Ultrafast primary processes in PS I from Synechocystis sp. PCC 6803: roles of P700 and A(0)

The excitation transport and trapping kinetics of core antenna-reaction center complexes from photosystem I of wild-type Synechocystis sp. PCC 6803 were investigated under annihilation-free conditions in complexes with open and closed reaction centers. For closed reaction centers, the long-component decay-associated spectrum (DAS) from global analysis of absorption difference spectra excited at 660 nm is essentially flat (maximum amplitude <10(-5) absorbance units). For open reaction centers, the long-time spectrum (which exhibits photobleaching maxima at approximately 680 and 700 nm, and an absorbance feature near 690 nm) resembles one previously attributed to (P700(+) - P700). For photosystem I complexes excited at 660 nm with open reaction centers, the equilibration between the bulk antenna and far-red chlorophylls absorbing at wavelengths >700 nm is well described by a single DAS component with lifetime 2.3 ps. For closed reaction centers, two DAS components (2.0 and 6.5 ps) are required to fit the kinetics. The overall trapping time at P700 ( approximately 24 ps) is very nearly the same in either case. Our results support a scenario in which the time constant for the P700 --> A(0) electron transfer is 9-10 ps, whereas the kinetics of the subsequent A(0) --> A(1) electron transfer are still unknown.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi: VIII. The 520 nm Light-induced Absorbance Change in the Wild-type and Mutant Strains 1

The 520 nm light-induced absorbance change in wild-type and 4 mutant strains of Chlamydomonas reinhardi was investigated. In the wild-type strain the absorbance change is composed of at least 2 components, P520 I and P520 II, sensitized by Systems I and II respectively. Some of the properties of these components can be studied by using the appropriate photosynthetic mutant strain. A group of mutant strains modified in the photochemical complex of System II shows only the P520 I absorbance change, whereas a mutant strain deficient in active P700 exhibits only the P520 II absorbance change. The possible relationship between these absorbance changes and the photosynthetic electron transport pathway is discussed.     

Near-IR absorbance changes and electrogenic reactions in the microsecond-to-second time domain in Photosystem I.

The back-reaction kinetics in Photosystem I (PS I) were studied on the microsecond-to-s time scale in cyanobacterial preparations, which differed in the number of iron-sulfur clusters to assess the contributions of particular components to the reduction of P700+. In membrane fragments and in trimeric P700-FA/FB complexes, the major contribution to the absorbance change at 820 nm (delta A820) was the back-reaction of FA- and/or FB- with lifetimes of approximately 10 and 80 ms (approximately 10% and 40% relative amplitude). The decay of photoinduced electric potential (delta psi) across a membrane with directionally incorporated P700-FA/FB complexes had similar kinetics. HgCl2-treated PS I complexes, which contain FA but no FB, retain both of these kinetic components, indicating that neither can be assigned uniquely to a specific acceptor. These results suggest that FA- reduces P700+ directly and argue for a rapid electron equilibration between FA and FB, which would eliminate their kinetic distinction in a back-reaction. In PsaC-depleted P700-Fx cores, as well as in P700-FA/FB complexes with chemically reduced FA and FB, the major contribution to the delta A820 and the delta psi decay is a biphasic back-reaction of F-X (approximately 400 microseconds and 1.5 ms) with some contribution from A-1 (approximately 10 microseconds and 100 microseconds), the latter of which is variable depending on experimental conditions. The delta A820 decay in a P700-A1 core devoid of all iron-sulfur clusters comprises two phases with lifetimes of 10 microseconds and 130 microseconds (2.7:1 ratio). The biexponential back-reaction kinetics found for each of the electron acceptors may be related to existence of different conformational states of the PS I complex. In all preparations studied, excitation at 532 nm with flash energies exceeding 10 mJ gives rise to formation of antenna 3Chl, which also contributes to delta A820 decay on the tens-of-microsecond time scale. A distinction between delta A820 components related to back-reactions and to 3Chl decay can be made by analysis of flash saturation dependencies and by measurements of kinetics with preoxidized P700.     

