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• W2058006951 abstract "A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of FX, FB, and FA was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and rubA genes were insertionally inactivated. In isolated PS I trimers, the decay of P700+ measured in the near-IR and the decay of A1– measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 μs. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700+ and PQ-9–. The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A1 binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the 3P700 product of P700+A0− charge recombination via the T0 spin level in those PS I complexes that do not contain a functional quinone. In those A1 sites that are occupied, the P700+Q– polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the A1 binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A1 site in the menB rubA mutant due to the absence of FX and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A1 site by washing. However, all A1 sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the A1 site makes the menB rubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I. A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of FX, FB, and FA was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and rubA genes were insertionally inactivated. In isolated PS I trimers, the decay of P700+ measured in the near-IR and the decay of A1– measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 μs. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700+ and PQ-9–. The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A1 binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the 3P700 product of P700+A0− charge recombination via the T0 spin level in those PS I complexes that do not contain a functional quinone. In those A1 sites that are occupied, the P700+Q– polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the A1 binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A1 site in the menB rubA mutant due to the absence of FX and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A1 site by washing. However, all A1 sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the A1 site makes the menB rubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I. Photosystem I is a multisubunit, pigment-protein complex that is found in the membranes of plants, algae, and cyanobacteria and that mediates the light-induced transfer of electrons from plastocyanin/cytochrome c6 to ferredoxin/flavodoxin. According to current understanding, light-induced charge separation results in the oxidation of the primary electron donor P700 (E′m +430 mV), a chlorophyll a/a′ heterodimer located on the luminal (inner) side of the membrane, and the reduction of the primary electron acceptor A0 (E′m approximately –1000 mV), a chlorophyll a monomer located in the interior of the membrane. The electron is passed to A1 (E′m approximately –800 mV), an alkyl-substituted menadione (2-methyl-1,4-naphthalenedione); to FX (E′m –705 mV), an interpolypeptide (4Fe-4S) cluster; and finally to FA (E′m –520 mV) and FB (E′m –580 mV), which are (4Fe-4S) clusters bound to the extrinsic subunit PsaC located on the stromal (cytoplasmic) side of the membrane. In most organisms, including Synechocystis sp. PCC 6803, the quinone in the A1 site is phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone), but in Euglena gracilis and Anacystis nidulans, the quinone is 5′-monohydroxyphylloquinone (1.Ziegler K. Maldener I. Lockau W. Z. Naturforsch. 