A novel, efficient method to derive bovine and mouse embryonic stem cells with in vivo differentiation potential by treatment with 5-azacytidine.

Pluripotent embryonic stem cells (ESCs) were first isolated nearly three decades ago from mice, yet efficient ESC isolation has been limited to rodents and primates to date. We report a novel and robust technique for isolating ESCs from mammalian pre-implantation embryos by altering the epigenotype of embryonic explants and using pressed zona pellucida-free blastocysts. We first examined this technique for murine ESC derivation. Compared with controls, murine ESCs were efficiently derived when explants were exposed to 1 µM 5-azacytidine, an epigenetic modifier that causes DNA demethylation (56.1% vs 31.6%; P < 0.01). Mouse ESCs stained positively for alkaline phosphatase, expressed markers of pluripotency including Oct4, Rex1 and SSEA1 and formed teratomas when injected into Severe Combined Immuno-Deficient (SCID) mice. The approach was subsequently used for bovine ESC derivation. In bovine a higher concentration of 5-azacytidine (5 µM) was required to elicit a response. This technique resulted in up to 18 times more efficient isolation of pluripotent cells than traditional methods (71.4% vs 4.0%; P < 0.001). These putative bovine ESCs expressed OCT4, REX1 mRNA and SSEA-1 and SSEA-4 proteins; and were able to form embryoid bodies in vitro and teratomas when injected in Severe Combined Immuno Deficient (SCID) mice. This is the first report on derivation of ESCs with both in vitro and in vivo differentiation potential in a livestock species.

Redox-linked structural changes in ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Class I RNRs are composed of two homodimeric proteins, alpha2 and beta2. The class Ia E. coli beta2 contains dinuclear, antiferromagnetically coupled iron centers and one tyrosyl free radical, Y122*/beta2. Y122* acts as a radical initiator in catalysis. Redox-linked conformational changes may accompany Y122 oxidation and provide local control of proton-coupled electron transfer reactions. To test for such redox-linked structural changes, FT-IR spectroscopy was employed in this work. Reaction-induced difference spectra, associated with the reduction of Y122* by hydroxyurea, were acquired from natural abundance, (2)H(4) tyrosine, and (15)N tyrosine labeled beta2 samples. Isotopic labeling led to the assignment of a 1514 cm(-1) band to the upsilon19a ring stretching vibration of Y122 and of a 1498 cm(-1) band to the upsilon7a CO stretching vibration of Y122*. The reaction-induced spectra also exhibited amide I bands, at 1661 and 1652 cm(-1). A similar set of amide I bands, with frequencies of 1675 and 1651 cm(-1), was observed when Y* was generated by photolysis in a pentapeptide, which matched the primary sequence surrounding Y122. This result suggests that reduction of Y122* is linked with structural changes at nearby amide bonds and that this perturbation is mediated by the primary sequence. To explain these data, we propose that a structural perturbation of the amide bond is driven by redox-linked electrostatic changes in the tyrosyl radical aromatic ring.

Evaluation of influent prefermentation as a unit process upon biological nutrient removal.

The objective of this NSF sponsored research was to provide a controlled comparison of identical continuous flow biological nutrient removal (BNR) processes both with and without prefermentation in order to provide a stronger, more quantitative, technical basis for design engineers to determine the potential benefits of prefermentation to EBPR in treating domestic wastewater. Specifically, this paper focused upon the potential impacts of primary influent prefermentation upon BNR processes treating septic domestic wastewater. This study can be divided into two distinct phases--an initial bench-scale phase which treated septic P-limited (TCOD:TP>40) wastewater and a subsequent pilot-scale phase which treated septic COD-limited (TCOD:TP<40) wastewater. The following conclusions can be drawn from the results obtained to date. Prefermentation increased both RBCOD, SBCOD and VFA content of septic domestic wastewater. Prefermentation resulted in increased biological P removal for a highly septic, non-P limited (TCOD:TP<40:1) wastewater. However, in septic, P-limited (TCOD:TP>40:1) wastewater, changes in net P removal due to prefermentation were suppressed by limited P availability, even though P release and PHA content were affected. Prefermentation increased specific anoxic denitrification rates for both COD and P-limited wastewaters, and in the pilot (COD-limited) study also coincided with greater system N removal.

Iron-sulfur clusters in type I reaction centers.

Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy. The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors. Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC. It is comprised of an interpolypeptide [4Fe-4S] cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal [4Fe-4S] clusters, F(A) and F(B), that are bound to the PsaC subunit. In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs. The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin. The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-). We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.

Photoinduced transient absorbance spectra of P840/P840(+) and the FMO protein in reaction centers of Chlorobium vibrioforme.