Adaptation of photosynthesis under iron deficiency in maize

This paper explores the effects of high light stress on Fe-deficient plants. Maize (Zea mays) plants were grown under conditions of Fe deficiency and complete nutrition. Attached, intact leaves of Fe-deficient and control plants were used for gas exchange experiments under suboptimal, optimal and photoinhibitory illumination. Isolated chloroplasts were used to study photosynthetic electron transport system, compromised by the induction of Fe deficiency. The reaction centers of PS II (measured as reduction of Q, the primary electron acceptor of P 680) and PS I (measured as oxidation of P 700) were estimated from the amplitude of light induced absorbance change at 320 and 700 nm, respectively. Plants were subjected to photoinhibitory treatment for different time periods and isolated chloroplasts from these plants were used for electron transport studies. Carbon dioxide fixation in control as well as in Fe-deficient plants decreased in response to high light intensities. Total chlorophyll, P 700 and Q content in Fe-deficient chloroplasts decreased, while Chl a/b ratio and Q/P 700 ratio increased. However, electron transport through PS II suffered more after photoinhibitory treatment as compared to electron transport through PS I or whole chain. Electron transfer through PS I+PS II, excluding the water oxidation complex showed a decrease in Fe-deficient plants. However, electron transport through this part of the chain did not suffer much as a result of photoinhibition, suggesting a defect in the oxidising side of PS II.     

Localization of electron transport inhibition in plastoquinone reactions

Reduction kinetics of P700 following a short flash are measured in spinach chloroplasts after oxidation of the electron carriers between the two photoreactions by far-red light. Three features of the kinetics allow us to localize simultaneously inhibition at different sites between photoreaction II and the reducing site of plastoquinol. These are the initial lag, the halftime, and the area under the transient of the P700 absorbance change, which indicate the electron transfer time from photoreaction II to the reducing site of plastoquinol, the rate of plastoquinol oxidation, and the number of electrons transferred to the special plastoquinone B functioning as secondary electron acceptor of photosystem II, respectively. As an additional diagnostic parameter for inhibition before and after the plastoquinone pool, the area under the transient of the P700 absorbance change is used after long flashes. This area is proportional to the amount of reduced plastoquinone as shown by the absorbance change at 265 nm. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) are compared with those of 2-bromo-4-nitrothymol, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol, and Illoxan as representatives for new classes of inhibitors. While 2-halogeno-4-nitrothymols inhibit the reduction of plastoquinone similarly to DCMU, their diphenyl ether derivatives inhibit selectively the oxidation of plastoquinol.     

Mutation of the putative hydrogen-bond donor to P700 of photosystem I

The primary electron donor of photosystem I (PS1), called P(700), is a heterodimer of chlorophyll (Chl) a and a'. The crystal structure of photosystem I reveals that the chlorophyll a' (P(A)) could be hydrogen-bonded to the protein via a threonine residue, while the chlorophyll a (P(B)) does not have such a hydrogen bond. To investigate the influence of this hydrogen bond on P(700), PsaA-Thr739 was converted to alanine to remove the H-bond to the 13(1)-keto group of the chlorophyll a' in Chlamydomonas reinhardtii. The PsaA-T739A mutant was capable of assembling active PS1. Furthermore the mutant PS1 contained approximately one chlorophyll a' molecule per reaction center, indicating that P(700) was still a Chl a/a' heterodimer in the mutant. However, the mutation induced several band shifts in the visible P(700)(+) - P(700) absorbance difference spectrum. Redox titration of P(700) revealed a 60 mV decrease in the P(700)/P(700)(+) midpoint potential of the mutant, consistent with loss of a H-bond. Fourier transform infrared (FTIR) spectroscopy indicates that the ground state of P(700) is somewhat modified by mutation of ThrA739 to alanine. Comparison of FTIR difference band shifts upon P(700)(+) formation in WT and mutant PS1 suggests that the mutation modifies the charge distribution over the pigments in the P(700)(+) state, with approximately 14-18% of the positive charge on P(B) in WT being relocated onto P(A) in the mutant. (1)H-electron-nuclear double resonance (ENDOR) analysis of the P(700)(+) cation radical was also consistent with a slight redistribution of spin from the P(B) chlorophyll to P(A), as well as some redistribution of spin within the P(B) chlorophyll. High-field electron paramagnetic resonance (EPR) spectroscopy at 330-GHz was used to resolve the g-tensor of P(700)(+), but no significant differences from wild-type were observed, except for a slight decrease of anisotropy. The mutation did, however, provoke changes in the zero-field splitting parameters of the triplet state of P(700) ((3)P(700)), as determined by EPR. Interestingly, the mutation-induced change in asymmetry of P(700) did not cause an observable change in the directionality of electron transfer within PS1.     