1989; 44: 468-472Crossref Scopus (16) Google Scholar), and in the red alga Cyanidium caldarium it is menaquinone-4 (MQ-4) 1The abbreviations used are: MQ-4, menaquinone-4; PS I, photosystem I; PS II, photosystem II; Car, carotenoid; Chl, chlorophyll; PhQ, phylloquinone; PQ-9, plastoquinone-9; Q, quinone (e.g. AQ, 9,10-anthraquinone, in the A1 site of the menB and menB rubA mutants); Fe/S, iron-sulfur cluster, either FX, FB, or FA; P700, chlorophyll a/a′ heterodimer that represents the primary electron donor; β-DM, n-dodecyl-β-d-maltopyranoside; CW, continuous wave; TR EPR, time-resolved or transient EPR; A, absorptive EPR signal, E, emissive EPR signal; DAS, decay-associated spectrum; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; mT, milliteslas. (2.Yoshida E. Nakamura A. Watanabe T. Anal. Sci. 2003; 19: 1001-1005Crossref PubMed Scopus (23) Google Scholar). Our approach to studying structural and functional relationships involving A1 is to replace the native quinone in PS I with quinones that have different thermodynamic and structural properties but are still able to mediate electron transfer from A0 to the Fe/S clusters. The replacement of the quinone can be accomplished either in vitro using chemical extraction and reconstitution protocols (3.Itoh S. Iwaki M. Ikegami I. Biochim. Biophys. Acta. 2001; 1507: 115-138Crossref PubMed Scopus (72) Google Scholar) or in vivo using genetic approaches (4.Johnson T.W. Golbeck J. Lenci F. CRC Handbook of Organic Photochemistry and Photobiology. CRC Press, Inc., Boca Raton, FL2003: 119-1-119-14Google Scholar). In the latter method, the menA or menB genes (5.Zybailov B. van der Est A. Zech S.G. Teutloff C. Johnson T.W. Shen G. Bittl R. Stehlik D. Chitnis P.R. Golbeck J.H. J. Biol. Chem. 2000; 275: 8531-8539Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 6.Johnson T.W. Shen G. Zybailov B. Kolling D. Reategui R. Beauparlant S. Vassiliev I.R. Bryant D.A. Jones A.D. Golbeck J.H. Chitnis P.R. J. Biol. Chem. 2000; 275: 8523-8530Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 7.Johnson T.W. Zybailov B. Jones A.D. Bittl R. Zech S. Stehlik D. Golbeck J.H. Chitnis P. in.J. Biol. Chem. 2001; 276: 31512-31521Google Scholar) or the menD or menE genes (8.Johnson W.T. Naithani S. Stewart C. Zybailov B. Daniel Jones A. Golbeck J.H. Chitnis P.R. in.Biochim. Biophys. Acta. 2003; 1557: 67-76Crossref PubMed Scopus (30) Google Scholar), which code for enzymes in the phylloquinone (PhQ) biosynthetic pathway, have been interrupted in Synechocystis sp. PCC 6803. In the absence of PhQ, PS I recruits plastoquinone-9 (PQ-9), which is normally associated with PS II, into the A1 site. When present in the quinone binding site of PS II, PQ-9 has a midpoint potential of 68 mV (9.Golbeck J.H. Kok B. Biochim. Biophys. Acta. 1979; 547: 347-360Crossref PubMed Scopus (71) Google Scholar) to 80 mV (10.Krieger A. Rutherford A.W. Johnson G.N. Biochim. Biophys. Acta. 1995; 1229: 193-201Crossref Scopus (146) Google Scholar), but when PQ-9 occupies the A1 site of PS I, it has an estimated midpoint potential of –670 mV (11.Semenov A.Y. Vassiliev I.R. van der Est A. Mamedov M.D. Zybailov B. Shen G. Stehlik D. Diner B.A. Chitnis P.R. Golbeck J.H. J. Biol. Chem. 2000; 275: 23429-23438Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). PQ-9 can be displaced both in vivo (7.Johnson T.W. Zybailov B. Jones A.D. Bittl R. Zech S. Stehlik D. Golbeck J.H. Chitnis P. in.J. Biol. Chem. 2001; 276: 31512-31521Google Scholar) and in vitro (12.Pushkar Y.N. Golbeck J.H. Stehlik D. Zimmermann H. J. Phys. Chem. B. 2004; 108: 9439-9450Crossref Scopus (45) Google Scholar) by a variety of substituted naphthoquinones, including authentic phylloquinone, thus allowing detailed spectroscopic analyses of this essential cofactor. In the experiments described in this paper, we extend our studies of PS I complexes that contain PQ-9 in the A1 site to a mutant in which the Fe/S clusters FX, FB, and FA are also missing. This was achieved in Synechococcus sp. PCC 7002 by interrupting the menB gene, which codes for 1,4-dihydroxy-2-naphthoate synthase (5.Zybailov B. van der Est A. Zech S.G. Teutloff C. Johnson T.W. Shen G. Bittl R. Stehlik D. Chitnis P.R. Golbeck J.H. J. Biol. Chem. 2000; 275: 8531-8539Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 6.Johnson T.W. Shen G. Zybailov B. Kolling D. Reategui R. Beauparlant S. Vassiliev I.R. Bryant D.A. Jones A.D. Golbeck J.H. Chitnis P.R. J. Biol. Chem. 2000; 275: 8523-8530Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 7.Johnson