treated PS II under the same conditions gives rise to two radical signals present in equal amounts and attributed to the Chl a(*)(+) and the Q(A)(*)(-) formed by light-induced charge separation. When the Cu(2+)-treated PS II is reduced by sodium ascorbate, at >/=77 K electron donation eliminates the donor-side radical leaving the Q(A)(*)(-) EPR signal. The data are explained as follows: (1) Cu(2+) and Zn(2+) have similar effects on PS II (although higher concentrations of Zn(2+) are required) causing the displacement of the non-heme Fe(2+). (2) In both cases chlorophyll is the electron donor at 200 K. It is proposed that the lack of a light-induced Q(A)(*)(-) signal in the unreduced Cu(2+)-treated sample is due to Cu(2+) acting as an electron acceptor from Q(A)(*)(-) at low temperature, forming the Cu(+) state and leaving the electron donor radical Chl a(*)(+) detectable by EPR. (3) The Cu(2+) in PS II is chemically reducible by ascorbate prior to illumination, and the metal can therefore no longer act as an electron acceptor; thus Q(A)(*)(-) is generated by illumination in such samples. (4) With dithionite, both the Cu(2+) and the quinone are reduced resulting in the presence of Q(A)(*)(-) in the dark. The suggested high redox potential of Cu(2+) when in the Fe(2+) site in PS II is in contrast to the situation in the bacterial reaction center where it has been shown in earlier work that the Cu(2+) is unreduced by dithionite. It cannot be ruled out however that Q(A)-Cu(2+) is formed and a magnetic interaction is responsible for the lack of the Q(A)(-) signal when no exogenous reductant is present. With this alternative possibility, the effects of reductants would be explained as the loss of Cu(2+) (due to formation of Cu(+)) leading to loss of the Cu(2+) from the Fe(2+) site due to the binding equilibrium. The quite different binding and redox behavior of the metal in the iron site in PS II compared to that of the bacterial reaction center is presumably a further reflection of the differences in the coordination of the iron in the two systems.     

Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides.

The photoreductive trapping of the transient, intermediate acceptor, I-, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I- was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 400, 587--599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301--304). The optical changes of I- showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I- were observed: one was a narrow singlet at g = 2.0035, deltaH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I- on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI-Q-Fe2+ and the singlet EPR signal is due to DI-Q2-Fe2+. : formula: (see text), where D is the primary donor (BChl)2+. The above model was supported by the following observations: (1) During the first illumination, sigmoidal kinetics of the formation of I- was observed. This is a direct consequence of the three-sequential reactions. (2) During the second and subsequent illuminations first-order (exponential) kinetics were observed for the formation of I-. This is due to the dark decay, k4, to the state DIQ2-Fe2+ formed after the first illumination. (3) Removal of the quinone resulted in first-order kinetics. In this case, only the first step, k1, is operative. (4) The observation of the doublet signal in reaction centers containing menaquinone but not ubiquinone is explained by the longer lifetime of the doublet species I-(Q-Fe2%) in reaction centers containing menaquinone. The value of tau2 was determined from kinetic measurements to be 0.01 s for ubiquinone and 4 s for menaquinone (T = 20 degrees C). The temperature and pH dependence of the dark electron transfer reaction I-(Q-Fe2+) yields I(Q2-Fe2+) was studied in detail. The activation energy for this process was found to be 0.42 eV for reaction centers containing ubiquinone and 0.67 eV for reaction centers with menaquinone. The activation energy and the doublet splitting were used to calculate the rate of electron transfer from I- to MQ-Fe2+ using Hopfield's theory for thermally activated electron tunneling. The calculated rate agrees well with the experimentally determined rate which provides support for electron tunneling as the mechanism for electron transfer in this reaction. Using the EPR doublet splitting and the activation energy for electron transfer, the tunneling matrix element was calculated to be 10(-3) eV. From this value the distance between I- and MQ- was estimated to be 7.5--10 A.     

