Role of acidic amino acid residues of PsaD subunit on limiting the affinity of photosystem I for ferredoxin.

The PsaD subunit of photosystem I is one of the central polypeptides for the interaction with ferredoxin, its acidic electron acceptor. In the cyanobacterium Synechocystis 6803, this role is partly performed by a sequence extending approximately from histidine 97 to arginine 119, close to the C-terminus. In the present work, acidic amino acids D100, E105, and E109 are shown to moderate the affinity of Photosystem I for ferredoxin. Most single replacements of these residues by neutral amino acids increased the affinity for ferredoxin, resulting in a dissociation constant as low as 0.015 microM for the E105Q mutant (wild-type K(D) = 0.4 microM). This is the first report on the limitation of photosystem I affinity for ferredoxin due to acidic amino acids from PsaD subunit. It highlights the occurrence of a negative control on the binding during the formation of transient complexes between electron carriers.

Multiple functions for the C terminus of the PsaD subunit in the cyanobacterial photosystem I complex.

PsaD subunit of Synechocystis sp PCC 6803 photosystem I (PSI) plays a critical role in the stability of the complex and is part of the docking site for ferredoxin (Fd). In the present study we describe major physiological and biochemical effects resulting from mutations in the accessible C-terminal end of the protein. Four basic residues were mutated: R111, K117, K131, and K135, and a large 36-amino acid deletion was generated at the C terminus. PSI from R111C mutant has a 5-fold decreased affinity for Fd, comparable with the effect of the C terminus deletion, and NADP+ is photoreduced with a 2-fold decreased rate, without consequence on cell growth. The K117A mutation has no effect on the affinity for Fd, but decreases the stability of PsaE subunit, a loss of stability also observed in R111C and the deletion mutants. The double mutation K131A/K135A does not change Fd binding and reduction, but decreases the overall stability of PSI and impairs the cell growth at temperatures above 30 degrees C. Three mutants, R111C, K117A, and the C-terminal deleted exhibit a higher content of the trimeric form of PSI, in apparent relation to the removal of solvent accessible positive charges. Various regions in the C terminus of cyanobacterial PsaD thus are involved in Fd strong binding, PSI stability, and accumulation of trimeric PSI.

Importance of the region including aspartates 57 and 60 of ferredoxin on the electron transfer complex with photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

Ferredoxin reduction by photosystem I has been studied by flash-absorption spectroscopy. Aspartate residues 20, 57, and 60 of ferredoxin were changed to alanine, cysteine, arginine, or lysine. On the one hand, electron transfer from photosystem I to all mutated ferredoxins still occurs on a microsecond time scale, with half-times of ferredoxin reduction mostly conserved compared to wild-type ferredoxin. On the other hand, the total amplitude of the fast first-order reduction varies largely when residues 57 or 60 are modified, in apparent relation to the charge modification (neutralized or inverted). Substituting these two residues for lysine or arginine induce strong effects on ferredoxin binding (up to sixfold increase in K(D)), whereas the same substitution on aspartate 20, a spatially related residue, results in moderate effects (maximum twofold increase in K(D)). In addition, double mutations to arginine or lysine were performed on both aspartates 57 and 60. The mutated proteins have a 15- to 20-fold increased K(D) and show strong modifications in the amplitudes of the fast reduction kinetics. These results indicate that the acidic area of ferredoxin including aspartates 57 and 60, located opposite to the C-terminus, is crucial for high affinity interactions with photosystem I.

The PsaE subunit is required for complex formation between photosystem I and flavodoxin from the cyanobacterium Synechocystis sp. PCC 6803.

