Reaction center triplet states in photosystem I and photosystem II

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.     

Recruitment of a foreign quinone into the A1 site of photosystem I. Consecutive forward electron transfer from A0 TO A1 to FX with anthraquinone in the A1 site as studied by transient EPR

In photosystem I (PS I), phylloquinone (PhQ) acts as a low potential electron acceptor during light-induced electron transfer (ET). The origin of the very low midpoint potential of the quinone is investigated by introducing anthraquinone (AQ) into PS I in the presence and absence of the iron-sulfur clusters. Solvent extraction and reincubation is used to obtain PS I particles containing AQ and the iron-sulfur clusters, whereas incubation of the menB rubA double mutant yields PS I with AQ in the PhQ site but no iron-sulfur clusters. Transient electron paramagnetic resonance spectroscopy is used to investigate the orientation of AQ in the binding site and the ET kinetics. The low temperature spectra suggest that the orientation of AQ in all samples is the same as that of PhQ in native PS I. In PS I containing the iron sulfur clusters, (i) the rate of forward electron transfer from the AQ*- to F(X) is found to be faster than from PhQ*- to F(X), and (ii) the spin polarization patterns provide indirect evidence that the preceding ET step from A0*- to quinone is slower than in the native system. The changes in the kinetics are in accordance with the more negative reduction midpoint potential of AQ. Moreover, a comparison of the spectra in the presence and absence of the iron-sulfur clusters suggests that the midpoint potential of AQ is more negative in the presence of F(X). The electron transfer from the AQ- to F(X) is found to be thermally activated with a lower apparent activation energy than for PhQ in native PS I. The spin polarization patterns show that the triplet character in the initial state of P700)*+AQ*- increases with temperature. This behavior is rationalized in terms of a model involving a distribution of lifetimes/redox potentials for A0 and related competition between charge recombination and forward electron transfer from the radical pair P700*+A0*-.     

Functional role of vitamin K in photosystem I of the cyanobacterium Synechocystis 6803

The function of vitamin K1 in the primary electron-transfer processes of photosystem I (PS I) was investigated in the cyanobacterium Synechocystis 6803. A preparation of purified PS I was found to contain two vitamin K1's per reaction center. One vitamin K1 was removed by extraction with hexane, and further extraction using hexane including 0.3% methanol resulted in a preparation devoid of vitamin K1. The hexane-extracted PS I was functional in the photoreduction of NADP+, but the PS I after extraction using hexane-methanol was totally inactive. Activity was restored by using exogenous vitamin K1 plus the hexane extract. Vitamin K3 would not substitute. The room temperature recombination kinetics of the PS I extracted with hexane were not significantly modified. However, following the removal of both vitamin K1's, the 20-ms recombination between P-700+ and P-430- was replaced by a dominant relaxation (t 1/2 = 30 ns) due to recombination of the primary biradical P-700+ A0- and a slower component originating from the P-700 triplet. This kinetic behavior was consistent with an interruption of forward electron transfer to the acceptor A1. Addition of either vitamin K1 or vitamin K3 to such preparations resulted in restoration of the slow kinetic phase (greater than 2 ms), indicating significant competition by the two exogenous quinones for electron transfer from A0-. In the case of vitamin K3, this change in the kinetics induced by vitamin K1, suggesting successful reconstitution of the acceptor site A1. These data support the hypothesis that acceptor A1 is vitamin K1 and is a component of the electron-transfer pathway for NADP+ reduction.     

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

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

The balance between primary forward and back reactions in bacterial photosynthesis.

The temperature dependence of the bacteriochlorophyll fluorescence and reaction center triplet yield in while cells of Rhodopseudomonas sphaeroides strain 2.4.1 and of the magnetic field-induced fluorescence increase are calculated, taking into account rate constants of losses in the antenna system and of charge separation and recombination in the reaction center. Triplet and singlet yield after recombination in the reaction center are described by the radical pair mechanism. Good fits of the theoretically calculated temperature dependence with published experimental results could be obtained, assuming that ks, the rate constant for recombination of the charges on the primary donor P+ and the reduced intermediate acceptor I- to the lowest excited singlet state P*I of the reaction center bacteriochlorophyll, is temperature-dependent via the Boltzmann factor Kso exp(-delta E/kT), where delta E is the energy difference between P*I and P+I- and kso is the frequency factor. kg and/or kt, the rate constants for recombination to the singlet ground and triplet states, respectively, were assumed to be temperature-independent, or temperature-dependent via their exothermicity factors ki = CiT-1/2 exp(-Ei/kT) with i = g, t. Depending on the particular choice for the temperature dependence of kg and kt, best fits were obtained for delta E = 45-75 meV and recombination rate constants at 300 K of ks = 0.4-0.8 ns-1, kg = 0.08-0.12 ns-1, and kt = 0.3-0.5 ns-1. The model predicts a lifetime of the radical pair P+I- that is somewhat larger than that of delayed fluorescence; a magnetic field increases both.     

