Biochemical and Biophysical Characterization of Photosystem Ifrom Phytoene Desaturase and �-Carotene DesaturaseDeletion Mutants of Synechocystis Sp. PCC 6803EVIDENCE FOR PsaA- AND PsaB-SIDE ELECTRON TRANSPORT IN CYANOBACTERIA*
Received for publication, January 21, 2005, and in revised form, March 4, 2005Published, JBC Papers in Press, March 9, 2005, DOI 10.1074/jbc.M500809200
James A. Bautista‡§, Fabrice Rappaport¶§, Mariana Guergova-Kuras¶, Rachel O. Cohen�,John H. Golbeck�**, Jamie Yehong Wang‡, Daniel Beal¶, and Bruce A. Diner‡ ‡‡
From the ‡Central Research and Development, Experimental Station, E. I. du Pont de Nemours & Co.,Wilmington, Delaware 19880-0173, ¶UMR 7141 CNRS/University Paris 6, Institut de Biologie Physico-Chimique,13 rue Pierre et Marie Curie, 75005 Paris, France, the �Department of Biochemistry and Molecular Biology and the**Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802
In photosystem I, oxidation of reduced acceptor A1�
through iron-sulfur cluster FX is biphasic with half-times of �5–30 ns (“fast” phase) and �150–300 ns (“slow”phase). Whether these biphasic kinetics reflect unidirec-tional electron transfer, involving only the PsaA-sidephylloquinone or bi-directional electron transfer, in-volving both the PsaA- and PsaB-side phylloquinones,has been the source of some controversy. Brettel(Brettel, K. (1988) FEBS Lett. 239, 93–98) and Joliot andJoliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38,11130–11136) have attributed to nearby carotenoidselectrochromic band shifts, accompanying A1 reduction,centered at �450 and 500–510 nm. As a test of theseassignments, we separately deleted in Synechocystis sp.PCC 6803 the genes that encode phytoene desaturase(encoded by crtP (pds)) and �-carotene desaturase (en-coded by crtQ (zds)). The pds� and zds� strains synthe-size phytoene and �-carotene, respectively, both ofwhich absorb to shorter wavelength than �-carotene.Compared with wild type, the mutant A1
�(FeS) �A1(FeS)� difference spectra, measured in cells and pho-tosystem I complexes, retain the electrochromic bandshift centered at 450 nm but show a complete loss of theelectrochromic band shifts centered at 500–510 nm.Thus, the latter clearly arise from �-carotene. In thewild type, the electrochromic band shift of the slowphase (centered at 500 nm) is shifted by 6 nm to the bluecompared with the fast phase (centered at 506 nm).Thus, the carotenoid pigments acting as electrochromicmarkers during the fast and slow phases of A1
� oxida-tion are different, indicating the involvement of boththe PsaA- and the PsaB-side phylloquinones in photo-system I electron transport.
The photosystem I (PS I)1 reaction center is a multisubunitintrinsic membrane protein complex that functions as a light-driven plastocyanin/ferredoxin oxidoreductase. At the core of PSI is a heterodimer of two highly homologous subunits, PsaA andPsaB, which contain the redox components involved in the firststeps of photoinduced electron transfer. Light energy initiatesprimary electron transfer such that the oxidizing equivalent re-sides on P700, a heterodimer of chlorophyll a (Chl a) and itsC132-epimer Chl a� (1–3). In chronological order, the electronacceptors are as follows: A0 (Chl a), A1 (phylloquinone or vitaminK1), FX (a [4Fe-4S] cluster), and the terminal electron acceptorsFA and FB (both [4Fe-4S] clusters). All are bound by PsaA andPsaB except for FA and FB, which are bound to subunit PsaC. ThePS I complex also includes two additional small extrinsicpolypeptides, PsaD and PsaE, which mediate the docking offerredoxin (4), the soluble electron carrier responsible for thereduction of NADP�, and PsaF, which facilitates the binding ofplastocyanin, the mobile electron carrier responsible for the re-reduction of the oxidized donor P700� (5–7).