The photoreduction of the oxidized and the semiquinone form of flavodoxin by photosystem I particles (PSI) from the wild type and a psaE deletion strain from the cyanobacterium Synechocystis sp. PCC 6803 was analyzed by flash-absorption spectroscopy to investigate a possible involvement of the PsaE subunit in this photoreduction process. The kinetics of the reduction of oxidized flavodoxin display a single-exponential component for both PSI preparations. Limiting electron transfer rates kobs of approximately 500 and approximately 900 s -1 are deduced for the wild type and PSI from the psaE-less mutant, respectively, indicating that the PsaE subunit is not important for this photoreduction process. In the case of wild-type PSI, the reduction of flavodoxin semiquinone is a biphasic process, displaying a fast first-order phase with a t1/2 of approximately 13 micro(s) which is then followed by a slower, concentration-dependent phase, for which a second-order rate constant k2 of 2.2 x 10(8) M-1 cm-1 is calculated. In contrast, photoreduction of the semiquinone by PSI from the psaE-less mutant is monoexponential, displaying only one second-order component with a second-order rate constant similar to those observed for wild-type PSI (k2 = 1.5 x 10(8) M-1 cm-1). The fast first-order component which is interpreted as an electron transfer process within a preformed complex between flavodoxin semiquinone and PSI is almost completely absent in the reduction of flavodoxin by the PsaE-less PSI. A similar loss of the fast phase is also observed for the photoreduction of flavodoxin semiquinone by PSI from a Synechococcus elongatus psaE-less mutant. Upon reconstitution of isolated PsaE to the PsaE-less PSI in vitro, approximately 80% of the fast first-order kinetic component is recovered, indicating that PsaE is required for high-affinity binding of the flavodoxin semiquinone to PSI. In addition, chemical cross-linking assays show that flavodoxin can no longer be cross-linked to PSI in detectable amounts when PsaE is missing on the reaction center. Taken together, these experiments indicate that the PsaE subunit is required for complex formation between PSI and flavodoxin but is not required for an efficient forward electron transfer from photosystem I to both forms of flavodoxin.

Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by plasmid shuffling.

The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedl gene (ssI0020) coding for the most abundant ferredoxin product was found to be expressed strongly as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (sIr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (sIr0150, negative control) or glucose availability (sII1382). fedl was found to be critical to Synechocystis PCC6803 viability in spite of sIr0150, sII1382 or flavodoxin induction, even after the addition of glucose that compensates for the loss of photosynthesis. Nevertheless, fedl could be deleted from all chromosome copies in cells propagating a fedl gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedl mutant alleles propagated on a second vector. Analysis of the fedl mutant strains generated after plasmid exchange showed that the C18-C85 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated that the length of the Fedl carboxy terminus is important for effective PSI/FedI interactions. The plasmid-shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms, as it is based on promiscuous plasmids.

Electron transfer in photosystem I reaction centers follows a linear pathway in which iron-sulfur cluster FB is the immediate electron donor to soluble ferredoxin.

Reaction centers of photosystem I contain three different [4Fe-4S] clusters named FX, FA, and FB. The terminal photosystem I acceptors (FA, FB) are distributed asymmetrically along the membrane normal, with one of them (FA or FB) being reduced from FX and the other one (FB or FA) reducing soluble ferredoxin. In the present work, kinetics of electron transfer has been measured in PSI from the cyanobacterium Synechocystis sp. PCC 6803 after inactivation of FB by treatment with HgCl2. Photovoltage measurements indicate that, in the absence of FB, reduction of FA by FX is still faster than the rate of FX reduction [(210 ns)-1]. Flash-absorption measurements show that the affinity of ferredoxin for HgCl2-treated PSI is only decreased by a factor of 3-4 compared to untreated photosystem I. The first-order rate of ferredoxin reduction by FA-, within the photosystem I/ferredoxin complex, has been calculated from measurements of P700+ decay. Compared to control PSI, this rate is several orders of magnitude smaller (6 s-1 versus 10(4)-10(6) s-1). Moreover, it is smaller than the rate of recombination from FA-, resulting in inefficient ferredoxin reduction (yield of 25%). After reconstitution of FB, about half of the reconstituted photosystem I reaction centers recover fast reduction of ferredoxin with kinetics similar to that of untreated photosystem I. These results support FB as the direct partner of ferredoxin and as the more distal cluster of photosystem I with respect to the thylakoid membrane, in accordance with a linear electron-transfer pathway FX-->FA-->FB-->ferredoxin.