Light-induced rapid absorption changes during photosynthesis. VII. Some reactions in sonicated chloroplasts

1. Spinach chloroplasts subjected to sonication show light-induced absorption changes at 700 mμ characteristic of the photooxidation of the chlorophyll component P700. The appearance of P700 absorption changes probably resulted from the release of plastocyanin thus interrupting the electron flow between pigment systems 1 and 2. The general features of the absorption-change transients are similar to those observed previously with digitonin-treated chloroplasts. The addition of 2 mM ascorbate or 10 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea had practically no effect on either the magnitude or the dark decay of the transient absorption change.

2. Phenazine methosulfate (PMS) (in the presence or in the absence of ascorbate) reduction appeared to be coupled to P700 photooxidation, as shown by the corresponding transients at 430 and 388 mμ. The absorbance changes at these two wavelengths indicate that the amount of PMS photoreduced was equivalent to that of P700 photooxidized. Higher PMS concentrations accelerate the dark decay of the P700 signal. When PMS alone is present, anaerobiosis caused the dark decay to become more rapid than in the presence of ascorbate.

3. Unlike PMS, other redox agents such as 2,6-dichlorophenolindophenol, N,N,N′,N′-tetramethyl-p-phenylenediamine or diaminodurol in the presence of excess ascorbate, did not noticeably affect the kinetics of the dark decay at 430 or 703 mμ, suggesting that these reduced species are not efficiently coupled to photooxidized P700.

4. The onset and decay rates of the P700 transient in the presence of PMS and excess ascorbate was insensitive to temperature between 25° and o°. However, when the chloroplast sample was frozen at temperatures ranging from −5° to −196°, all reactions ceased. When the frozen (−196°) sample was brought back to the room temperature, the reaction was restored completely. Fresh broken chloroplasts behave similarly. Digitonin-treated chloroplasts persisted down to about −25° but with diminishing magnitude and slower decay.     

Steady-state and transient polarized absorption spectroscopy of photosystem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus

Core antenna and reaction centre of photosystem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(-), 683-685(-), 696-697(-), and 711(-) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700(+)A(1)(-) or (3)P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P(A), whereas the cation is localized most likely on P(B).     

P700+- and 3P700-induced quenching of the fluorescence at 760 nm in trimeric Photosystem I complexes from the cyanobacterium Arthrospira platensis