In addition to these redox cofactors, carotenoids and otherChls a are also bound within the PS I complex. A 2.5 Å x-raycrystallographic structure of the trimeric PS I reaction centerfrom the thermophilic cyanobacterium Thermosynechococcuselongatus was recently reported (2). Resolved in each monomerof the crystal structure were 22 carotenoids, 96 chlorophylls a,2 phylloquinones, and 3 [4Fe-4S] clusters. The two phylloqui-none molecules are symmetrically located with respect to theC2 symmetry axis of the protein heterodimer. Whether thePsaB-side quinone as well as the PsaA-side quinone are activefor electron transfer has been the subject of considerable recentdebate. Optical studies have shown that the reoxidation of A1
�
is biphasic (t1⁄2 of 5–30 ns and 150–300 ns) (8, 9), but theassignments and interpretations of these two kinetic phasesare still controversial (10). To explain the biphasic kinetics,Brettel and co-workers (8, 10, 11) originally proposed a modelwhere rapid equilibration between A1 and FX would give rise tothe faster phase of A1
� reoxidation, whereas the depletion ofthis quasi-equilibrium by electron transfer to FA/FB would give
* This work was supported by the National Research Initiative of theUnited States Department of Agriculture Cooperative State Research,Education and Extension Service, Grant 2001-35318-11270 (toB. A. D.), the National Science Foundation Grant MCB-0117079 (toJ. H. G.), the CNRS, and the College de France (to F. R. and M. G.-K.).This paper is contribution 8518 of the Central Research and Develop-ment Department of the E. I. du Pont de Nemours & Co. The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.‡‡ To whom correspondence should be addressed: Central Research
and Development, Experimental Station, E. I. du Pont de Nemours &Co., Wilmington, DE 19880-0173. Tel.: 302-695-2494; Fax: 302-695-9183; E-mail: [email protected].
1 The abbreviations used are: PS I, photosystem I; PS II, photosystemII; A0, primary electron acceptor; A1, secondary electron acceptor;DCPIP, dichlorophenolindophenol; �-DM, n-dodecyl �-D-maltoside;DCMU, 3-(3�,4�-dichlorophenyl)-1,1-dimethylurea; FX, FA, FB, [4Fe-4S]clusters; HPLC, high performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; P700, primary electron donor of PS I;PhQA, phylloquinone bound by PsaA; Vit, vitamin; WT, wild type; Zds,�-carotene desaturase (encoded by crtQ (zds)); Chl, chlorophyll.
rise to the slower phase. The same phylloquinone was proposedto participate in both phases. Joliot and Joliot (12) showed,however, that the relative amplitudes of the fast and slowphases were insensitive to the membrane potential, inconsist-ent with the attribution of the fast phase to the formation ofequilibrated state, A1
�FX ^ A1FX�, an electrogenic electron
transfer step (13). They suggested that the biphasic kineticscould come from either of the following two models: (i) a singlephylloquinone molecule in two different conformational stateshaving different A1
� reoxidation rates, or (ii) two differentphylloquinones, one on each side of the reaction center andoxidized by FX at different rates. The former would constitute aunidirectional pathway, and the latter would be bidirectional.The x-ray structure reveals no obvious differences between thebinding pockets of the two phylloquinones nor a difference inthe phylloquinone-FX edge-to-edge distances (6.8 Å). Recently,Guergova-Kuras et al. (14), using site-directed mutants ofChlamydomonas reinhardtii, provided the first experimentalsupport for the participation of both the PsaA-side and PsaB-side quinones in electron transfer to FX. Site-directed replace-ment of the tryptophan directly �-stacked to the PsaA-sidephylloquinone with phenylalanine (PsaA-W693F) selectivelyslowed the slower phase of A1
� reoxidation, whereas the samemutation of the corresponding tryptophan on the PsaB side(PsaB-W673F) selectively slowed the faster phase. Xu and co-workers (10), using the cyanobacterium Synechocystis sp. PCC6803, confirmed the room temperature optical results on thePsaA-W693F and the PsaB-W673F mutations, but althoughtransient EPR studies provided experimental evidence forPsaA-side electron transfer, there was no indication of PsaB-side electron transfer. Similarly, Cohen et al. (15) constructedsite-directed mutants in which the axial methionine ligands tochlorophylls eC-A3 and eC-B3 were changed to leucine, andthey found an effect on the transient EPR signal only on thePsaA-side mutant. They concluded that electron transfer oc-curs primarily along the PsaA branch in cyanobacteria, al-though a minor fraction of electron transfer along the PsaBbranch could not be excluded. Fairclough and co-workers (16),however, constructed a site-directed replacement with histi-dine of a methionine ligand to chlorophyll eC-A3 in C. rein-hardtii, PsaA-M684H, that still allows photoautotrophicgrowth despite having blocked electron transfer to the PsaAbranch phylloquinone. This observation implies that the PsaBbranch is active for electron transfer and that its activity issufficient to support photoautotrophic growth. This mutationand the corresponding PsaB-side ligand replacement, PsaB-M664H, were constructed by Fairclough et al. (16) and byRamesh et al. (17). Both mutations block or slow electron trans-fer from A0
� to A1 on their respective branches, implying thatin C. reinhardtii both PsaA and PsaB branches are active inelectron transport.