T.W. Zybailov B. Jones A.D. Bittl R. Zech S. Stehlik D. Golbeck J.H. Chitnis P. in.J. Biol. Chem. 2001; 276: 31512-31521Google Scholar, 11.Semenov A.Y. Vassiliev I.R. van der Est A. Mamedov M.D. Zybailov B. Shen G. Stehlik D. Diner B.A. Chitnis P.R. Golbeck J.H. J. Biol. Chem. 2000; 275: 23429-23438Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), as well as the rubA gene, which codes for a membrane-bound rubredoxin (13.Shen G. Antonkine M.L. van der Est A. Vassiliev I.R. Brettel K. Bittl R. Zech S.G. Zhao J. Stehlik D. Bryant D.A. Golbeck J.H. J. Biol. Chem. 2002; 277: 20355-20366Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14.Shen G. Zhao J. Reimer S.K. Antonkine M.L. Cai Q. Weiland S.M. Golbeck J.H. Bryant D.A. J. Biol. Chem. 2002; 277: 20343-20354Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). We isolated PS I complexes from the resulting menB rubA mutant and evaluated the kinetics of charge recombination between P700+ and PQ-9–. The structural and functional properties of PQ-9 correspond to those in the menB mutant. We additionally show that these PS I complexes can reversibly incorporate 9,10-anthraquinone into the A1 binding site more efficiently than in the menB mutant. Construction of the menB and menB rubA Mutants—The menB mutant was constructed in Synechocystis sp. PCC 6803 as described previously (6.Johnson T.W. Shen G. Zybailov B. Kolling D. Reategui R. Beauparlant S. Vassiliev I.R. Bryant D.A. Jones A.D. Golbeck J.H. Chitnis P.R. J. Biol. Chem. 2000; 275: 8523-8530Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Wild type and mutant strains of Synechococcus sp. PCC 7002 were grown as described previously (14.Shen G. Zhao J. Reimer S.K. Antonkine M.L. Cai Q. Weiland S.M. Golbeck J.H. Bryant D.A. J. Biol. Chem. 2002; 277: 20343-20354Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15.Frigaard N.-U. Sakuragi Y. Bryant D.A. Methods Mol. Biol. 2004; 274: 325-340PubMed Google Scholar). A DNA fragment containing the menB gene was cloned and sequenced from the genome of Synechococcus sp. PCC 7002 (GenBank™ accession number AY563042). This menB gene was identified by the high sequence similarity (89%) of its product with that of sll1127 (menB) of Synechocystis sp. PCC 6803. The accC1 gene, which was derived from plasmid pMS266 after restriction digestion with PstI and which confers gentamicin resistance, was inserted into the unique PstI site of the menB coding region. This construct was used to transform cells of the kanamycin-resistant rubA mutant of Synechococcus sp. PCC 7002 as described in Ref. 14.Shen G. Zhao J. Reimer S.K. Antonkine M.L. Cai Q. Weiland S.M. Golbeck J.H. Bryant D.A. J. Biol. Chem. 2002; 277: 20343-20354Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar. Full segregation of the menB::aacC1 and menB alleles was verified by PCR analysis. Preparation of PS I Complexes—Cells of Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 were broken using a French pressure cell operated at 4 °C at 120 megapascals. Thylakoid membranes were solubilized using n-dodecyl-β-d-maltopyranoside (β-DM), and PS I trimers were isolated by sucrose density ultracentrifugation according to previously published procedures (14.Shen G. Zhao J. Reimer S.K. Antonkine M.L. Cai Q. Weiland S.M. Golbeck J.H. Bryant D.A. J. Biol. Chem. 2002; 277: 20343-20354Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Quinone and Chlorophyll Analysis—Quinones were extracted from 20 μl of a solution of PS I complexes with 400 μl of acetone/methanol (7:2, v/v) by vigorous vortexing for 3 min. After centrifugation and filtration through a PTFE filter membrane with a 0.2-μm pore size (Whatman International Ltd., Maldstone, UK), the extract in the organic solvent phase was injected directly into an Agilent Technology 1100 series HPLC system equipped with a reverse-phase SUPELCO Discovery® C18 column (25 cm × 4.6 mm, 5 μm). Separation and elution were performed with a linear gradient of solvent A (100% methanol) and B (100% isopropyl alcohol) according to the following protocol: 80%:20% (v/v) solvent A/B for 10 min, a linear gradient from 80%:20% (v/v) to 20%:80% (v/v) of solvent A/B in 30 min, and 20%:80% (v/v) solvent A/B for 5 min. The flow rate was 0.75 ml min–1. Detection of eluates was performed with a diode array detector (Agilent 1100 series). MQ-4 and PQ-9 were quantified using extinction coefficients of 18.9 mm–1 cm–1 at 270 nm (16.Dunphy P.J. Brodie A.F. Methods Enzymol. 