Long-lived charge-separated states in bacterial reaction centers isolated from Rhodobacter sphaeroides

We studied the accumulation of long-lived charge-separated states in reaction centers isolated from Rhodobacter sphaeroides, using continuous illumination, or trains of single-turnover flashes. We found that under both conditions a long-lived state was produced with a quantum yield of about 1%. This long-lived species resembles the normal P(+)Q(-) state in all respects, but has a lifetime of several minutes. Under continuous illumination the long-lived state can be accumulated, leading to close to full conversion of the reaction centers into this state. The lifetime of this accumulated state varies from a few minutes up to more than 20 min, and depends on the illumination history. Surprisingly, the lifetime and quantum yield do not depend on the presence of the secondary quinone, Q(B). Under oxygen-free conditions the accumulation was reversible, no changes in the normal recombination times were observed due to the intense illumination. The long-lived state is responsible for most of the dark adaptation and hysteresis effects observed in room temperature experiments. A simple method for quinone extraction and reconstitution was developed.     

Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR

The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

Low-temperature electron transfer in photosystem II: a tyrosyl radical and semiquinone charge pair

The states induced by illumination at 7 K in the oxygen-evolving enzyme (PSII) from Thermosynechococcus elongatus were studied by EPR. In the S(0) and S(1) redox states, two g approximately 2 EPR signals, a split signal and a g = 2.03 signal, respectively, were generated by illumination with visible light. These signals were comparable to those already reported in plant PSII in terms of their g value, shape, and stability at low temperatures. We report that the formation and decay of these signals correlate with EPR signals from the semiquinone of the first quinone electron acceptor, Q(A)(-). The light-induced EPR signals from oxidized side-path electron donors (Cyt b(559), Car, and Chl(Z)) were also measured, and from these and the signals from Q(A)(-), estimates were made of the proportion of centers involved in the formation of the g approximately 2 signals (approximately 50% in S(0) and 40% in S(1)). Comparisons with the signals generated in plant PSII indicated approximately similar yields for the S(0) split signal. A single laser flash at 7 K induced more than 75% of the maximum split and g = 2.03 EPR signal observed by continuous illumination, with no detectable oxidation of side-path donors. The matching electron acceptor side reactions, the high quantum yield, and the relatively large proportion of centers involved support earlier suggestions that the state being monitored is Tyr(Z)(*)Q(A)(-), with the g approximately 2 EPR signals arising from Tyr(Z)(*) interacting magnetically with the Mn complex. The current picture of the photochemical reactions occurring in PSII at low temperatures is reassessed.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr(Z)) and side-path electron donors (Car/Chl(Z)/Cyt(b)(559)) can be oxidized by P(680)(+) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z) EPR signal were independent of the treatment of K(3)Fe(CN)(6), whereas formation and decay of the Car(+)/Chl(Z)(+) EPR signal correlated with the reduction and recovery of the Fe(3+) EPR signal of the non-heme iron in K(3)Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe(3+) reduction, the oxidation of non-heme iron by K(3)Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe(3+) EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr(Z) oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K(3)Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe(3+) state is probably not the intermediate species for the turnover of quinone reduction.     

Molecular interference of Cd(2+) with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+). In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites. The number of sites for Cd(2+) binding and the possible nature of these sites are discussed.     

Cu2+ site in photosynthetic bacterial reaction centers from Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis

The interaction of metal ions with isolated photosynthetic reaction centers (RCs) from the purple bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis has been investigated with transient optical and magnetic resonance techniques. In RCs from all species, the electrochromic response of the bacteriopheophytin cofactors associated with Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer is slowed in the presence of Cu(2+). This slowing is similar to the metal ion effect observed for RCs from Rb. sphaeroides where Zn(2+) was bound to a specific site on the surface of the RC [Utschig et al. (1998) Biochemistry 37, 8278]. The coordination environments of the Cu(2+) sites were probed with electron paramagnetic resonance (EPR) spectroscopy, providing the first direct spectroscopic evidence for the existence of a second metal site in RCs from Rb. capsulatus and Rps. viridis. In the dark, RCs with Cu(2+) bound to the surface exhibit axially symmetric EPR spectra. Electron spin echo envelope modulation (ESEEM) spectral results indicate multiple weakly hyperfine coupled (14)N nuclei in close proximity to Cu(2+). These ESEEM spectra resemble those observed for Cu(2+) RCs from Rb. sphaeroides [Utschig et al. (2000) Biochemistry 39, 2961] and indicate that two or more histidines ligate the Cu(2+) at the surface site in each RC. Thus, RCs from Rb. sphaeroides, Rb. capsulatus, and Rps. viridis each have a structurally analogous Cu(2+) binding site that is involved in modulating the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer process. Inspection of the Rps. viridis crystal structure reveals four potential histidine ligands from three different subunits (M16, H178, H72, and L211) located beneath the Q(B) binding pocket. The location of these histidines is surprisingly similar to the grouping of four histidine residues (H68, H126, H128, and L211) observed in the Rb. sphaeroides RC crystal structure. Further elucidation of these Cu(2+) sites will provide a means to investigate localized proton entry into the RCs of Rb. capsulatus and Rps. viridis as well as locate a site of protein motions coupled with electron transfer.     

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q(B) site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q(B) sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q(A) to Q(B) electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q(B) site inhibitors, DCMU and atrazine. In the sensitive centers, the S(2)Q(A)(-) state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S(2)Q(A)(-) state than the S(1)Q(A) state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S(2)-multiline electron paramagnetic resonance (EPR) signal and the 'split' EPR signal, originating from the S(2)Y(Z) state showed no significant changes upon binding of phenolic inhibitors at the Q(B) site. We thus propose a working model where Q(A) redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q(B) niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q(B) site.     

Semiquinone-iron complex of photosystem II: EPR signals assigned to the low-field edge of the ground state doublet of QA•-Fe2+ and QB•-Fe2+

The quinone-iron complex of the electron acceptor complex of Photosystem II was studied by EPR spectroscopy in Thermosynechococcus elongatus. New g ～ 2 features belonging to the EPR signal of the semiquinone forms of the primary and secondary quinone, i.e., Q(A)(?-)Fe(2+) and Q(B)(?-)Fe(2+), respectively, are reported. In previous studies, these signals were missed because they were obscured by the EPR signal arising from the stable tyrosyl radical, TyrD(?). When the TyrD(?) signal was removed, either by chemical reduction or by the use of a mutant lacking TyrD, the new signals dominated the spectrum. For Q(A)(?-)Fe(2+), the signal was formed by illumination at 77 K or by sodium dithionite reduction in the dark. For Q(B)(?-)Fe(2+), the signal showed the characteristic period-of-two variations in its intensity when generated by a series of laser flashes. The new features showed relaxation characteristics comparable to those of the well-known features of the semiquinone-iron complexes and showed a temperature dependence consistent with an assignment to the low-field edge of the ground state doublet of the spin system. Spectral simulations are consistent with this assignment and with the current model of the spin system. The signal was also present in Q(B)(?-)Fe(2+) in plant Photosystem II, but in plants, the signal was not detected in the Q(A)(?-)Fe(2+) state.     

Stability of the S₃and S₂state intermediates in photosystem II directly probed by EPR spectroscopy