All of the carotenoids in the electron density map derivedfrom the x-ray crystal structure of PS I were modeled as �-car-otene (2). Seventeen were in an all-trans configuration. Twowere modeled as 9-cis; another showed a 13-cis configuration,and two were shown with 2-cis double bonds: 9,9�-di-cis and9,13�-di-cis (for numbering, see Fig. 1 and Ref. 18). The carot-enoids are arranged in six clusters with each cluster containing2, 3, or 6 carotenoids. (see Fig. 9 of Ref. 3). In cyanobacteria andin other carotenoid-producing organisms, carotenoids are syn-thesized from isoprenoid precursors. The first step specific forcarotenoid biosynthesis is the head-to-head-condensation ofgeranylgeranyl pyrophosphate (see Fig. 1). This reaction iscatalyzed by phytoene synthase and forms phytoene, a C40
carotenoid with three conjugated double bonds. Phytoene de-saturase (encoded by crtP (pds)) catalyzes the double dehydro-
genation of phytoene to form �-carotene, which possesses sevenconjugated double bonds. Another enzyme, �-carotene desatu-rase (encoded by crtQ (zds)), is responsible for the dehydro-genation of �-carotene to form lycopene, a carotenoid with 11conjugated double bonds. Lycopene cyclase catalyzes the for-mation of �-ionylidene rings at both ends of lycopene to form�-carotene, still with 11 conjugated double bonds but with theterminal double bonds being part of the rings. �-Carotene canthen be hydroxylated in the rings by specific �-carotene hy-droxylases to form �-cryptoxanthin, isocryptoxanthin, and/orzeaxanthin (Fig. 1).
Brettel (11) and Joliot and Joliot (12) proposed that thereduction of the PS I phylloquinone electron acceptor, A1, pro-duces electrochromic band shifts centered at 450 and 500–510nm of one or more nearby carotenoid molecules. According tothe x-ray crystal structure, several carotenoids with differentorientations and configurations and modeled as �-carotene areindeed present in the immediate vicinity of each of the twophylloquinone molecules (Fig. 2). If both branches of the elec-tron transport chain were active, then each of the phylloquino-nes should show electrochromic shifts of two different sets ofcarotenoids, the absorption spectra of which might be slightlydifferent. To examine this possibility, we have altered the ca-rotenoid biosynthetic pathway to replace �-carotene with carot-enoids having shorter �-conjugation, the absorbance bands ofwhich are blue-shifted compared with �-carotene. This replace-ment should shift the carotenoid electrochromism to shorterwavelengths, allowing the WT carotenoid band shifts associ-ated with A1
� formation to be clearly identified.The results presented here show that associated with each of
the two phases of the A1� reoxidation is the loss of electrochromic
band shifts on two different sets of carotenoid molecules withdifferent absorbance spectra. These spectral differences are con-sistent with the differences in configuration of the carotenoidsclose to phylloquinones on the PsaA and PsaB branches as seenin the x-ray crystal structure. These results therefore support amodel for electron transport in PS I in which both the PsaA- andPsaB-side phylloquinones participate in electron transfer to FX.
FIG. 1. The biosynthetic pathway leading to ringed carote-noids in cyanobacteria starting from geranylgeranyl pyrophos-phate (GGPP). The molecules are depicted in the all-trans configura-tion. The standard numbering is given in the last structure.
Carotenoids Sense PsaA- and PsaB-side Electron Transport 20031
Construction of Mutants—The genes for phytoene desaturase (pds)and �-carotene desaturase (zds) were deleted separately in the genome ofWT Synechocystis sp. PCC 6803 and replaced by a kanamycin resistancecassette. A PCR copy of the gene to be deleted (pds or zds) that included500 bases upstream and downstream of the gene was ligated onto apGEM-T plasmid vector (Promega A1360) using T4 DNA ligase andamplified in XL1-Blue Escherichia coli competent cells (Stratagene200249). In this resulting plasmid, the first and last 50 bases of the genetogether with the ligated pGEM-T plasmid vector between them werecopied by PCR using primers designed to give unique restriction enzymesites to the termini of the PCR product. The PCR product was digestedwith the appropriate restriction enzymes and then ligated using T4 DNAligase to a kanamycin resistance cassette with the same sticky ends as thePCR product. This final plasmid construct was then amplified in XL1-Blue E. coli competent cells and used to transform the WT Synechocystissp. PCC 6803 strain. Transformation of Synechocystis sp. PCC 6803 WTstrain was as described in Nixon et al. (19). Successful transformants wereselected on Petri plates containing BG-11 medium, 5 mM glucose, 50 �g/mlkanamycin and grown in very dim light (�0.1 �mol photons m�2 s�1) at30 °C. Kanamycin-resistant colonies representing successful transfor-mants were replated seven times in BG-11 plates containing 5 mM glucoseand 200 �g/ml kanamycin to obtain full segregation. Deletion mutationswere confirmed by extracting total pigments from the cells and analyzingfor the complete absence of �-carotene by HPLC (see below). The cellswere also unable to produce �-carotene under nonselective conditions(without kanamycin).
