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.     

Incorporation of a high potential quinone reveals that electron transfer in Photosystem I becomes highly asymmetric at low temperature

Photosystem I (PS I) has two nearly identical branches of electron-transfer co-factors. Based on point mutation studies, there is general agreement that both branches are active at ambient temperature but that the majority of electron-transfer events occur in the A-branch. At low temperature, reversible electron transfer between P(700) and A(1A) occurs in the A-branch. However, it has been postulated that irreversible electron transfer from P(700) through A(1B) to the terminal iron-sulfur clusters F(A) and F(B) occurs via the B-branch. Thus, to study the directionality of electron transfer at low temperature, electron transfer to the iron-sulfur clusters must be blocked. Because the geometries of the donor-acceptor radical pairs formed by electron transfer in the A- and B-branch differ, they have different spin-polarized EPR spectra and echo-modulation decay curves. Hence, time-resolved, multiple-frequency EPR spectroscopy, both in the direct-detection and pulse mode, can be used to probe the use of the two branches if electron transfer to the iron-sulfur clusters is blocked. Here, we use the PS I variant from the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, to incorporate 2,3-dichloro-1,4-naphthoquinone (Cl(2)NQ) into the A(1A) and A(1B) binding sites. The reduction midpoint potential of Cl(2)NQ is approximately 400 mV more positive than that of phylloquinone and is unable to transfer electrons to the iron-sulfur clusters. In contrast to previous studies, in which the iron-sulfur clusters were chemically reduced and/or point mutations were used to prevent electron transfer past the quinones, we find no evidence for radical-pair formation in the B-branch. The implications of this result for the directionality of electron transfer in PS I are discussed.     

PsaC subunit of photosystem I is oriented with iron-sulfur cluster F(B) as the immediate electron donor to ferredoxin and flavodoxin.

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.     

Deciphering the 820 nm signal: redox state of donor side and quantum yield of Photosystem I in leaves

By recording leaf transmittance at 820 nm and quantifying the photon flux density of far red light (FRL) absorbed by long-wavelength chlorophylls of Photosystem I (PS I), the oxidation kinetics of electron carriers on the PS I donor side was mathematically analyzed in sunflower (Helianthus annuus L.), tobacco (Nicotiana tabacum L.) and birch (Betula pendula Roth.) leaves. PS I donor side carriers were first oxidized under FRL, electrons were then allowed to accumulate on the PS I donor side during dark intervals of increasing length. After each dark interval the electrons were removed (titrated) by FRL. The kinetics of the 820 nm signal during the oxidation of the PS I donor side was modeled assuming redox equilibrium among the PS I donor pigment (P700), plastocyanin (PC), and cytochrome f plus Rieske FeS (Cyt f + FeS) pools, considering that the 820 nm signal originates from P700⁺ and PC⁺. The analysis yielded the pool sizes of P700, PC and (Cyt f + FeS) and associated redox equilibrium constants. PS I density varied between 0.6 and 1.4 μmol m⁻². PS II density (measured as O₂ evolution from a saturating single-turnover flash) ranged from 0.64 to 2.14 μmol m⁻². The average electron storage capacity was 1.96 (range 1.25 to 2.4) and 1.16 (range 0.6 to 1.7) for PC and (Cyt f + FeS), respectively, per P700. The best-fit electrochemical midpoint potential differences were 80 mV for the P700/PC and 25 mV for the PC/Cyt f equilibria at 22 °C. An algorithm relating the measured 820 nm signal to the redox states of individual PS I donor side electron carriers in leaves is presented. Applying this algorithm to the analysis of steady-state light response curves of net CO₂ fixation rate and 820 nm signal shows that the quantum yield of PS I decreases by about half due to acceptor side reduction at limiting light intensities before the donor side becomes oxidized at saturating intensities. Footnote: This revised version was published online in August 2006 with corrections to the Cover Date.     

Secondary pair charge recombination in photosystem I under strongly reducing conditions: temperature dependence and suggested mechanism.

Photoinduced electron transfer in photosystem I (PS I) proceeds from the excited primary electron donor P700 (a chlorophyll a dimer) via the primary acceptor A0 (chlorophyll a) and the secondary acceptor A1 (phylloquinone) to three [4Fe-4S] clusters, Fx, FA, and FB. Prereduction of the iron-sulfur clusters blocks electron transfer beyond A1. It has been shown previously that, under such conditions, the secondary pair P700+A1- decays by charge recombination with t1/2 approximately 250 ns at room temperature, forming the P700 triplet state (3P700) with a yield exceeding 85%. This reaction is unusual, as the secondary pair in other photosynthetic reaction centers recombines much slower and forms directly the singlet ground state rather than the triplet state of the primary donor. Here we studied the temperature dependence of secondary pair recombination in PS I from the cyanobacterium Synechococcus sp. PCC6803, which had been illuminated in the presence of dithionite at pH 10 to reduce all three iron-sulfur clusters. The reaction P700+A1- --> 3P700 was monitored by flash absorption spectroscopy. With decreasing temperature, the recombination slowed down and the yield of 3P700 decreased. In the range between 303 K and 240 K, the recombination rates could be described by the Arrhenius law with an activation energy of approximately 170 meV. Below 240 K, the temperature dependence became much weaker, and recombination to the singlet ground state became the dominating process. To explain the fast activated recombination to the P700 triplet state, we suggest a mechanism involving efficient singlet to triplet spin evolution in the secondary pair, thermally activated repopulation of the more closely spaced primary pair P700+A0- in a triplet spin configuration, and subsequent fast recombination (intrinsic rate on the order of 10(9) s(-1)) forming 3P700.     