The kinetics of photoinduced absorbance changes in the 400-ns to 100-ms time range were studied between 770 and 1025 nm in reaction center core (RCC) complexes isolated from the green sulfur bacterium Chlorobium vibrioforme. A global, multiple stretched-exponential analysis shows the presence of two distinct but strongly overlapping spectra. The spectrum of the 70-micros component consists of a broad bleaching with two minima at 810 and 825 nm and a broad positive band at wavelengths greater than 865 nm and is assigned to the decay of (3)Bchl a of the Fenna-Matthews-Olson (FMO) protein. The contribution of the 70-micros component correlates with the amount of FMO protein in the isolated RCC complex. The spectrum of the 1.6-micros component has a sharp bleaching at 835 nm, a maximum at 805 nm, a broad positive band at wavelengths higher than 865 nm, and a broad negative band at wavelengths higher than 960 nm. When the RCC is incubated with inorganic iron and sulfur, the 1.6-micros component is replaced by a component with a lifetime of approximately 40 micros, consistent with the reconstruction of the F(X) cluster. We propose that the 1.6-micros component results from charge recombination between P840(+) and an intermediate electron acceptor operating between A(0) and F(X). Our studies in Chlorobium RCCs show that approaches that employ a single wavelength in the measurement of absorption changes have inherent limitations and that a global kinetic analysis at multiple wavelengths in the near-infrared is required to reliably separate absorption changes due to P840/P840(+) from the decay of (3)Bchl a in the FMO protein.

The bound electron acceptors in green sulfur bacteria: resolution of the g-tensor for the F(X) iron-sulfur cluster in Chlorobium tepidum.

The photosynthetic reaction center (RC) of green sulfur bacteria contains two [4Fe-4S] clusters named F(A) and F(B), by analogy with photosystem I (PS I). PS I also contains an interpolypeptide [4Fe-4S] cluster named F(X); however, spectroscopic evidence for an analogous iron-sulfur cluster in green sulfur bacteria remains equivocal. To minimize oxidative damage to the iron-sulfur clusters, we studied the sensitivity of F(A) and F(B) to molecular oxygen in whole cells of Chlorobium vibrioforme and Chlorobium tepidum and obtained highly photoactive membranes and RCs from Cb. tepidum by adjusting isolation conditions to maximize the amplitude of the F(A)(-)/F(B)(-) electron paramagnetic resonance signal at g = 1.89 (measured at 126 mW of microwave power and 14 K) relative to the P840(+) signal at g = 2.0028 (measured at 800 microW of microwave power and 14 K). In these optimized preparations we were able to differentiate F(X)(-) from F(A)(-)/F(B)(-) by their different relaxation properties. At temperatures between 4 and 9 K, isolated membranes and RCs of Cb. tepidum show a broad peak at g = 2.12 and a prominent high-field trough at g = 1.76 (measured at 126 mW of microwave power). The complete g-tensor of F(X)(-), extracted by numerical simulation, yields principal values of 2.17, 1.92, and 1. 77 and is similar to F(X) in PS I. An important difference from PS I is that because the bound cytochrome is available as a fast electron donor in Chlorobium, it is not necessary to prereduce F(A) and F(B) to photoaccumulate F(X)(-).

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

Recruitment of a foreign quinone into the A(1) site of photosystem I. I. Genetic and physiological characterization of phylloquinone biosynthetic pathway mutants in Synechocystis sp. pcc 6803.

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters.

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.

Establishment of pluripotent cell lines from vertebrate species--present status and future prospects.

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse and chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals.

Role of D1-His190 in the proton-coupled oxidation of tyrosine YZ in manganese-depleted photosystem II.

To further characterize the role of D1-His190 in the oxidation of tyrosine Y(Z) in photosystem II, the pH dependence of P(680)(*)()(+) reduction was measured in H190A and Mn-depleted wild-type PSII particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803. Measurements were conducted in the presence and absence of imidazole and other small organic bases. In H190A PSII particles, rapid reduction of P(680)(*)()(+) attributed to electron transfer from Y(Z) increased dramatically above pH 9, with an apparent pK(A) of approximately 10.3. In the presence of ethanolamine and imidazole, this dramatic increase occurred at lower pH values, with the efficiency of Y(Z) oxidation correlating with the solution pK(A) value of the added base. We conclude that the pK(A) of Y(Z) is approximately 10.3 in D1-H190A PSII particles. In Mn-depleted wild-type PSII particles, P(680)(*)()(+) reduction was accelerated by all exogenous bases examined (substituted imidazoles, histidine, Tris, and 1,4-diazabicyclo[2.2.2]octane). We conclude that Y(Z) is solvent accessible in Mn-depleted wild-type PSII particles and that its pK(A) is near that of tyrosine in solution. In Mn-depleted wild-type PSII particles, over 80% of the kinetics of P(680)(*)()(+) reduction after a flash could be described by three kinetic components. The individual rate constants of these components varied slightly with pH, but their relative proportions varied dramatically with pH, showing apparent pK(A) values of 7.5 and 6.25 (6.9 and 5.8 in the presence of Ca(2+) and Mg(2+) ions). An additional pK(A) value (pK(A) < 4.5) may also be present. To describe these data, we propose (1) the pK(A) of His190 is 6.9-7.5, depending on buffer ions, (2) the deprotonation of Y(Z) is facilitated by the transient formation of a either a hydrogen bond or a hydrogen-bonded water bridge between Y(Z) and D1-His190, and (3) when protonated, D1-His190 interacts with nearby residues having pK(A) values near 6 and 4. Because Y(Z) and D1-His190 are located near the Mn cluster, these residues may interact with the Mn cluster in the intact system.