PS I Preparation—WT Synechocystis sp. PCC 6803 cells were grownin liquid BG-11 containing 5 mM glucose medium as described (20–22).The mutants were grown in BG-11 containing 5 mM glucose in very dimlight (�0.1 �mol photons m�2 s�1). Isolation of the thylakoid mem-branes from these cells was performed as described previously (20, 21).The thylakoid membranes were suspended at a concentration of 1mg/ml chlorophyll in Buffer A (25% glycerol, 20 mM CaCl2, 5 mM MgCl2,50 mM MES, pH 6.0). After addition of a one-ninth volume of 10% (w/v)n-dodecyl �-D-maltoside (�-DM) (final concentration of 1% (w/v)), thesuspension was stirred for 20 min and then centrifuged at 55,000 rpmin a 70Ti rotor (Beckman-Coulter) for 30 min. The supernatant wasthen loaded onto an �500-ml weak anion exchange Toyopearl DEAE-650S (Supelco) column pre-equilibrated with Buffer B (Buffer A con-taining 0.03% �-DM) and washed with Buffer B containing 19 mM
MgSO4. Elution of PS I complex from the column was performed byusing 500 ml of Buffer B, forming an increasing salt gradient from 19 to30 mM MgSO4. At the end of the gradient, the salt concentration wasincreased to 50 mM MgSO4. The eluent fractions containing PS I werepooled and concentrated in a Millipore concentrator utilizing a YM 100Diaflo ultrafiltration membrane. The concentrated samples were thendesalted by passing them through a gel filtration column (Econo-Pac 10DG, Bio-Rad) pre-equilibrated with Buffer B. The samples were thenfrozen in liquid N2 and stored at �80 °C until use.
Pigment Analysis—The chlorophyll content of the cells was deter-mined by extraction with 100% methanol and using the 79.24 ml/mgextinction coefficient of Lichtenthaler (23) at 665 nm. For total pigmentanalysis, PS I complexes were extracted with a 50:50 acetone/methanol
mixture under dim light. The mixture was centrifuged for 2 min(10,000 � g), and a 50-�l aliquot of the supernatant was immediatelyfiltered (0.45 �m, Millex-LH PTFE) and injected onto a reverse phaseHPLC column (Zorbax-ODS, 4.6 mm x 15 cm, Rockland Technologies)equilibrated with methanol/ethyl acetate (68:32, v/v). The separationwas performed in isocratic mode using the same solvent system (24) ata flow rate of 0.25 ml/min for 60 min at room temperature. The relativechlorophyll and carotenoid concentrations were determined by meas-uring the area of their respective peaks in the HPLC and dividing by theextinction coefficients according to Giorgi et al. (24).
EPR Spectroscopy—X-band (9.4 GHz) EPR studies were performedusing a Bruker ECS-106 spectrometer and a standard mode resonator(ST 8615) equipped with a slotted port for light entry. Cryogenic tem-peratures were maintained with a liquid helium cryostat and an ITC-4temperature controller (Oxford, UK). The microwave frequency wasmeasured with a Hewlett-Packard 5340A frequency counter, and themagnetic field was calibrated using �,��-diphenyl-�-picrylhydrazyl asthe standard. Sample temperatures were monitored by a calibratedthermocouple located 3 mm beneath the bottom of the quartz sampletube and referenced to liquid N2. The spectra of FA
�/FB� in WT and
mutant PS I complexes were obtained as described previously (25).Glycine buffer (1 M) was added to bring the pH to 10.0 (100 mM finalconcentration), and sodium dithionite was added to a final concentra-tion of 50 mM. The sample was incubated in darkness on ice for 30 min,after which the sample was frozen in liquid N2 and the EPR spectrumwas recorded at 15 K.