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).     

Selective extraction of antenna chlorophylls, carotenoids and quinones from photosystem I reaction center

By the ether treatment of lyophilized PSI pigment-protein complexes, all the carotenoids and the secondary acceptor phylloquinone (A1), and more than 90% of the Chl were removed to yield the PSI complex with 9-11 molecules of Chl per reaction-center unit. The complexes retained the primary electron donor and acceptor (P700 and A0), in addition to three FeS clusters (F(X), F(A) and F(B)), and showed an activity of highly efficient electron transfer when phylloquinone was reconstituted. The methods for the preparation and the characterization of the ether-extracted PSI complexes are reviewed in this article. We also review the studies done with this PSI preparation on (1) the identification of the absorption and fluorescence spectra of P700, (2) the nano- and picosecond reaction of A0 and A1, (3) the energy-gap dependency of the reaction rate between A0 and the artificial quinones reconstituted at the A1 site, (4) the direct excitation of P700 followed by the ultra-fast electron transfer from P700 to A0, and (5) the de- and re-stabilization of the PSI structure by the removal and reconstitution, respectively, of antenna Chl in the presence of certain lipids.     

Removal of PsaF alters forward electron transfer in photosystem I: evidence for fast reoxidation of QK-A in subunit deletion mutants of Synechococcus sp. PCC 7002

Recent studies of point mutations in photosystem I have suggested that the two kinetic phases of phylloquinone reoxidation represent electron transfer in the two branches of cofactors. This interpretation implies that changes in the relative amplitudes of the two kinetic phases represent a change in the extent of electron transfer in the two branches. Using time-resolved electron paramagnetic resonance (EPR), this issue is investigated in subunit deletion mutants of Synechococcus sp. PCC 7002. The spin-polarized EPR signals of P(700)(+)A(1)(-) and P(700)(+)FeS(-), both at room temperature and in frozen solution, are altered by deletion of PsaF and/or PsaE, and the differences from the wild type are much more pronounced in PS I complexes isolated from the mutants using Triton X-100 rather than n-dodecyl beta-d-maltopyranoside. The changes in the transient EPR data for the mutant complexes are consistent with a significant fraction of reaction centers showing (i) faster electron transfer from A(1)(-) to F(X), (ii) slower forward electron transfer from A(0)(-) to A(1), and (iii) slightly altered quinone hyperfine couplings, possibly as a result of a change in the hydrogen bonding. The fraction of fast electron transfer and its dependence on the isolation procedure are estimated approximately from simulations of the room temperature EPR data. The results are discussed in terms of possible models for the electron transfer. It is suggested that the detergent-induced fraction of fast electron transfer is most likely due to alteration of the environment of the quinone in the PsaA branch of cofactors and is not the result of a change in the directionality of electron transfer.     

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.     

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.     

Photoinhibition of Photosystem I electron transfer activity in isolated Photosystem I preparations with different chlorophyll contents

Photoinhibition of the light-induced Photosystem I (PS I) electron transfer activity from the reduced dichlorophenol indophenol to methyl viologen was studied. PS I preparations with Chl/P700 ratios of about 180 (PS I-180), 100 (PS I-100) and 40 (PS I(HA)-40) were isolated from spinach thylakoid membranes by the treatments with Triton X-100, followed by sucrose density gradient centrifugation and hydroxylapatite column chromatography. White light irradiation (1.1 × 10⁴E m^–2 s^–1) of PS I-180 for 2 hours bleached 50% of the chlorophyll and caused a 58% decrease in the electron transfer activity with virtually no loss of the primary donor, P700. The flash-induced absorbance change showed the decay phase with a half time of about 10 s that was attributed to the P700 triplet, suggesting that the photoinhibitory light treatment caused the destruction of the PS I acceptor(s), F_x and possibly A₁. PS I-100 was similarly photobleached by the irradiation and the electron transfer activity decreased. There was, however, no apparent photoinhibition of the electron transport activity in PS I(HA)-40. Photoinhibition similar to that seen in PS I-180 also occurred in membrane fragments that were isolated without any detergent from a PS II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. PS I-180 was not photoinhibited under anaerobic conditions. The production of superoxide and fatty acid hydroperoxide during white light irradiation was significantly greater in PS I-180 than in PS I(HA)-40. The mechanism of photoinhibition in PS I preparations is discussed in relation to the formation of toxic oxygen molecules.Abbreviations A₀,A₁ primary and secondary electron acceptors of PS I - CD circular dichroism - DCPIP 2,6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur centers A, B, X - HA hydroxylapatite - LHCI lightharvesting complex of PS I - MDA malondialdehyde - MV methyl viologen - Na-Asc sodium L-ascorbate - P700 primary electron donor of PS I - PFD photon flux density - PS I-A and PS I-B psaA and psaB gene products - TBA thiobarbituric acid     

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   

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.     