The 5 K absorption spectrum of Photosystem I (PS I) trimers from Arthrospira platensis (old name: Spirulina platensis) exhibits long-wavelength antenna (exciton) states absorbing at 707 nm (called C707) and at 740 nm (called C740). The lowest energy state (C740) fluoresces around 760 nm (F760) at low temperature. The analysis of the spectral properties (peak position and line width) of the lowest energy transition (C740) as a function of temperature within the linear electron-phonon approximation indicates a large optical reorganization energy of approximately 110 cm(-1) and a broad inhomogeneous site distribution characterized by a line width of approximately 115 cm(-1). Linear dichroism (LD) measurements indicate that the transition dipole moment of the red-most state is virtually parallel to the membrane plane. The relative fluorescence yield at 760 nm of PS I with P700 oxidized increases only slightly when the temperature is lowered to 77 K, whereas in the presence of reduced P700 the fluorescence yield increases nearly 40-fold at 77 K as compared to that at room temperature (RT). A fluorescence induction effect could not be resolved at RT. At 77 K the fluorescence yield of PS I trimers frozen in the dark in the presence of sodium ascorbate decreases during illumination by about a factor of 5 due to the irreversible formation of (P700+)F(A/B-) in about 60% of the centers and the reversible accumulation of the longer-lived state (P700+)FX-. The quenching efficiency of different functionally relevant intermediate states of the photochemistry in PS I has been studied. The redox state of the acceptors beyond A(0) does not affect F760. Direct kinetic evidence is presented that the fluorescence at 760 nm is strongly quenched not only by P700+ but also by 3P700. Similar kinetics were observed for flash-induced absorbance changes attributed to the decay of 3P700 or P700+, respectively, and flash-induced fluorescence changes at 760 nm measured under identical conditions. A nonlinear relationship between the variable fluorescence around 760 nm and the [P700red]/[P700total] ratio was derived from titration curves of the absorbance change at 826 nm and the variable fluorescence at 760 nm as a function of the redox potential imposed on the sample solution at room temperature before freezing. The result indicates that the energy exchange between the antennae of different monomers within a PS I trimer stimulates quenching of F760 by P700+.     

Photoinduced changes in adsorption at 800 nm related to photosystem I functioning]

The spectra and kinetics of light-induced absorbance changes in the near-infrared region of subchloroplast fragments enriched by P700 were studied. An increase in absorbancy within the region of 725--900 nm upon illumination was characterized by a maximum around 810 nm and by "shoulders" around 760 and 870 nm. Similar effects of thermal inactivation and low temperatures on the duration of dark recovery of light-induced absorbance changes at 700 nm and within the region of 725--900 nm suggest that the absorbance changes in the near-infrared region are due to photooxidation of P700. The values of P700 differential extinction coefficients at 810 nm are 8,2.10(3) M-1.cm-1 for digitonin fragments and 7,7.10(3) M-1.cm-1 for fragments prepared with the use of diethyl ester. It was shown that the value of midpoint oxidation-reduction potential measured for the absorbance changes at 810 nm (+492 mv) is higher than that measured at 700 nm (+475 mv).     

The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature.

The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.     

Evidence for an electrogenic and a non-electrogenic component in the slow phase of the P515 response in chloroplasts

The flash-induced P515 absorbance change in intact chloroplasts consists of a fast and a slow phase. There is disagreement in the literature over the origin of the slow phase. Here we argue that the flash-induced slow phase in P515 absorbance change is composed of two different components. One component is most probably due to the electrogenic Q-cycle associated with the cytochrome b/f complex. The second component has decay kinetics that are much slower than the electrogenic reactions. We suggest that the second component is due to a non-electrogenic reaction.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DQH₂ durohydroquinone - MV methylviologen - P515 Absorbance change at 518 nm     

Species-specific differences of the spectroscopic properties of P700: analysis of the influence of non-conserved amino acid residues by site-directed mutagenesis of photosystem I from Chlamydomonas reinhardtii

We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the (3P700-P700) and (P700+.-P700) absorbance and CD difference spectra. The main bleaching band in the Qy region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the (3P700-P700), the (P700+.-P700) absorbance difference and in the (P700+.-P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra.     

Nature of light-induced absorbance changes at 682 m{micro} and 702 m{micro} in photosynthesis of Anacystis nidulans

Light-induced absorbance changes in the region around the redabsorption band of chlorophyll a were measured in cells andlamella fragments of Anacystis nidulans. In both materials,absorbance decreases were observed at 702 mµ and 682 mµ.(The pigments are designated as P₇₀₀ and P₆₈₀.) The nature ofP₆₈₀ was investigated with special reference to its relationshipto P₇₀₀. In the cells, light absorbed by chlorophyll a causedan absorbance decrease at 682 mµ; Simultaneous illuminationwith light absorbed by phycocyanin caused a partial recoveryof the absorbance decrease. Similar results were observed withthe light-induced absorbance change at 702 mµ. This indicatesthat P₆₈₀ is also an electron carrier in the electron transportchain and occupies a place between the two photoreactions. Inlamella fragments, both the light-induced reversible absorbancechanges of P₆₈₀ and P₇₀₀ appeared in the presence of an electrondonor system; i.e., ascorbate and 2,6-dichlorophenolindophenolor N,N,N',N'-tetramethyl-l,4-phenylenediamine. The experimentsin which the oxidation-reduction potential of the reaction mediumwas changed showed that both P₆₈₀ and P₇₀₀ are one-electroncarriers, having a normal oxidation-reduction potential of 0.44v (assuming that the normal oxidation-reduction potential ofthe ferricyanide-ferrocyanide system is 0.409 v). A possibilitywas suggested that the absorbance change observed at 682 mµis another expression of the oxidation-reduction reaction ofP₇₀₀). (Received October 30, 1968; )     

Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes

The reaction of hydrogen peroxide and certain aromatic aldehydes with cytochrome P450BM3-F87G results in the covalent modification of the heme cofactor of this monooxygenase. Analysis of the resulting heme by electronic absorption spectrophotometry indicates that the reaction in the BM3 isoform is analogous to that in P450(2B4), which apparently occurs via a peroxyhemiacetal intermediate [Kuo et al., Biochemistry, 38 (1999) 10511]. It was observed that replacement of the Phe-87 in the P450BM3 by the smaller glycyl residue was essential for the modification to proceed, as the wild-type enzyme showed no spectral changes under identical conditions. The kinetics of this reaction were examined by stopped-flow spectrophotometry with 3-phenylpropionaldehyde and 3-phenylbutyraldehyde as reactants. In each case, the process of heme modification was biphasic, with initial bleaching of the Soret absorbance, followed by an increase in absorbance centered at 430 nm, consistent with meso-heme adduct formation. The intermediate formed during phase I also showed an increased absorbance between 700 and 900 nm, relative to the native heme and the final product. Phase I showed a linear dependence on peroxide concentration, whereas saturation kinetics were observed for phase II. All of these observations are consistent with a mechanism involving radical attack at the gamma-meso position of the heme cofactor, resulting in the intermediate formation of an isoporphyrin, the deprotonation of which produces the gamma-meso-alkyl heme derivative.     

Effects of Dark Adaptation on Light-Induced Electron Transport through Photosystem I in the Cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Kinetics of the redox reactions in the reaction center (P700) of photosystem I (PSI) of the cyanobacterium Synechocystis sp. PCC 6803 have been studied by EPR spectroscopy. The redox kinetics were recorded based on accumulation of the EPRI signal when the final signal was the sum of individual signals produced in response to illumination of the cells. After prolonged (more than 3 sec) dark intervals between illuminations, the kinetic curve of the EPR signal from P700+ was multiphasic. After a sharp increase in the signal amplitude at the beginning of illumination (phase I), the amplitude rapidly (for 0.1-0.2 sec) decreased (phase II). Then the signal amplitude gradually increased (phase III) until the steady rate of electron transfer was established. With short-term (1 sec) dark intervals between the flashes and also in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the kinetics of the light-induced increase in the EPR signal from P700+ were monophasic. Inhibition with iodoacetamide of electron transport on the acceptor side of PSI under anaerobic conditions or an increase in the amount of respiration substrates on addition of glucose into a suspension of DCMU-treated wild-type cells increased the level of P700 reduction in phase III. The findings suggest that the kinetic curve of the EPR signal from P700+ is determined by both the electron entrance onto P700+ on the donor side of PSI and activity of electron acceptors of PSI.     

A novel P700 redox kinetics probe for rapid,non‐intrusive and whole‐tissue determination of photosystem II functionality,and the stoichiometry of the two photosystems in vivo