ESEEM study of the phyllosemiquinone radical A1.- in 14N- and 15N-labeled photosystem I.

The phyllosemiquinone radical of the photosystem I reaction center has been studied by electron spin echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of ESEEM data of the semiquinone in 14N- and 15N-labeled PSI and numerical simulations demonstrate the existence of two protein nitrogen nuclei coupled to the semiquinone. One of the 14N couplings is characterized by a quadrupolar coupling constant e2qQ/4h of 0.77 MHz, an asymmetry parameter eta of 0.18, and a hyperfine coupling tensor with an almost pure isotropic hyperfine coupling, i.e. (Axx, Ayy, Azz) = (1.3, 1.3, 1.5 MHz). The second nitrogen coupling is characterized by a quadrupolar coupling constant e2qQ/4h of 0.45 MHz, an asymmetry parameter eta of 0.85, and a weak hyperfine coupling tensor with a dominant anisotropic part, i.e. (Axx, Ayy, Azz) = (-0.2, -0.2, 1.5 MHz). On the basis of a comparison of the 14N-ESEEM data with 14N-NQR and 14N-ESEEM data from the literature, the first coupled nitrogen is assigned to the indole nitrogen of a tryptophan residue. The coupling of the second nitrogen is much weaker and therefore more difficult to assign. However, the simulated spectrum best describes an amino nitrogen of a histidine, although the amide group of an asparagine or glutamine cannot be ruled out. The possible origins of teh nitrogen hyperfine coupling are discussed in terms of the amino acid residues thought to be close to the semiquinone in PSI.

A new non-heme iron environment in Paracoccus denitrificans adenylate kinase studied by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy.

Adenylate kinase from the Gram-negative bacterium Paracoccus denitrificans (AKden) has structural features highly similar to those of the enzyme from Gram-positive organisms. Atomic absorption spectroscopy of the recombinant protein, which is a dimer, revealed the presence of two metals, zinc and iron, each binding most probably to one monomer. Under oxidizing conditions, the electron paramagnetic resonance (EPR) spectrum of AKden at 4.2 K consists of features at g = 9.23, 4.34, 4.21, and 3.68. These features are absent in the ascorbate-reduced protein and are characteristic of a S = 5/2 spin system in a rhombic environment with E/D = 0.24 and are assigned to a non-heme Fe3+ (S = 5/2) center. The zero-field splitting parameter D (D = 1.4 +/- 0.2 cm-1) was estimated from the temperature dependence of the EPR spectra. These EPR characteristic as well as the difference absorption spectrum (oxidized minus reduced) of AKden are similar to those reported for the non-heme iron protein rubredoxin. Nevertheless, the redox potential of the Fe2+/Fe3+ couple in AKden was measured at +230 +/- 30 mV, which is more positive than the redox potential of the non-heme iron in rubredoxin. Binding of cyanide converts the iron from the high-spin (S = 5/2) to the low-spin (S = 1/2) spin state. The EPR spectrum of the non-heme Fe3+(S = 1/2) in the presence of cyanide has g values of 2.45, 2.18, and 1.92 and spin-Hamiltonian parameters R/lambda = 7. 4 and R/mu = 0.56. The conversion of the non-heme iron to the low-spin (S = 1/2) state allowed the study of its local environment by electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM data revealed the existence of 14N or 15N nuclei coupled to the low-spin iron after addition of KC14N or KC15N respectively. This demonstrated that iron in AKden has at least one labile coordination position that can be easily occupied by cyanide. Other possible magnetic interactions with nitrogen(s) from the protein are discussed.

Functional reconstitution of photosystem I reaction center from cyanobacterium Synechocystis sp PCC6803 into liposomes using a new reconstitution procedure.