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*-.     

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).     

Interferon (IFN)-like antiviral effect is induced by unspecific cross-linking of cell surface receptors

Photosystem I preparations were irradiated with UV to destroy vitamin K₁ in situ. The depletion of vitamin K₁ resulted in inactivation of NADP⁺ photoreduction and introduction of a 220 ms component in the flash generated P700⁺ re-reduction at room temperature. The photoreduction of the terminal FeS centers FA and FB in control and vitamin K₁-depleted preparations at 7 K were comparable. The data confirm that vitamin K₁ is functionally implicated in primary electron transfer reactions in PS I at physiological temperature, and that the anomalous results at cryogenic temperature may be explicable in terms of a by-pass of the vitamin K₁ acceptor site or heterogeneity introduced into the photosystem by quinone removal.     

A kinetic assessment of the sequence of electron transfer from F(X) to F(A) and further to F(B) in photosystem I: the value of the equilibrium constant between F(X) and F(A)

The x-ray structure analysis of photosystem I (PS I) crystals at 4-A resolution (Schubert et al., 1997, J. Mol. Biol. 272:741-769) has revealed the distances between the three iron-sulfur clusters, labeled F(X), F(1), and F(2), which function on the acceptor side of PS I. There is a general consensus concerning the assignment of the F(X) cluster, which is bound to the PsaA and PsaB polypeptides that constitute the PS I core heterodimer. However, the correspondence between the acceptors labeled F(1) and F(2) on the electron density map and the F(A) and F(B) clusters defined by electron paramagnetic resonance (EPR) spectroscopy remains controversial. Two recent studies (Diaz-Quintana et al., 1998, Biochemistry. 37:3429-3439;, Vassiliev et al., 1998, Biophys. J. 74:2029-2035) provided evidence that F(A) is the cluster proximal to F(X), and F(B) is the cluster that donates electrons to ferredoxin. In this work, we provide a kinetic argument to support this assignment by estimating the rates of electron transfer between the iron-sulfur clusters F(X), F(A), and F(B). The experimentally determined kinetics of P700(+) dark relaxation in PS I complexes (both F(A) and F(B) are present), HgCl(2)-treated PS I complexes (devoid of F(B)), and P700-F(X) cores (devoid of both F(A) and F(B)) from Synechococcus sp. PCC 6301 are compared with the expected dependencies on the rate of electron transfer, based on the x-ray distances between the cofactors. The analysis, which takes into consideration the asymmetrical position of iron-sulfur clusters F(1) and F(2) relative to F(X), supports the F(X) --> F(A) --> F(B) --> Fd sequence of electron transfer on the acceptor side of PS I. Based on this sequence of electron transfer and on the observed kinetics of P700(+) reduction and F(X)(-) oxidation, we estimate the equilibrium constant of electron transfer between F(X) and F(A) at room temperature to be approximately 47. The value of this equilibrium constant is discussed in the context of the midpoint potentials of F(X) and F(A), as determined by low-temperature EPR spectroscopy.     

Biogenesis of iron-sulfur clusters in photosystem I: holo-NfuA from the cyanobacterium Synechococcus sp. PCC 7002 rapidly and efficiently transfers [4Fe-4S] clusters to apo-PsaC in vitro

The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, M?ssbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro.     

Electrogenic reduction of the primary electron donor P700 by plastocyanin in photosystem I complexes

An electrometric technique was used to investigate electron transfer between spinach plastocyanin (Pc) and photooxidized primary electron donor P700 in photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803. In the presence of Pc, the fast unresolvable kinetic phase of membrane potential generation related to electron transfer between P700 and the terminal iron–sulfur acceptor F_B was followed by additional electrogenic phases in the microsecond and millisecond time scales, which contribute approximately 20% to the overall electrogenicity. These phases are attributed to the vectorial electron transfer from Pc to the protein-embedded chlorophyll dimer P700⁺ within the PsaA/PsaB heterodimer. The observed rate constant of the millisecond kinetic phase exhibited a saturation profile at increasing Pc concentration, suggesting the formation of a transient complex between Pc and PS I with the dissociation constant K_d of about 80 μM. A small but detectable fast electrogenic phase was observed at high Pc concentration. The rate constant of this phase was independent of Pc concentration, indicating that it is related to a first-order process.