1971; 18: 407-461Crossref Scopus (84) Google Scholar) and 15.2 mm–1 cm–1 at 254 nm (17.Dilley R.A. Crane F. Anal. Biochem. 1963; 5: 531-541Crossref PubMed Scopus (23) Google Scholar), respectively. The HPLC assignments were confirmed by mass spectrometry using atmospheric pressure chemical ionization with a Perspective Biosystems Mariner time-of-flight mass spectrometer operated in the negative ion mode. Chlorophylls and carotenoids were extracted from whole cells with 100% methanol and from thylakoids with 80% (v/v) acetone. The optical density was measured using a Cary 14 spectrophotometer, and the chlorophyll concentration was calculated according to MacKinney (18.MacKinney G. J. Biol. Chem. 1941; 140: 315-322Abstract Full Text PDF Google Scholar) and Lichtenthaler (19.Lichtenthaler H.K. in.Methods Enzymol. 1987; 148: 350-382Crossref Scopus (9395) Google Scholar). Anthraquinone Exchange into the menB and menB rubA Mutants—A 100-fold molar excess of 9,10-anthraquinone (10 μl of 0.034 m 9,10-anthraquinone in Me2SO) was added to PS I complexes (150 μl in Tris-HCl buffer pH 8.3 containing 0.2% Triton X-100) isolated from the menB and menB rubA mutant strains of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, respectively. The incubation was carried out at room temperature (2–4 h) with vigorous stirring. In an additional step, the PS I complexes were washed twice by ultrafiltration with 150 μl of buffer solution to remove the excess 9,10-anthraquinone as well as exchanged PQ-9. The washed PS I complexes were resuspended in 150 μl of buffer and stored at –80 °C. Time-resolved Optical Spectroscopy in the Near-IR—Optical studies in the near-IR were conducted using a laboratory-built, time-resolved spectrophotometer. The high frequency roll-off amplifier described in Ref. 20.Vassiliev I.R. Jung Y.S. Mamedov M.D. Semenov A.Y. Golbeck J.H. Biophys. J. 1997; 72: 301-315Abstract Full Text PDF PubMed Scopus (83) Google Scholar was not used to ensure resolution of the kinetic phases in the submicrosecond range. A tunable titanium-sapphire laser (Schwartz Electro-Optics, Orlando, FL) was pumped at 532 nm by using a 5-watt, frequency-doubled CW YAG laser (Millenia® Series; Spectra Physics) and provided the 820-nm measuring beam. The actinic flash was provided by a frequency-doubled, Nd-YAG laser (Quanta-Ray DCR-11, Spectra Physics). For most studies, the excitation flash intensity was adjusted to ∼2 mJ cm–2, which is just sufficient to saturate P700 under the conditions employed. For studies of the dependence of the signal intensity on the excitation flash energy, the energy was varied from 1 mJ cm–2 to 80 mJ cm–2. The flash energy was adjusted by the timing of the Q-switch and by the use of neutral density filters. PS I complexes were diluted under anaerobic conditions with 50 mm Tris-HCl buffer, pH 8.3, to a final concentration of 50 μg/ml Chl (∼0.5 μm P700). Sodium ascorbate and 1,6-dichlorophenolindophenol were added to final concentrations of 2 mm and 5 μm, respectively. β-DM was added to a final concentration of 0.04% (w/v) to reduce light scattering. A differential extinction coefficient of 8000 m–1 cm–1 at 820 nm was used for calculations of P700+/P700 concentration. Time-resolved Optical Spectroscopy in the Visible—Optical studies in the visible wavelength range were conducted using a laboratory-built, pump-probe spectrometer described in Ref. 21.Cohen R.O. Shen G. Golbeck J.H. Xu W. Chitnis P.R. Valieva A.I. van der Est A. Pushkar Y. Stehlik D. Biochemistry. 