Photosystem I reaction center from the cyanobacterium Synechocystis sp PCC6803 was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. Liposomes prepared by reversephase evaporation were treated with various amounts of different detergents and protein incorporation was analyzed at each step of the solubilization process. After detergent removal the activities of the resulting proteoliposomes were measured. The most efficient reconstitution was obtained by insertion of the protein complex into preformed liposomes destabilized by saturating amounts of octylglucoside. In the presence of N-methylphenazonium methosulfate and ascorbic acid, liposomes containing the reaction center catalyzed a light-dependent net H+ uptake as measured by the 9-aminoacridine fluorescence quenching and the pH meter. An important benefit of the new reconstitution procedure is that it produces a homogeneous population of large-size proteoliposomes with a low ionic permeability and with a majority inwardly directed H+ transport activity. In optimal conditions, a light-induced delta pH of about 1.8 units could be sustained at 20 degrees C in the presence of valinomycin. In the absence of valinomycin, a "back-pressure" effect of an electrical transmembrane potential decreased both the rate and the extent of the H+ transport. The reaction center was also co-reconstituted with F0F1 H(+)-ATPases from chloroplasts and from the thermophilic bacterium, PS3. The co-reconstituted system was shown to catalyze a light-dependent phosphorylation which could only be measured in the presence of a high concentration of PSI (low lipid/PSI ratios) while no delta pH could be detected.

Mutagenesis of photosystem I in the region of the ferredoxin cross-linking site: modifications of positively charged amino acids.

The psaD gene isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been mutated in the region encoding a cross-linking site for ferredoxin. A glucose tolerant strain of Synechocystis 6803 was first deleted for psaD, and the resulting PS-I was characterised by EPR and flash absorption spectroscopy. The major modification related to the absence of the PsaD subunit is the disappearance of the first order reduction of ferredoxin which is replaced by a second order reaction. Reconstitution of the deleted PS-I with the purified PsaD polypeptide restored 80% of the fast photoreduction of ferredoxin. The deletion of PsaD has no apparent effect on the main biochemical features of the resulting depleted PS-I complex, with the exception of minor modifications to the FA/FB centers. The deleted strain was transformed by a series of psaD genes mutated at three conserved residues, all located close to the ferredoxin cross-linking site. The resulting photosystem I complexes were extensively studied by flash absorption spectroscopy. Unexpectedly, the change of Lys 106 involved in the cross-linking of ferredoxin for an uncharged amino acid has almost no effect (mutation K106A). However, the functional consequences of more drastic substitutions of either Lys 106 or Arg 111 indicate a role for these two basic amino acids in the binding and submicrosecond reduction of ferredoxin. Various mutations of the unique His at position 97 show that this amino acid is involved in the increased affinity of PS-I for ferredoxin when the pH is lowered. This histidine could be central in regulating in vivo the rate of ferredoxin reduction as a precise sensor of the local proton concentration.

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.

1H and 15N NMR sequential assignment, secondary structure, and tertiary fold of [2Fe-2S] ferredoxin from Synechocystis sp. PCC 6803.

The [2Fe-2S] ferredoxin extracted from Synechocystis sp. PCC 6803 was studied by 1H and 15N nuclear magnetic resonance. Sequence-specific 1H and 15N assignment of amino acid residues far from the paramagnetic cluster (distance higher than 8 A) was performed. Interresidue NOE constraints have allowed the identification of several secondary structure elements: one beta sheet composed of four beta strands, one alpha helix, and two alpha helix turns. The analysis of interresidue NOEs suggests the existence of a disulfide bridge between the cysteine residues 18 and 85. Such a disulfide bridge has never been observed in plant-type ferredoxins. Structure modeling using the X-PLOR program was performed with or without assuming the existence of a disulfide bridge. As a result, two structure families were obtained with rms deviations of 2.2 A. Due to the lack of NOE connectivities resulting from the paramagnetic effect from the [2Fe-2S] cluster, the structures were not well resolved in the region surrounding the [2Fe-2S] cluster, at both extremities of the alpha helix and the C and N terminus segments. In contrast, when taken separately, the beta sheet and the alpha helix were well defined. This work is the first report of a structure model of a plant-type [2Fe-2S] Fd in solution.