The cysteine-proximal aspartates in the Fx-binding niche of photosystem I. Effect of alanine and lysine replacements on photoautotrophic growth, electron transfer rates, single-turnover flash efficiency, and EPR spectral properties.

The FX electron acceptor in Photosystem I (PS I) is a highly electronegative (Em = -705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566PsaB and D557PsaB on electron transfer through FX by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566APsaB, D557APsaB, D566KPsaB, and D557KPsaB) or in pairs (D557APsaB/D566APsaB and D557KPsaB/D566APsaB). All mutants except for D557KPsaB/D566APsaB grew photoautotrophically, but the growth of D557KPsaB and D557APsaB/D566APsaB was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557KPsaB and D557APsaB/D566APsaB relative to the wild type and the other mutants. Nevertheless, the rates of NADP+ photoreduction in PS I complexes from all mutants were no less than 75% of that of the wild type. The kinetics of back-reaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors FA and FB in PS I complexes from all mutants. The EPR spectrum of FX was identical to that in the wild type in all but the single and double D566APsaB mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PS I rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through FX.

Photosystem I is indispensable for photoautotrophic growth, CO2 fixation, and H2 photoproduction in Chlamydomonas reinhardtii.

Certain Chlamydomonas reinhardtii mutants deficient in photosystem I due to defects in psaA mRNA maturation have been reported to be capable of CO2 fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L. , and Mets, L. J. (1995) Nature 376, 438-441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364-367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of either psaA or psaC; these activities could be restored to the F8-psaADelta mutant by reintroduction of psaA. We observed limited O2 photoevolution in mutants lacking photosystem I; use of 18O2 indicated that this O2 evolution is coupled to O2 uptake (i.e. respiration) rather than CO2 fixation or H2 evolution. We conclude that the reported instances of CO2 fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I.

Electrometrical study of electron transfer from the terminal FA/FB iron-sulfur clusters to external acceptors in photosystem I.

An electrometrical technique was used to investigate electron transfer between the terminal iron-sulfur centers F(A)/F(B) and external electron acceptors in photosystem I (PS I) complexes from the cyanobacterium Synechococcus sp. PCC 6301 and from spinach. The increase of the relative contribution of the slow components of the membrane potential decay kinetics in the presence of both native (ferredoxin, flavodoxin) and artificial (methyl viologen) electron acceptors indicate the effective interaction between the terminal 14Fe-4S] cluster and acceptors. The finding that FA fails to donate electrons to flavodoxin in F(B)-less (HgCl2-treated) PS I complexes suggests that F(B) is the direct electron donor to flavodoxin. The lack of additional electrogenicity under conditions of effective electron transfer from the F(B) redox center to soluble acceptors indicates that this reaction is electrically silent.

Role of D1-His190 in proton-coupled electron transfer reactions in photosystem II: a chemical complementation study.

Recent models for water oxidation in photosystem II propose that His190 of the D1 polypeptide facilitates electron transfer from tyrosine YZ to P680+ by accepting the hydroxyl proton from YZ. To test these models, and to further define the role of D1-His190 in the proton-coupled electron transfer reactions of PSII, the rates of P680+ reduction, YZ oxidation, QA- oxidation, and YZ* reduction were measured in PSII particles isolated from several D1-His190 mutants constructed in the cyanobacterium Synechocystis sp. PCC 6803. These measurements were conducted in the absence and presence of imidazole and other small organic bases. In all mutants examined, the rates of P680+ reduction, YZ oxidation, and YZ* reduction after a single flash were slowed dramatically and the rate of QA- oxidation was accelerated to values consistent with the reduction of P680+ by QA- rather than by YZ. There appeared to be little correlation between these rates and the nature of the residue substituted for D1-His190. However, in nearly all mutants examined, the rates of P680+ reduction, YZ oxidation, and YZ* reduction were accelerated dramatically in the presence of imidazole and other small organic bases (e.g., methyl-substituted imidazoles, histidine, methylamine, ethanolamine, and TRIS). In addition, the rate of QA- oxidation was decelerated substantially. For example, in the presence of 100 mM imidazole, the rate of electron transfer from YZ to P680+ in most D1-His190 mutants increased 26-87-fold. Furthermore, in the presence of 5 mM imidazole, the rate of YZ* reduction in the D1-His190 mutants increased to values comparable to that of Mn-depleted wild-type PSII particles in the absence of imidazole. On the basis of these results, we conclude that D1-His190 is the immediate proton acceptor for YZ and that the hydroxyl proton of YZ remains bound to D1-His190 during the lifetime of YZ*, thereby facilitating the reduction of YZ*.