2004; 43: 4741-4754Crossref PubMed Scopus (88) Google Scholar. Measuring flashes were supplied by a xenon lamp and selected by a ¾ meter monochrometer incorporating a 10-cm × 10-cm interference grating blazed at 350 nm. The bandwidth of the measuring flash was 5 nm, and the data were recorded in 5-nm intervals from 400 to 600 nm. Excitation flashes were provided by a Q-switched, Nd:YAG laser (Brilliant®, Quantel S. A., Les Ulis Cedex, France) equipped with an optical parametric oscillator (Vibrant Arrow 355, type II crystal) tuned to 685 nm. The photodiodes were protected with cyan subtractive dichroic filters (Edmund H52-536). The sample was placed in a 10 × 10-mm quartz cuvette perpendicular to the direction of the excitation flash. An identical sample was placed in a 10 × 10-mm quartz cuvette in the reference beam. Each data point represents the average of 16 measurements taken at a flash spacing of 20 s. An extinction coefficient of 242,000 m–1 cm–1 at 515 nm is used for calculations of 3Car/1Car concentration (22.Bensasson R. Land E.J. Biochim. Biophys. Acta. 1973; 325: 175-181Crossref PubMed Scopus (108) Google Scholar). It should be noted that this value is taken from pulse radiolysis measurements of β-carotene in hexane, and hence the extinction coefficient of 3Car/1Car in PS I may differ considerably. Time-resolved Optical Spectroscopy in the Near-UV—Optical studies in the UV were conducted using a pulse probe spectrometer described in Ref. 23.Diner B.A. McIntosh L. Photosynthesis: Molecular Biology of Energy Capture. 297. Academic Press Inc., San Diego1998: 337-360Google Scholar. The monochromator slit was fixed at 4 mm, equivalent to a bandwidth of 8 nm. Excitation flashes were provided by a xenon flashlamp filtered by Schott and Kodak Wratten 34 filters. The photodiodes were protected with Corion Solar Blind UV-transmitting filters. The optical path length of the cuvette was 1 cm. Each data point represents the average of eight measurements, taken with a flash spacing of 20 s. A background measurement was obtained similarly, except that the sample was shielded from the detecting flash to allow for correction of the actinic flash artifact. The absorbance shown represents the difference between the two measurements. The differential extinction coefficient of PQ-9–/PQ-9 at the peak in the UV is reported as 13,000 m–1 cm–1 in solution (22.Bensasson R. Land E.J. Biochim. Biophys. Acta. 1973; 325: 175-181Crossref PubMed Scopus (108) Google Scholar). Data Analysis—Multiexponential fits of the optical kinetic data were performed using the Marquardt algorithm in Igor Pro version 3.14 (Wavemetrics Inc., Lake Oswego, OR) running on a Macintosh computer. For global analyses in the visible region, individual kinetics were analyzed first. The results of these analyses were used for fitting the whole set of data to global lifetimes, and the best solution was chosen based on the analysis of χ2, standard errors of the parameters, and the residuals. In several instances, several closely spaced kinetic components were required to fit the data at the longer times. A stretched multiexponential fitting routine was employed in such cases (24.Vassiliev I.R. Antonkine M.L. Golbeck J.H. Biochim. Biophys. Acta. 2001; 1507: 139-160Crossref PubMed Scopus (103) Google Scholar); the stretch parameter, βi, assumes a value between 0 and 1. This equation represents a robust solution of a general equation for kinetics with a distributed time constant; in the case when βi = 1, the equation turns into a sum of simple exponentials. CW EPR Spectroscopy of the Mutant PS I Complexes at X-Band— EPR spectra of PS I complexes isolated from the wild type, the menB mutant, and the menB rubA mutant were obtained using a Bruker ECS-106 spectrometer equipped with an Oxford temperature controller and cryostat. The instrument conditions for F–A and F–B were as follows: microwave power, 20 milliwatts; temperature, 15 K; and modulation amplitude, 10 G. The sample was suspended to 0.6 mg/ml Chl in 50 mm Tris buffer, pH 8.3, containing 10 mm sodium ascorbate and 30 μm 2,6-dichlorophenol-indophenol. The instrument conditions for