Evidence for asymmetric electron transfer in cyanobacterial photosystem I: analysis of a methionine-to-leucine mutation of the ligand to the primary electron acceptor A0

The X-ray crystal structure of photosystem I (PS I) depicts six chlorophyll a molecules (in three pairs), two phylloquinones, and a [4Fe-4S] cluster arranged in two pseudo C2-symmetric branches that diverge at the P700 special pair and reconverge at the interpolypeptide FX cluster. At present, there is agreement that light-induced electron transfer proceeds via the PsaA branch, but there is conflicting evidence whether, and to what extent, the PsaB branch is active. This problem is addressed in cyanobacterial PS I by changing Met688(PsaA) and Met668(PsaB), which provide the axial ligands to the Mg2+ of the eC-A3 and eC-B3-chlorophylls, to Leu. The premise of the experiment is that alteration or removal of the ligand should alter the midpoint potential of the A0-/A0 redox pair and thereby result in a change in the forward electron-transfer kinetics from A0- to A1. In comparison with the wild type, the PsaA-branch mutant shows: (i) slower growth rates, higher light sensitivity, and reduced amounts of PS I; (ii) a reduced yield of electron transfer from P700 to the FA/FB iron-sulfur clusters at room temperature; (iii) an increased formation of the 3P700 triplet state due to P700(+)A0- recombination; and (iv) a change in the intensity and shape of the polarization patterns of the consecutive radical pair states P700(+)A1- and P700(+)FX-. The latter changes are temperature dependent and most pronounced at 298 K. These results are interpreted as being due to disorder in the A0 binding site, which leads to a distribution of lifetimes for A0- in the PsaA branch of cofactors. This allows a greater degree of singlet-triplet mixing during the lifetime of the radical pair P700(+)A0-, which changes the polarization patterns of P700(+)A1- and P700(+)FX-. The lower quantum yield of electron transfer is also the likely cause of the physiological changes in this mutant. In contrast, the PsaB-branch mutant showed only minor changes in its physiological and spectroscopic properties. Because the environments of eC-A3 and eC-B3 are nearly identical, these results provide evidence for asymmetric electron-transfer activity primarily along the PsaA branch in cyanobacterial PS I.     

Photosystem I photochemistry: A new kinetic phase at low temperature

A new phase of charge recombination between the oxidized primary electron donor of photosystem I (P700⁺) and a reduced acceptor has been detected by flash absorption spectroscopy in PS I particles at low temperature. It occurs under highly reducing conditions (the secondary electron acceptors F_A and F_B and one or possibly two ‘more primary’ acceptors being prereduced) with a t_1/2 of about 20 μs between 10 and 80 K.     

Singlet oxygen production in photosystem II and related protection mechanism

High-light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II (PSII) and causes photo-oxidative stress. In the PSII reaction centre, singlet oxygen is generated by the interaction of molecular oxygen with the excited triplet state of chlorophyll (Chl). The triplet Chl is formed via charge recombination of the light-induced charge pair. Changes in the midpoint potential of the primary electron donor P(680) of the primary acceptor pheophytin or of the quinone acceptor Q(A), modulate the pathway of charge recombination in PSII and influence the yield of singlet oxygen formation. The involvement of singlet oxygen in the process of photoinhibition is discussed. Singlet oxygen is efficiently quenched by beta-carotene, tocopherol or plastoquinone. If not quenched, it can trigger the up-regulation of genes, which are involved in the molecular defence response of photosynthetic organisms against photo-oxidative stress.     

Nanosecond electron transfer kinetics in photosystem I following substitution of quinones for vitamin K1 as studied by time resolved EPR.

Room temperature transient EPR spectra of photosystem I (PS I) particles from Synechocystis 6803 are presented. Native PS I samples and preparations depleted in the A1-acceptor site by solvent extraction and then reconstituted with the quinones (Q) vitamin K1 (VK1), duroquinone (DQ and DQd12) and naphthoquinone (NQ) have been studied. Sequential electron transfer to P700+A1- (FeS) and P700+A1 (FeS)- is recovered only with VK1. With DQ and NQ electron transfer is restored to form the radical pair P700+Q- as specified by a characteristic electron spin polarization (ESP)-pattern, but further electron transfer is either slowed down or blocked. A qualitative analysis of the K-band spectrum suggests that the orientation of reconstituted NQ in PS I is different from the native acceptor A1 = VK1.     