We sought a rapid, non‐intrusive, whole‐tissue measure of the functional photosystem II (PS II) content in leaves. Summation of electrons, delivered by a single‐turnover flash to P700⁺ (oxidized PS I primary donor) in continuous background far‐red light, gave a parameter S in absorbance units after taking into account an experimentally determined basal electron flux that affects P700 redox kinetics. S was linearly correlated with the functional PS II content measured by the O₂ yield per single‐turnover repetitive flash in Arabidopsis thaliana expressing an antisense construct to the PsbO (manganese‐stabilizing protein in PS II) proteins of PS II (PsbO mutants). The ratio of S to z_max (total PS I content in absorbance units) was comparable to the PS II/PS I reaction‐center ratio in wild‐type A. thaliana and in control Spinacea oleracea. Both S and S/z_max decreased in photoinhibited spinach leaf discs. The whole‐tissue functional PS II content and the PS II/photosystem I (PS I) ratio can be non‐intrusively monitored by S and S/z_max, respectively, using a quick P700 absorbance protocol compatible with modern P700 instruments.     

Influence of the axial ligands on the spectral properties of P700 of photosystem I: a study of site-directed mutants

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)(*)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., K?ss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)(*)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).     

Properties of partially purified photosynthetic reaction centers from scenedesmus mutant 6E and Anabaena variabilis grown in the presence of diphenylamine

When membrane fragments of Anabaena variabilis grown in the presence of diphenylamine (designated diphenylamine-Anabaena) are treated with Triton X-100 and subjected to sucrose density gradient centrifugation, a bluish-green membrane fragment enirched in P700 is obtained. This high-P700 fragment, denoted HP700, contains three P700 molecules per 100 chlorophyll a molecules and reduces NADP at a rate that is approximately nine times higher than that of HP700 fragments prepared from normally cultured Anabaena by the use of Triton X-100 following extraction with organic solvents. An HP700 fragment has also been isolated from a carotenoidless Scenedesmus mutant 6E, by the use of Triton X-100 and sucrose density gradient centrifugation.

Both HP700 fragments show the characteristic rapid absorbance changes of P700 upon illumination. The fluorescence properties of the HP700 fragments at −196° are different from those of the original membrane fragments. At −196° the long wavelength fluorescence peak is located at a shorter wavelength (724 mμ) in the diphenylamine-Anabaena HP700 fragment and is lower in intensity than that observed with the membrane fragment. Long wavelength fluorescence at −196° is low in the flurorescence spectra of the membrane fragments of Scenedesmus mutant 6E and is barely observable in the HP700 fragment. The fluorescence spectra of the HP700 fragments of both diphenylamine-Anabaena and Scenedesmus mutant 6E at −196° show a shoulder or peak at 700 nm. The data on fluorescence properties of the HP700 fragments suggest that 730 nm fluorescence does not originate from P700.     

Nonmonotonic Redox Conversions of P700 in Leaves Irradiated with Far-Red Light Originate from Variable Contributions of Several Alternative Electron Transport Pathways during the Induction Period

The origin of nonmonotonic changes in the redox state of P700, the primary electron donor of PSI, was investigated on predarkened barley (Hordeum vulgare L.) leaves exposed to far-red light. To accomplish this, the relaxation kinetics of absorbance changes at 830 nm, reflecting the dark reduction of P700⁺, were measured at different stages of the induction curve. The onset of far-red light resulted in rapid oxidation of P700, which was followed by its partial reduction and subsequent slow oxidation of P700 to a steady-state level. This steady-state level was usually attained within 10 s under far-red light. The relative contribution of the slow kinetic component of P700⁺ reduction decreased in parallel with the transient photoreduction of P700⁺ and increased upon a subsequent stage of P700 photooxidation. The contribution of the middle component to the dark reduction of P700⁺ increased monotonically with the length of far-red light irradiation. The relative amplitude of the fast component of P700⁺ reduction increased sharply during the first 3 s of irradiation and decreased upon longer light exposures. The rates of fast and slow components of dark reduction of P700⁺ remained constant upon illumination of dark-adapted leaves with far-red light for 1 s and longer periods. Thus, nonmonotonic changes in the redox state of P700 in barley leaves exposed to far-red light reflect variable contributions of few alternative electron transport pathways characterized by different rates of electron donation to PSI. The results show the principle possibility of switching-over between alternative pathways of PSI-related electron transfer within one complex of this photosystem. Such switching may occur irrespective of active operation or inhibition of ferredoxin-dependent electron transport.