F–X were as follows: microwave power, 80 milliwatts; temperature, 6 K; and modulation amplitude, 32 G. The sample was suspended to 0.6 mg/ml Chl in 100 mm glycine buffer, pH 10.0, containing 10 mm sodium hydrosulfite. Transient EPR Spectroscopy at X-band and Q-Band—Low temperature, X-band (9-GHz) transient EPR experiments were performed with a laboratory-built spectrometer using a Bruker ER046 XK-T microwave bridge equipped with an ER-4118X-MD-5W1 dielectric ring resonator and using an Oxford CF935 helium gas flow cryostat (25.van der Est A. Hager-Braun C. Leibl W. Hauska G. Stehlik D. Biochim. Biophys. Acta. 1998; 1409: 87-98Crossref PubMed Scopus (48) Google Scholar). The loaded Q value for this dielectric ring resonator was about 3000, equivalent to a rise time of τr = Q/(2π *νmw) ≈ 50 ns. Q-band (35-GHz) transient EPR spectra of the samples were measured with the same set-up except that a Bruker ER 056 QMV microwave bridge, equipped with a home-built cylindrical resonator, was used. All samples contained 1 mm sodium ascorbate as an external electron donor and were frozen in the dark. The samples were illuminated using a Spectra Physics Nd-YAG laser system operating at the second harmonic (533 nm) and a repetition rate of 10 Hz. Construction of the menB rubA Mutant in Synechococcus sp. PCC 7002—The menB gene of Synechococcus sp. PCC 7002 was inactivated by inserting the aacC1 gene, conferring gentamicin resistance, from plasmid pMS266 into the unique PstI site within the coding sequence of the gene (Fig. 1, a and b). After transformation of this construction into the rubA mutant of Synechococcus sp. PCC 7002, segregation of the menB::aacC1 and menB alleles in the rubA mutant strain was analyzed by PCR. As expected for the parental strain (Fig. 1a), the PCR using the designed primers resulted in a product of 1.0 kb (Fig. 1c, lane 1). In the transformed rubA strain, however, the 1.0-kb product is absent, and a new product of 2.1 kb was detected (Fig. 1c, lane 2). The difference in the sizes of the PCR products from the parental and transformant strains corresponds to the size of the inserted 1.1-kb gentamicin resistance cartridge (Fig. 1, a and b). The data show that the transformed rubA mutant strain is homozygous for the menB::aacC1 allele. HPLC Pigment Analysis of Synechococcus sp. PCC 7002— Authentic PhQ and PQ-9 elute at 22 and 36 min, respectively, using the HPLC protocol described under “Materials and Methods.” When the pigment extracts from whole cells and PS I complexes from wild-type Synechococcus sp. PCC 7002 were analyzed, no UV-absorbing compounds were detected at 22 min. Further examination of the chromatogram led to the discovery of a new peak eluting at 14 min that showed an intense UV absorption with maxima at 248, 263, 270, and 332 nm. The spectrum is consistent with a 1,4-naphthoquinoid compound having alkyl substitutions at the C2 and C3 positions (16.Dunphy P.J. Brodie A.F. Methods Enzymol. 1971; 18: 407-461Crossref Scopus (84) Google Scholar). Mass spectroscopic analysis showed that the compound has an m/z of 444 as opposed to an m/z of 450 for PhQ. This difference is most easily explained by the presence of a geranylgeranyl tail with four fully unsaturated isoprenoid units, which is characteristic of MQ-4. Thus, Synechococcus sp. PCC 7002 probably synthesizes 2-methyl-3-all-trans-tetraisoprenyl-1,4-naphthalenedione (MQ-4) instead of 2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-1,4-naphthalenedione (PhQ). The transient EPR spectra of wild-type PS I complexes isolated from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 were identical at both X- and Q-bands, indicating that the difference in the degree of unsaturation of the C3 tail has no detectable influence on the orientation of the 1,4-naphthalenedione head group in the A1 site (data not shown). When the pigment extracts from whole cells of the menB rubA mutant were analyzed, no UV-absorbing material eluted at 14 min. This indicates that the menB homolog indeed encodes 1,4-dihydroxynaphthoate