Absorption studies of Photosystem I photochemistry in the absence of vitamin K-1

Flash-induced absorption changes were studied on different timescales (nanosecond to millisecond) and at different temperatures (10 to 278 K) in highly enriched spinach PS I particles lacking vitamin K-1 and in which the electron transfer from the primary acceptor to the secondary acceptors was blocked. At all temperatures, the initial absorption change at 820 nm was followed by a fast decay (t_1/2 ≈ 47 ns at 278 K and ≈ 82 ns at 10 K) which is attributed to the decay of the primary radical pair (P-700⁺-A⁻₀). A slower phase of absorption decay is attributed to the P-700 triplet state, which was formed as a result of the biradical recombination, with a yield of about 30% at 278 K and about 75% at 10 K. Under air, the ³P-700 state decayed with a t_1/2 of about 50 μs at 278 K, whereas in the absence of oxygen it decayed with t_1/2 ≈ 560 μs. At 278 K, this yield was shown to depend on the presence of a magnetic field, with a maximum around 60 G. The ³P-700 decay halftime was nearly independent of temperature in the absence of oxygen (t_1/2 ≈ 1 ms at 10 K). The implications for the mechanisms involved in this decay are discussed. Addition of vitamin K-1 to these particles resulted in a decrease in the amplitude of the fast submicrosecond decay and a concomitant increase in the amplitude of a slow phase, indicating an efficient transfer from A⁻₀ to vitamin K-1. However, most functional properties of the acceptor A₁ were not reconstituted under these conditions.     

A light-induced spin-polarized triplet detected by EPR in photosystem II reaction centers

A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680+, and the reduced intermediate pheophytin, I-, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3-5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor Q(A)

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron--quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D|=0.0286 cm(-1), |E|=0.0044 cm(-1) was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T=100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron--quinone primary acceptor complex.     

Assembly of photosystem I. II. Rubredoxin is required for the in vivo assembly of F(X) in Synechococcus sp. PCC 7002 as shown by optical and EPR spectroscopy

The rubA gene was insertionally inactivated in Synechococcus sp. PCC 7002, and the properties of photosystem I complexes were characterized spectroscopically. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, F(X), F(A), and F(B), are missing in whole cells, thylakoids, and photosystem (PS) I complexes of the rubA mutant. The flash-induced decay kinetics of both P700(+) in the visible and A(1)- in the near-UV show that charge recombination occurs between P700(+) and A(1)- in both thylakoids and PS I complexes. The spin-polarized EPR signal at room temperature from PS I complexes also indicates that forward electron transfer does not occur beyond A(1). In agreement, the spin-polarized X-band EPR spectrum of P700(+) A(1)- at low temperature shows that an electron cycle between A(1)- and P700(+) occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a relatively large fraction of the electrons promoted are irreversibly transferred to [F(A)/F(B)]. The electron spin polarization pattern shows that the orientation of phylloquinone in the PS I complexes is identical to that of the wild type, and out-of-phase, spin-echo modulation spectroscopy shows the same P700(+) to A(1)- center-to-center distance in photosystem I complexes of wild type and the rubA mutant. In contrast to the loss of F(X), F(B), and F(A), the Rieske iron-sulfur protein and the non-heme iron in photosystem II are intact. It is proposed that rubredoxin is specifically required for the assembly of the F(X) iron-sulfur cluster but that F(X) is not required for the biosynthesis of trimeric P700-A(1) cores. Since the PsaC protein requires the presence of F(X) for binding, the absence of F(A) and F(B) may be an indirect result of the absence of F(X).     

Location of the iron-sulfur clusters FA and FB in photosystem I: an electron paramagnetic resonance study of spin relaxation enhancement of P700+.

Photosystem I (PS I) mediates electron-transfer from plastocyanin to ferredoxin via a photochemically active chlorophyll dimer (P700), a monomeric chlorophyll electron acceptor (A0), a phylloquinone (A1), and three [4Fe-4S] clusters (FX/A/B). The sequence of electron-transfer events between the iron-sulfur cluster, FX, and ferredoxin is presently unclear. Owing to the presence of a 2-fold symmetry in the PsaC protein to which the iron-sulfur clusters F(A) and F(B) are bound, the spatial arrangement of these cofactors with respect to the C2-axis of symmetry in PS I is uncertain as well. An unequivocal determination of the spatial arrangement of the iron-sulfur clusters FA and FB within the protein is necessary to unravel the complete electron-transport chain in PS I. In the present study, we generate EPR signals from charge-separated spin pairs (P700+-FredX/A/B) in PS I and characterize them by progressive microwave power saturation measurements to determine the arrangement of the iron-sulfur clusters FX/A/B relative to P700. The microwave power at half saturation (P1/2) of P700+ is greater when both FA and FB are reduced in untreated PS I than when only FA is reduced in mercury-treated PS I. The experimental P1/2 values are compared to values calculated by using P700-FA/B crystallographic distances and assuming that either FA or FB is closer to P700+. On the basis of this comparison of experimental and theoretical values of spin relaxation enhancement effects on P700+ in P700+ [4Fe-4S]- charge-separated pairs, we find that iron-sulfur cluster FA is in closer proximity to P700 than the FB cluster.     