The stability of the S(3) and S(2) states of the oxygen evolving complex in photosystem II (PSII) was directly probed by EPR spectroscopy in PSII membrane preparations from spinach in the presence of the exogenous electron acceptor PpBQ at 1, 10, and 20 °C. The decay of the S(3) state was followed in samples exposed to two flashes by measuring the split S(3) EPR signal induced by near-infrared illumination at 5 K. The decay of the S(2) state was followed in samples exposed to one flash by measuring the S(2) state multiline EPR signal. During the decay of the S(3) state, the S(2) state multiline EPR signal first increased and then decreased in amplitude. This shows that the decay of the S(3) state to the S(1) state occurs via the S(2) state. The decay of the S(3) state was biexponential with a fast kinetic phase with a few seconds decay half-time. This occurred in 10-20% of the PSII centers. The slow kinetic phase ranged from a decay half-time of 700 s (at 1 °C) to ~100 s (at 20 °C) in the remaining 80-90% of the centers. The decay of the S(2) state was also biphasic and showed quite similar kinetics to the decay of the S(3) state. Our experiments show that the auxiliary electron donor Y(D) was oxidized during the entire experiment. Thus, the reduced form of Y(D) does not participate to the fast decay of the S(2) and S(3) states we describe here. Instead, we suggest that the decay of the S(3) and S(2) states reflects electron transfer from the acceptor side of PSII to the donor side of PSII starting in the corresponding S state. It is proposed that this exists in equilibrium with Y(Z) according to S(3)Y(Z) ? S(2)Y(Z)(?) in the case of the S(3) state decay and S(2)Y(Z) ? S(1)Y(Z)(?) in the case of the S(2) state decay. Two kinetic models are discussed, both developed with the assumption that the slow decay of the S(3) and S(2) states occurs in PSII centers where Y(Z) is also a fast donor to P(680)(+) working in the nanosecond time regime and that the fast decay of the S(3) and S(2) states occurs in centers where Y(Z) reduces P(680)(+) with slower microsecond kinetics. Our measurements also demonstrate that the split S(3) EPR signal can be used as a direct probe to the S(3) state and that it can provide important information about the redox properties of the S(3) state.     

Inhibition of tyrosine Z photooxidation after formation of the S3 state in Ca(2+)-depleted and Cl(-)-depleted photosystem II.

Ca2+ and Cl- are obligatory cofactors in photosystem II (PS-II), the oxygen-evolving enzyme of plants. The sites of inhibition in both Ca(2+)- and Cl(-)-depleted PS-II were compared using EPR and flash absorption spectroscopies to follow the extent of the photooxidation of the redox-active tyrosine (TyrZ) and of the primary electron donor chlorophyll (P680) and their subsequent reduction in the dark. The inhibition occurred after formation of the S3 state in Ca(2+)-depleted PS-II. In Cl(-)-depleted photosystem II, the inhibition occurred after formation of the S3 state in about half of the centers and probably after S2TyrZ+ formation in the remaining centers. After the S3 state was formed in Ca(2+)- and Cl(-)-depleted photosystem II, electron transfer from TyrZ to P680 was inhibited. This inhibition is discussed in terms of electrostatic constraints resulting from S3 formation in the absence of Ca2+ and Cl-.     

Localization of light-induced structural changes in bacterial photosynthetic reaction centers

Light-induced structural changes in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated using two approaches. Cu²⁺ was used as a paramagnetic structural probe. The EPR spectrum of Cu²⁺ incorporated into the metal-depleted reaction centers was affected by 1,10-phenanthroline, an electron transfer inhibitor substituting Q_B, which suggests a localization of Cu²⁺ in a vicinity of the Q _B ^– site. However, the spectrum was not influenced by low temperature (77 K) illumination of the sample which suggests that the copper ion position is not exactly the same as that of the iron ion. Freezing the reaction centers under illumination in the presence of potassium ferricyanide and 1,10-phenanthroline caused a change in the shape of the Cu²⁺ EPR spectrum in comparison to that of a sample frozen in darkness. These data indicate a change of the Cu²⁺ ligand symmetry owing to light-induced structural changes which are probably located near the acceptor side of the reaction center. Partial trypsinolysis of reaction centers was also used to locate the structural changes. Trypsin treatment in the dark and under illumination resulted in different peptide patterns as detected by gel electrophoresis and reverse-phase high-performance liquid chromatography. Partial amino-acid sequence analysis of a number of peptides, characteristic of either light- or dark-treated reaction centers, showed that they originated from the acceptor sides of the H and M subunits. The occurrence of light-induced structural differences in the H-subunit is consistent with the suggestion that it may be involved in regulating electron transfer in this part of the reaction center.     