synthase in Synechococcus sp. PCC 7002 and that it is required for the synthesis of MQ-4. The absence of MQ-4 was confirmed in pigment extracts from PS I complexes; however, a peak appeared at 36 min with an m/z of 748 characteristic of PQ-9. This observation is in agreement with a previous study in which the interruption of PhQ biosynthesis in Synechocystis sp. PCC 6803 results in the incorporation of PQ-9 into the PS I (6.Johnson T.W. Shen G. Zybailov B. Kolling D. Reategui R. Beauparlant S. Vassiliev I.R. Bryant D.A. Jones A.D. Golbeck J.H. Chitnis P.R. J. Biol. Chem. 2000; 275: 8523-8530Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). These results indicate that the interruption of 1,4-dihydroxy-2-naphthoate synthase in Synechococcus sp. PCC 7002 similarly results in the incorporation of PQ-9 into PS I. Low Temperature CW EPR Spectroscopy in PS I Complexes from the menB rubA Mutant—The FX, FA, and FB iron-sulfur clusters were not detected in PS I complexes isolated from the menB rubA mutant when measured by low temperature CW X-band EPR spectroscopy (data not shown). The iron-sulfur clusters were similarly not detected when PS I complexes were treated with sodium hydrosulfite at pH 10.0 in an attempt to reduce FA− and FB−; additionally, no spectrum was obtained by illuminating the same sample during freezing in an attempt to photoaccumulate FX−. These results are identical to those for PS I complexes isolated from the rubA mutant and indicate that all three (4Fe-4S) clusters are missing from the PS I complexes of the menB rubA mutant as expected (13.Shen G. Antonkine M.L. van der Est A. Vassiliev I.R. Brettel K. Bittl R. Zech S.G. Zhao J. Stehlik D. Bryant D.A. Golbeck J.H. J. Biol. Chem. 2002; 277: 20355-20366Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14.Shen G. Zhao J. Reimer S.K. Antonkine M.L. Cai Q. Weiland S.M. Golbeck J.H. Bryant D.A. J. Biol. Chem. 2002; 277: 20343-20354Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Flash-induced Absorbance Changes in the Near-IR and UV Regions—Fig. 2 (top) shows decay kinetics measured at 820 nm in PS I complexes isolated from the menB rubA mutant. Under the conditions employed, the absorbance change after a saturating flash corresponds primarily to the decay of P700+. The best fit to the data results in kinetic phases with lifetimes (stretch parameters) of 1.9 μs (0.92), 10.3 μs (0.97), and 315 μs (0.74). The longest kinetic phase represents the major component to the overall signal amplitude (∼75%), and the stretch parameter indicates that this value encompasses a broader distribution of lifetimes than the faster components. Fig. 2 (bottom) shows decay kinetics measured at 315 nm in PS I complexes isolated from the menB rubA mutant. In this spectral region, the flash-induced absorbance change corresponds primarily to the decay of PQ-9–. The best fit to the data results in kinetic phases with lifetimes (stretch parameters) of 15 μs (1.00), 392 μs (1.00), and a long lived residual. (Due to the time resolution of the spectrometer, it could not be determined whether the 1.9-μs kinetic phase measured in the near-IR is also present in the near-UV.) The similarity in the lifetimes of the two slower kinetic components in the near-IR and near-UV suggests that these two kinetic phases can be assigned to the same process, namely charge recombination between P700+ and PQ-9–. The total absorbance change (excluding the unassigned 1.9-μs component) in the near-IR (Fig. 2, top) corresponds to 223 nm P700+, which is equivalent to 251 Chl/P700 given that the total Chl a content in the sample was 56 μm (50 μg/ml Chl a). Similarly, the total absorption change in the near-UV (Fig. 2, bottom) corresponds to 46 nm PQ-9–, which is equivalent to 239 Chl/P700, given that the total Chl a content in the sample was 11 μm" @default.
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• W2058006951 title "Recruitment of a Foreign Quinone into the A1 Site of Photosystem I" @default.
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