Kinetics of charge separation and A0- --> A1 electron transfer in photosystem I reaction centers

The charge separation P700*A(0) --> P700(+)A(0)(-) and the subsequent electron transfer from the primary to secondary electron acceptor have been studied by subtracting absorption difference profiles for cyanobacterial photosystem I (PS I) complexes with open and closed reaction centers. Samples were excited at 660 nm, which lies toward the blue edge of the core antenna absorption spectrum. The resulting PS I kinetics were analyzed in terms of the relevant P700, P700(+), A(0), and A(0)(-) absorption spectra. In our kinetic model, the radical pair P700(+)A(0)(-) forms with 1.3 ps rise kinetics after creation of electronically excited P700*. The formation of A(1)(-) via electron transfer from A(0)(-) requires approximately 13 ps. The kinetics of the latter step are appreciably faster than previously estimated by other groups (20--50 ps).     

Full replacement of the function of the secondary electron acceptor phylloquinone(= vitamin K1) by non-quinone carbonyl compounds in green plant photosystem I photosynthetic reaction centers

One-carbonyl quinonoid compounds, fluorenone (fluoren-9-one), anthrone, and their derivatives are introduced into spinach photosystem (PS) I reaction centers in place of the intrinsic secondary electron acceptor phylloquinone (= vitamin K1). Anthrone and 2-nitrofluorenone fully mediated the electron-transfer reaction between the reduced primary electron acceptor chlorophyll A0- and the tertiary electron acceptor iron-sulfur centers. It is concluded that the PS I phylloquinone-binding site has a structure that enables various compounds with different molecular structures to function as the secondary acceptor and that the reactions of incorporated compounds are mainly determined by their redox properties rather than by their molecular structure. Carbonyl groups increase the binding affinity of the quinone/quinonoid compounds but do not seem to be essential to their function. The quinonoid compounds as well as quinones incorporated into the PS I phylloquinone-binding sites are estimated to function at redox potentials more negative than in organic solvents.     

Analysis of the spin-polarized electron spin echo of the [P700+ A1-] radical pair of photosystem I indicates that both reaction center subunits are competent in electron transfer in cyanobacteria, green algae, and higher plants

The decay of the light-induced spin-correlated radical pair [P700+ A1-] and the associated electron spin echo envelope modulation (ESEEM) have been studied in either thylakoid membranes, cellular membranes, or purified photosystem I prepared from the wild-type strains of Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii, and Spinaceae oleracea. The decay of the spin-correlated radical pair is described in the wild-type membrane by two exponential components with lifetimes of 2-4 and 16-25 micros. The proportions of the two components can be altered by preillumination of the membranes in the presence of reductant at temperatures lower than 220 K, which leads to the complete reduction of the iron-sulfur electron acceptors F(A), F(B), and F(X) and partial photoaccumulation of the reduced quinone electron acceptor A1A-. The "out-of-phase" (OOP) ESEEM attributed to the [P700+ A1-] radical pair has been investigated in the three species as a function of the preillumination treatment. Values of the dipolar (D) and the exchange (J) interactions were extracted by time-domain fitting of the OOP-ESEEM. The results obtained in the wild-type systems are compared with two site-directed mutants of C. reinhardtii [Santabarbara et al. (2005) Biochemistry 44, 2119-2128], in which the spin-polarized signal on either the PsaA- or PsaB-bound electron transfer pathway is suppressed so that the radical pair formed on each electron transfer branch could be monitored selectively. This comparison indicates that when all of the iron-sulfur centers are oxidized, only the echo modulation associated with the A branch [P700+ A1A-] radical pair is observed. The reduction of the iron-sulfur clusters and the quinone A1 by preillumination treatment induces a shift in the ESEEM frequency. In all of the systems investigated this observation can be interpreted in terms of different proportions of the signal associated with the [P700+ A1A-] and [P700+ A1B-] radical pairs, suggesting that bidirectionality of electron transfer in photosystem I is a common feature of all species rather than being confined to green algae.     

Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.