Q-band (34 GHz) EPR studies were performed using a Bruker ER5106 QT-W1 resonator equipped with a port for sample illumination.Cryogenic temperatures were maintained with a Bruker ER4118CVliquid nitrogen cryostat and a Bruker ER4121 temperature control unit.The microwave frequency was measured with a Bruker ER035M NMRgaussmeter. The magnetic field was calibrated with �,�-bisdiphenylene-�-phenylallyl complexed 1:1 with benzene, which has no detectable ganisotropy at 34 GHz. Photoaccumulation of A1
� and FX� in all PS I
samples was carried out as described in Ref. 26. The pH of the samplewas adjusted to 10.0 with glycine buffer (1 M) (final concentration of 100mM), and sodium dithionite was added to a final concentration of 50 mM.After incubation for 20 min in the dark on ice, the sample was frozen inliquid nitrogen and placed in the microwave cavity. A pre-illuminationspectrum was then recorded at 205 K. The sample was then illuminatedwith a 20-milliwatt He-Ne laser for 40 min at 205 K. The laser was thenturned off, and the light-induced spectrum was recorded at the sametemperature. The light-minus-dark difference spectrum at 205 Kshowed primarily A1
�, as signals from FX
� and the other iron-sulfurclusters were only detectable at very low temperatures (less than 20 K).
Optical Spectroscopy—Charge recombination kinetics from FA�/FB
� toP700� nm were measured at 435 nm in a laboratory-built flash-detectionspectrophotometer similar to that originally described by Joliot et al. (27).PS I complexes at a Chl a concentration of 14 �g/ml were suspended in 25mM Tris-HCl (pH 8.3), 10 �M sodium ascorbate, plus 4 �M DCPIP (or 250�M sodium ascorbate without DCPIP), and 0.03% �-DM. Under theseconditions, P700 is fully reduced, and all electron acceptors are oxidizedprior to excitation (26). Charge separation was induced by a saturatingflash (30 mJ) provided by a linear flash lamp pulsed dye laser (model
FIG. 2. The electron transfer cofac-tors from the x-ray crystallographicstructure of PS I from T. elongatus at2.5 Å resolution (2). The figure showsthe clusters of carotenoids near the phyl-loquinones PhyQA and PhyQB. The�-carotenefam carotenoids are color-codedas follows: red, cis; yellow, all-trans. Thephytyl tails of the chlorophylls have beenomitted for simplicity. Note the greaternumber of carotenoid cis double bonds onthe PsaA side as compared with the PsaBside. The coordinates of the structurewere taken from the Brookhaven ProteinData Bank (entry 1JBO).
Carotenoids Sense PsaA- and PsaB-side Electron Transport20032
LFDL-3, Cynosure Inc.) using rhodamine 590 perchlorate (Exciton). Thereduction of P700� by recombination was measured at 435 nm usingdetecting flashes provided by an EG & G FX199U flash lamp and passedthrough a Jobin-Yvon HL300 monochromator.
Measurements of the kinetics of A1� oxidation in the nanosecond to
microsecond time scale were performed on whole cells and isolated PSI complexes in a flash-detection spectrophotometer as described previ-ously (14, 28). Whole cells were suspended in 20 mM HEPES (pH 7.2),20% (w/v) Ficoll, and 5 �M carbonyl cyanide (4-(trifluoromethoxy)phe-nyl)hydrazone. The PS I complexes were suspended at a concentrationof 40 �g/ml under the same conditions as for charge recombination,except that the sodium ascorbate and DCPIP concentrations were 10mM and 40 �M, respectively. Decay-associated spectra of the kineticphases were derived from a global multiexponential fit of the kineticcomponents obtained at each wavelength, using the program MEXFIT(29). Charge separation was induced by a 5-ns (full width at half-maximal) light pulse at 700 nm using a Nd:YAG pumped LDS 698 dyeexciting �70% of the reaction centers. Absorbance changes were fol-lowed from 5 ns to 20 �s using detecting flashes provided by an OPOContinuum (OPO Panther, type II) from 300 to 540 nm, frequencydoubled for wavelengths less than 410 nm. For detection in the UV, afluorescent glass (Sumita Optical Glass, Lumilass-B) was used to con-vert the UV photons to the blue where the blocking filters performbetter.
Measurements of the variable fluorescence yield in whole cells wereused to quantify functional PS II relative to the WT using a Joliot-typeflash detection spectrophotometer as described previously (30). Cellswere suspended in 50 mM HEPES/KOH (pH 7.5) at a concentrationequivalent to an OD730 � 0.9 and were incubated for 10 min in the darkin 0.3 mM p-benzoquinone and 0.3 mM potassium ferricyanide prior tothe start of the experiment. The cells were then incubated in the darkat room temperature in the presence of 20 �M DCMU and 20 mM
NH2OH, the latter added �30 s before the start of the measurement.The variable fluorescence yield of PS II was then measured by detectingflashes following each of 20 saturating light flashes (18 Hz).