Laser-flash kinetic analysis of the fast electron transfer from plastocyanin and cytochrome c6 to photosystem I. Experimental evidence on the evolution of the reaction mechanism.

The reaction mechanism of electron transfer from the interchangeable metalloproteins plastocyanin (Pc) and cytochrome c6 (Cyt) to photooxidized P700 in photosystem I (PSI) has been studied by laser-flash absorption spectroscopy using a number of evolutionarily differentiated organisms such as cyanobacteria (Anabaena sp. PCC 7119 and Synechocystis sp. PCC 6803), green algae (Monoraphidium braunii), and higher plants (spinach). PSI reduction by Pc or Cyt shows different kinetics depending on the organism from which the photosystem and metalloproteins are isolated. According to the experimental data herein reported, three different kinetic models are proposed by assuming either an oriented collisional reaction mechanism (type I), a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer (type II), or rearrangement of the reaction partners within the complex before electron transfer takes place (type III). Our findings suggest that PSI was able to first optimize its interaction with positively charged Cyt and that the evolutionary replacement of the ancestral Cyt by Pc, as well as the appearance of the fast kinetic phase in the Pc/PSI system of higher plants, would involve structural modifications in both the donor protein and PSI.

Laser flash absorption spectroscopy study of ferredoxin reduction by photosystem I: spectral and kinetic evidence for the existence of several photosystem I-ferredoxin complexes.

The existence of three first-order phases has been previously reported for the reduction of soluble ferredoxin by photosystem I (PSI), both from the cyanobacterium Synechocystis sp. PCC 6803 (at pH 8 and in the presence of salts) [Sétif, P. Q. Y., & Bottin, H. (1994) Biochemistry 33, 8495-8504]. The spectra of these three phases (t1/2 < 1 microsecond, = 13-20 and 103-123 microseconds) have been measured between 460 and 600 nm. All of them are fully consistent with electron transfer from (FA,FB)-, the terminal 4Fe-4S acceptors of PSI, to ferredoxin. Though the three spectra deviate significantly from the spectrum that can be calculated independently for this process, their sum closely matches the calculated spectrum. A detailed examination of these deviations indicates that the intermediate (13-20 microseconds) and slow (103-123 microseconds) first-order phases are associated with two distinct ferredoxin-binding sites on PSI. Under the same conditions, a fourth phase of negative amplitude is also observed in the 460-600 nm region. It is ascribed to reoxidation of reduced ferredoxin by an unknown species. The kinetic properties of this process show that it is triggered by collision of free ferredoxin with a preformed PSI-ferredoxin complex. Taking this reaction into account, it is shown that the relative proportions of the three first-order phases of ferredoxin reduction do not depend upon the ferredoxin concentration, indicating that the different sites of ferredoxin binding are mutually exclusive. The kinetics of ferredoxin reduction were also studied at pH 5.8, in the absence of salts. Under these conditions, the affinity of ferredoxin for PSI is much higher than at pH 8 (dissociation constant approximately 0.05 microM versus 0.6 microM) and the kinetics of ferredoxin reduction are much faster (a major submicrosecond phase and a single first-order microsecond phase with t1/2 approximately 9 microseconds), whereas a third, slower first-order phase is essentially absent. Two similar first-order components are found for the reduction of spinach ferredoxin by PSI from Synechocystis at pH 8, though the apparent dissociation constant for the latter system is larger (approximately 5 microM). Despite the different affinities of spinach and Synechocystis ferredoxins for the cyanobacterial PSI, similar second-order rate constants are found in both cases at pH 8 [(2-6) x 10(8) M-1 s-1].

Laser flash absorption spectroscopy study of ferredoxin reduction by photosystem I in Synechocystis sp. PCC 6803: evidence for submicrosecond and microsecond kinetics.