Electrogenicity accompanies photoreduction of the iron-sulfur clusters F(A) and F(B) in photosystem I.

Photovoltage responses accompanying electron transfer on the acceptor side of photosystem I (PS I) were investigated in proteoliposomes containing PS I complexes from the cyanobacterium Synechococcus sp. PCC 6301 using a direct electrometrical technique. The relative contributions of the F(X) --> F(B) and the F(X) --> F(A) electron transfer reactions to the overall electrogenicity were elucidated by comparing the sodium dithionite-induced decrease in the magnitude of the total photoelectric responses in control and in F(B)-less (HgCl2-treated) PS I complexes. The results obtained suggest that the electrogenesis on the acceptor side of PS I is related to electron transfers between both F(X) and F(A) and F(A) and F(B). Based on the electrogenic nature of the latter reaction in PS I complexes, we conclude that F(A) rather than F(B) is the acceptor proximal to F(X).

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.

Electrogenic reduction of the primary electron donor P700+ in photosystem I by redox dyes.

The kinetics of reduction of the photo-oxidized primary electron donor P700+ by redox dyes N,N,N',N'-tetramethyl-p-phenylendiamine, 2,6-dichlorophenol-indophenol and phenazine methosulfate was studied in proteoliposomes containing Photosystem I complexes from cyanobacteria Synechocystis sp. PCC 6803 using direct electrometrical technique. In the presence of high concentrations of redox dyes, the fast generation of a membrane potential related to electron transfer between P700 and the terminal iron-sulfur clusters F(A)/F(B) was followed by a new electrogenic phase in the millisecond time domain, which contributes approximately 20% to the overall photoelectric response. This phase is ascribed to the vectorial transfer of an electron from the redox dye to the protein-embedded chlorophyll of P700+. Since the contribution of this electrogenic phase in the presence of artificial redox dyes is approximately equal to that of the phase observed earlier in the presence of cytochrome c6, it is likely that electrogenic reduction of P700+ in vivo occurs due to vectorial electron transfer within RC molecule rather than within the cytochrome c6-P700 complex.

Strains of synechocystis sp. PCC 6803 with altered PsaC. I. Mutations incorporated in the cysteine ligands of the two [4Fe-4S] clusters FA and FB of photosystem I.

Two [4Fe-4S] clusters, FA and FB, function as terminal electron carriers in Photosystem I (PS I), a thylakoid membrane-bound protein-pigment complex. To probe the function of these two clusters in photosynthetic electron transport, site-directed mutants were created in the transformable cyanobacterium Synechocystis sp. PCC 6803. Cysteine ligands in positions 14 or 51 to FB and FA, respectively, were replaced with aspartate, serine, or alanine, and the effect on the genetic, physiological, and biochemical characteristics of PS I complexes from the mutant strains were studied. All mutant strains were unable to grow photoautotrophically, and compared with wild type, mixotrophic growth was inhibited under normal light intensity. The mutant cells supported lower rates of whole-chain photosynthetic electron transport. Thylakoids isolated from the aspartate and serine mutants have lower levels of PS I subunits PsaC, PsaD, and PsaE and lower rates of PS I-mediated substrate photoreduction compared with the wild type. The alanine and double aspartate mutants have no detectable levels PsaC, PsaD, and PsaE. Electron transfer rates, measured by cytochrome c6-mediated NADP+ photoreduction, were lower in purified PS I complexes from the aspartate and serine mutants. By measuring the P700(+) kinetics after a single turnover flash, a large percentage of the backreaction in the aspartate and serine mutants was found to be derived from A1 and FX, indicating an inefficiency at the FX --> FA/FB electron transfer step. The alanine and double aspartate mutants failed to show any backreaction from [FA/FB]-. These results indicate that the various mutations of the cysteine 14 and 51 ligands to FB and FA affect biogenesis and electron transfer differently depending on the type of substitution, and that the effects of mutations on biogenesis and function can be biochemically separated and analyzed.

Strains of Synechocystis sp. PCC 6803 with altered PsaC. II. EPR and optical spectroscopic properties of FA and FB in aspartate, serine, and alanine replacements of cysteines 14 and 51.

A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1. 944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.