RESULTS
Gene Knock-out Mutations—The crtP (pds) and crtQ (zds)genes were deleted independently in Synechocystis sp. PCC6803 as described under “Materials and Methods.” The pds�
and zds� mutants were difficult to select as the gene deletionscaused the mutants to be acutely light-sensitive. This photo-sensitivity was most probably due to the inability of the caro-tenoids with shorter conjugated chains to quench chlorophylltriplet states, which in the presence of light and oxygen lead tothe formation of toxic singlet excited O2 (1�g) (31). Conse-quently, to drive genomic segregation to completion, the pds�
and zds� mutants were selected on BG-11 containing 5 mM
glucose plates prepared with fresh 200 �g/ml kanamycin andgrown under very dim light (�0.1 �mol photons m�2 s�1). Thereduced light intensity resulted in a very low rate of cellgrowth. These knock-out mutations also resulted in the com-plete loss of the PS II reaction centers in both mutant strains,as indicated by the absence of fluorescence induction in thepresence of DCMU and hydroxylamine (see “Materials andMethods”). Several failed attempts at the biochemical isolationof PS II complexes from thylakoid membranes (21) also indi-cated the absence of PS II. The absence of PS II is likely aconsequence of the inability to assemble PS II complexes,where only phytoene or �-carotene is available as a replacementfor �-carotene.
The PS I concentration of the mutants, on a per chlorophyllbasis, was close to that of the WT strain, as equivalent loadingof detergent-solubilized thylakoid membranes (based on chlo-rophyll content) onto the DEAE column leads to similar recov-eries of PS I complexes. However, the amount of chlorophyllisolated from the pds� and zds� strains (chlorophyll/cell) wasapproximately one-half that of the WT strain from cultures ofthe same volume (18 liters) and optical density (OD730 � 1.5).In Fig. 3a, the absorption spectrum of the WT PS I complex iscompared with that of the pds� and zds� strains followingnormalization to the chlorophyll Qy absorption band. Particu-
larly noticeable in the absorption spectra of the mutants is theloss of absorption in the 450–525 nm region, indicating the lossof �-carotene biosynthesis in the mutants. Consistent with thisspectrum and the loss of �-carotene and its derivatives, the PSI complexes isolated from the pds� and zds� strains are bluerin color than their WT homologues. In the zds� strain spectrum(Fig. 3a), however, there is an enhanced absorption in the350–425 nm region, which is attributed to the presence of the7-conjugated double bond carotenoid �-carotene and its deriv-atives (Fig. 3b). �-Carotene absorbs maximally at �400 nm insolvents with low polarizability (32). The pds� cores, on theother hand, show an absorbance spectrum that is essentiallyidentical to that of pure Chl a, indicating the lack of synthesisof both �-carotene and �-carotene. HPLC analysis (see below)confirmed these observations. Instead of �-carotene or �-caro-tene, the pds� strain produces the 3-conjugated double bondcarotenoid phytoene. Phytoene absorbs maximally at �280 nmin solvents with low polarizability (32). In the red region, themaximum absorption corresponding to the Qy transitions of thechlorophylls in the WT PS I complexes peaks at 680.6 nm. Forthe �-carotene desaturase and phytoene desaturase mutants,the maximum absorption of the Qy transition was shifted to678.8 and 679.0, respectively. The possible reasons for this willbe discussed below. Fig. 3b shows the spectral subtractions ofthe Qy-normalized absorbance spectra, WT minus pds� andzds� minus pds�. The former yields the absorbance spectra of�-carotene, together with its isomers and hydroxy derivatives,that are bound to the WT PS I complex, whereas the latter, thatof �-carotene and its isomers, that are bound to the zds� PS Icomplex. The maximum height of the zds� carotenoid absorb-ance spectrum is �35% that of the WT carotenoid absorbancespectrum. As the extinction coefficients of �- and �-carotene arepractically equivalent, the loss of absorption indicates that
FIG. 3. a, absorption spectra of the WT, zds�, and pds� PS I com-plexes after normalization at the Qy absorbance maximum. The insetshows an expansion of the region of maximum absorption of the Qyoptical transitions. b, subtractions of the Qy-normalized spectra of thePS I complexes of WT minus pds� and of zds� minus pds�, revealingthe absorbance spectra of the carotenoids present in the WT and zds�
strains.
Carotenoids Sense PsaA- and PsaB-side Electron Transport 20033
some of the carotenoid-binding sites in the PS I complexes ofthe zds� strain are devoid of carotenoids. This finding is alsoconfirmed by the HPLC analysis below. Also noticeable in thesespectral subtractions is the difference in shape of the cumula-tive absorbance of the carotenoids in the WT (mainly �-caro-tene) versus that in the zds� strain (all �-carotene). The absorb-ance peaks of �-carotene (385, 406, and 432 nm) in the spectralsubtractions are sharper and better defined, whereas those of�-carotene (434, 468, 496 nm) are less so. This is mainly be-cause linear carotenoids tend to have sharper absorption peaksthan carotenoids with cyclic ends (32).