Delayed fluorescence from the photosynthetic reaction center measured by electronic gating of the photomultiplier

The decay of the delayed fluorescence (920 nm) of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides R26 in the P(+)Q(A)(-) charge-separated state (P and Q(A) are the primary donor and quinone, respectively) has been monitored in a wide (100 ns to 100 ms) time range. The photomultiplier (Hamamatsu R3310-03) was protected from the intense prompt fluorescence by application of gating potential pulses (-280 V) to the first, third, and fifth dynodes during the laser pulse. The gain of the photomultiplier dropped transiently by a factor of 1 x 10(6). The delayed fluorescence showed a smooth but nonexponential decay from 100 ns to 1 ms that was explained by the relaxation of the average free energy between P* and P(+)Q(A)(-) changing from -580 to -910 meV. This relaxation is due to the slow protein response to charge separation and can be described by a Kohlrausch relaxation function with time constant of 65 micros and a stretching exponent of alpha = 0.45.     

The effect of temperature on the formation and decay of the multiline EPR signal species associated with photosynthetic oxygen evolution

We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S₂ state gives rise to this multiline signal; we find that the S₁ state can be fully advanced to the S₂ state at temperatures as low as 160 K. The S₂ state is capable of further advancement at temperatures above about 210 K, but not below that temperature.     

Light-induced voltage changes associated with electron and proton transfer in photosystem II core complexes reconstituted in phospholipid monolayers.

We have measured light-induced voltage changes (electrogenic events) in photosystem II (PSII) core complexes oriented in phospholipid monolayers. These events are compared to those measured in the functionally and structurally closely related reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides. In both systems we observed a rapid (< 100 ns) light-induced increase in voltage associated with charge separation. In PSII reaction centers it was followed by a decrease (decay) of approximately 14% of the charge-separation voltage and a time constant of approximately 500 microseconds. In bacterial reaction centers this decay was approximately 9% of the charge-separation voltage, and the time constant was approximately 200 microseconds. The decay was presumably associated with a structural change. In bacterial reaction centers, in the presence of excess water-soluble cytochrome c2+, it was followed by a slower increase of approximately 30% of the charge-separation voltage, associated with electron transfer from the cytochrome to the oxidized donor, P+. In PSII reaction centers, after the decay the voltage remained on the same level for > or = 0.5 s. In PSII reaction centers the electron transfer Q-AQB-->QA Q-B contributed with an electrogenicity of < or = 5% of that of the charge separation. In bacterial reaction centers this electrogenicity was < or = 2% of the charge-separation electrogenicity. Proton transfer to Q2-B in PSII reaction centers contributed with approximately 5% of the charge-separation voltage, which is approximately a factor of three smaller than that observed in bacterial reaction centers.     

Modulation of primary radical pair kinetics and energetics in photosystem II by the redox state of the quinone electron acceptor Q(A)

Time-resolved photovoltage measurements on destacked photosystem II membranes from spinach with the primary quinone electron acceptor Q(A) either singly or doubly reduced have been performed to monitor the time evolution of the primary radical pair P680(+)Pheo(-). The maximum transient concentration of the primary radical pair is about five times larger and its decay is about seven times slower with doubly reduced compared with singly reduced Q(A). The possible biological significance of these differences is discussed. On the basis of a simple reversible reaction scheme, the measured apparent rate constants and relative amplitudes allow determination of sets of molecular rate constants and energetic parameters for primary reactions in the reaction centers with doubly reduced Q(A) as well as with oxidized or singly reduced Q(A). The standard free energy difference DeltaG degrees between the charge-separated state P680(+)Pheo(-) and the equilibrated excited state (Chl(N)P680)* was found to be similar when Q(A) was oxidized or doubly reduced before the flash (approximately -50 meV). In contrast, single reduction of Q(A) led to a large change in DeltaG degrees (approximately +40 meV), demonstrating the importance of electrostatic interaction between the charge on Q(A) and the primary radical pair, and providing direct evidence that the doubly reduced Q(A) is an electrically neutral species, i.e., is doubly protonated. A comparison of the molecular rate constants shows that the rate of charge recombination is much more sensitive to the change in DeltaG degrees than the rate of primary charge separation.