The kinetics of reduction of soluble ferredoxin by photosystem I (PSI), both purified from the cyanobacterium Synechocystis sp. PCC 6803, were investigated by flash-absorption spectroscopy between 460 and 600 nm. Most experiments were made with isolated monomeric PSI reaction centers prepared with the detergent beta-dodecyl maltoside. Analysis of absorption transients, in parallel at 480 and 580 nm and under several conditions, shows the existence of three different first-order components in the presence of ferredoxin (t1/2 approximately 500 ns, 20 microseconds, and 100 microseconds). A second-order phase of ferredoxin reduction is also present [k = (2-5) x 10(8) s-1 at pH 8 and at moderate ionic strength]. Similar first-order kinetic components were found with membranes from Synechocystis, with dissolved crystals of trimeric PSI reaction centers from Synechococcus, and also when ferredoxin from Synechocystis is replaced by ferredoxin from Chlamydomonas reinhardtii. The three first-order phases exhibit similar, though not identical, spectra which are consistent with electron transfer from the [4Fe-4S] centers of PSI to the [2Fe-2S] center of ferredoxin and are all attributed to reduction of ferredoxin bound to PSI. At pH 8 and at moderate ionic strength, the dissociation constants associated with each of these components are also similar, with a global value varying between 0.2 and 0.8 microM in different cyanobacterial preparations. The presence of three exponential components is discussed assuming homogeneity of the two partners and using the estimated values for the shortest possible distance of approach of soluble ferredoxin from the different iron-sulfur centers of PSI. It is concluded that the 500-ns phase corresponds to electron transfer from either FA- or FB-, the terminal iron-sulfur acceptors of PSI, to ferredoxin and that the immediate electron donor to ferredoxin is reduced within less than 500 ns. The presence of at least two different types of PSI-ferredoxin complex, all competent in electron transfer, is also deduced from the kinetic behavior.

Identification of the amino acids involved in the functional interaction between photosystem I and ferredoxin from Synechocystis sp. PCC 6803 by chemical cross-linking.

Ferredoxin isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been chemically cross-linked to purified photosystem I from the same organism. The reaction was catalyzed by N-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of N-hydroxysulfosuccinimide. A short reaction time and neutral pH values can be used in the presence of the two reagents, ensuring the integrity of both of the proteins and the iron-sulfur cluster of the ferredoxin. The only covalent complex detected comprised ferredoxin and the photo-system I (PSI)-D subunit, as identified by antibodies probing after electrophoresis. Electron paramagnetic resonance measurements of this covalent complex have shown that the cross-linked ferredoxin was entirely photoreducible by photosystem I and that the molar ratio of ferredoxin to PSI was close to 1. Extensive sequencing of the peptides obtained after proteolysis of the purified cross-linked product led to the identification of a covalent bond between glutamic acid 93 of ferredoxin and lysine 106 of the PSI-D subunit.

Structural organization of the iron-sulfur centers in Synechocystis 6803 photosystem I. EPR study of oriented thylakoid membranes and analysis of the magnetic interactions.

The structural properties of the iron-sulfur centers of photosystem I (PSI) from the cyanobacterium Synechocystis 6803 have been investigated by EPR spectrometry. The stoichiometry of centers A, B, and X, determined by EPR intensity measurements, gives direct evidence for center X being a [4Fe-4S] center in the native system and for the core reaction center protein being a dimer. The directions of the magnetic axes of centers A, B, and X were accurately determined by EPR experiments carried out on membrane fragments oriented on thin Mylar films. These directions are very similar to those previously reported for plants and algae. To get a detailed description of the relative arrangement of A and B, the magnetic interactions between these centers have been analyzed through numerical simulations of X-band and Q-band EPR spectra. The relative orientation of the magnetic axes deduced from these simulations is fully consistent with that given by oriented multilayer experiments. Numerical simulations of X-band and Q-band EPR spectra given by spinach PSI lead to a very similar set of structural parameters, which demonstrates that the functional unit of PSI is highly conserved in all photosynthetic organisms. Moreover, the results of these studies indicate that the A-B direction is close to the membrane normal, which supports a sequential electron transfer mechanism between the iron-sulfur centers in PSI.

Ferredoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803.

The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.

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.

Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: selection of mutants defective in photosynthesis.

Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Kmr) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Kmr gene into an AvaII restriction site of the gene psbD-1. All others harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.