Pigment Analysis—The PS I complex in the x-ray crystalstructure is a trimer with each monomer binding 96 Chls a and22 carotenoid molecules. Fig. 4a shows an HPLC of the totalpigment extract of WT PS I complex run on a reverse phasecolumn. Three peaks for carotenoids are observed together witha peak for Chl a. The absorbance maxima of all of the carote-noid peaks are at 450 nm (not shown), suggesting that thedifferent carotenoids found in PS I all have the same extent ofcarbon-carbon �-electron conjugation. Because the methodused for elution was reverse phase HPLC, we expected that themost nonpolar molecules will elute last. We assigned the peakeluting at �30 min to �-carotene not only because of its positionin the elution profile but also because of its relative abundancecompared with the other carotenoid peaks. This assignmentwas also confirmed by the observation of the same retentiontime and absorption spectrum for a �-carotene standard (notshown). Noticeable in this peak is a shoulder at �32 min thatwe assigned to several forms of cis-�-carotene. The absorbancespectrum of the sample eluting at that time showed a “cis-band,” a feature present at �140 nm to the blue of the red-mostabsorption band of carotenoids. Because of the C2h symmetrypresent in all-trans-carotenoids, this transition was absent orless prominent in all-trans-carotenoids but became more visible
in cis-carotenoids because trans-to-cis isomerization breaks theC2h symmetry (33, 34). We assigned the peak eluting at �10min to all-trans-zeaxanthin, and this was confirmed by anidentical retention time and absorption spectrum of a standardzeaxanthin sample (not shown). Zeaxanthin (Fig. 1, bottom) isisoelectronic with �-carotene and contains two hydroxyl groupsthat are present in each of the terminal �-ionylidene rings,which makes it relatively more polar causing it to elute muchearlier than �-carotene. The peak eluting at �17 min wasassigned to a mixture of cis-�-cryptoxanthin and/or isocrypto-xanthin, which were also found in substantial amounts in PS Ipreparations of Synechococcus elongatus (35). These two caro-tenoids only differ from each other by the position of the hy-droxyl group on the ring (Fig. 1, bottom) and were expected tohave identical absorption spectra (18, 36). For simplicity, themention of �-carotenefam (�-carotene family) will henceforthrepresent all the carotenoids (zeaxanthin, �-cryptoxanthin,and isocryptoxanthin and their cis-trans isomers) “isoelec-tronic” (i.e. the same number of �-conjugated double bonds)with �-carotene.
Fig. 4b shows the HPLC chromatogram of the total pigmentextract for the zds� PS I complex. The absorption spectra of allthe carotenoid peaks have their absorption maxima at �400nm (not shown), which indicates that the carotenoids in thiscomplex are isoelectronic. From the biosynthetic pathway ofcarotenoids for cyanobacteria shown in Fig. 1, it is most likelythat these carotenoids are isomeric �-carotenes, which wouldaccumulate in the absence of the zds gene. However, the pos-sibility of a derivative form of �-carotene with cyclohexenyl or�-ionylidene rings cannot be completely discounted as lycopenecyclase is still present in this mutant and could use �-caroteneas a substrate. These derivatives of �-carotene would also showabsorption spectra similar to acyclic or linear �-carotene be-cause the extent of their �-electron conjugation would not beaffected by the cyclization of the ends of the molecule. Forsimplicity, the mention of �-carotenefam (�-carotene family) inthis paper represents all the isoelectronic carotenoids with 7�-conjugated double bonds produced in this mutant. The mainpeak eluting at �29 min is all-trans-�-carotene, and the smallshoulder before that eluting at �27 min is a cis-�-carotene. Aminor peak eluting at �33 min is assigned to another form of�-carotene, perhaps another cis, where the cis configuration isat or near the end of the �-electron conjugation. The cis-band inthis form has a lower intensity than in central cis-carotenoids(34).
For the pds� mutant complex (Fig. 4c), only one distinctpigment peak is present in addition to Chl a. The peak elutingat �30 min was assigned to phytoene (maximum absorbance at288 nm) (not shown). The earlier peak at �5 min is due to theacetone/methanol solvent front in which the pigment extractswere dissolved before injection into the HPLC.
Table I summarizes the approximate stoichiometry of thechlorophyll and carotenoids extracted from the WT and mutantPS I complexes. The number of carotenoids present here perWT complex (�26, normalized to 96 chlorophylls) is slightlygreater than the 22 reported in the Thermosynechococcus elon-
FIG. 4. HPLCs of total pigment extracts from PS I complexes ofthe WT (a), zds� (b), and pds� strains (c). The pigments wereextracted, and the HPLC was run as described under “Materials andMethods.” The chromatograms were detected at 450, 400, and 288 nm,respectively, for the WT, zds�, and pds� strains.
TABLE ICarotenoid composition of WT and mutant PSI complexes
Strain Carotenoids Mole ratio normalizedto 96 chlorophylls
WT �-Carotene (trans and cis) 22.1�-Cryptoxanthin/isocryptoxanthin 3.1Zeaxanthin 0.9
zds� �-Carotene (trans- and central cis) 10.4�-Carotene (terminal cis- or di-cis) 1.3
pds� Phytoene 3.3
Carotenoids Sense PsaA- and PsaB-side Electron Transport20034
gatus crystal structure (2), possibly reflecting differences in thepreparations. Noticeable in Table I is the reduced amount ofcarotenoid in the mutant PS I complexes. Because the nativeprotein evolved binding �-carotenefam, �-carotenefam and phy-toene were not likely to produce a tight fit in the carotenoid-binding sites as the more saturated carotenoids were nonpla-nar at the ends because of more sp3-hybridized carbon centerswith tetrahedral geometry and greater rotational degrees offreedom. The carotenoids are therefore more loosely bound andmay either not have bound in vivo or may have been lost duringthe purification process.
X- and Q-band EPR—According to the x-ray crystallographicstructure of the PS I reaction center of T. elongatus (2), severalcarotenoids are located in the immediate vicinity of each of thephylloquinones of PS I (Fig. 2). To check if a change in thenumber and structure of carotenoids in PS I has had an effecton the EPR spectra of the electron acceptors, X-band and Q-band EPR spectra were recorded. X-band (9.4 GHz) EPR spec-troscopy was performed on the WT and mutant (pds� and zds�)PS I complexes to examine iron-sulfur clusters FA
�/FB�. The
pH of the samples was adjusted to 10.0, and sodium dithionitewas added to a final concentration of 50 mM (see “Materials andMethods”), and the EPR spectrum was recorded at 15 K. The gvalues and the relative spin concentrations of reduced FA (g �2.05, 1.94, and 1.85) and FB (g � 2.07, 1.92, and 1.88) in thepds� and zds� PS I mutants were identical to those of the WT(Fig. 5). Similar spectra were recorded when the samples wereadjusted to pH 8.3 in the presence of sodium ascorbate andilluminated during freezing to 15 K (data not shown). Thus, theabsence of and/or replacement of �-carotenefam with carote-noids of shorter conjugated length in PS I did not affect eitherthe ability to form FA
�/FB� or the electronic structures of the
FA�/FB
� iron-sulfur clusters at low temperature.Fig. 6 shows the Q-band (34 GHz) EPR spectra of the WT and
the mutant PS I complexes after photoaccumulation at 205 K,a technique that generates A1
� and FX� in a background of
chemically prereduced FA�/FB
�. Note that at 205 K, signalsfrom the iron-sulfur clusters are not detectable, hence thespectra shown are mainly due to A1
� and perhaps a smallamount of A0
�. The WT and mutant spectra very closely re-sembled each other. At 34 GHz, the field-dependent g anisot-ropy dominated the spectrum of A1
�, which allowed for thepartial resolution of the turning points at gxx � 2.0062 andgzz � 2.0021, values that agree with previous studies (26,37–39). Because the gyy component of the tensor is hidden bythe methyl proton hyperfine lines that arise from the high spindensity at carbon 2 of the phylloquinone anion radical (A1
�), itis hard to determine its exact value. To better resolve the valueof the gyy tensor, PS I from Synechocystis sp. PCC 6803 cellsgrown in 92% D2O were used in a similar experiment (37) tosuppress the intensity of the proton hyperfine couplings. Deu-teration resulted in the narrowing of the line widths of the gcomponents and revealed a value of 2.0051 for the gyy compo-nent in WT PS I (37). The Q-band EPR spectra indicated thatthe g tensor values and the hyperfine couplings for phyllo-semiquinone were virtually identical in the WT and mutant
FIG. 7. Kinetics of A1� oxidation in the WT (f), zds� (‚), and
pds� (E) PS I complexes monitored at 380 (left) and 480 nm(right) following an actinic flash exciting 70% of the reactioncenters. The PS I complexes were suspended as described under “Ma-terials and Methods.”
FIG. 5. EPR spectra of reduced FA and FB in the WT, zds�, andpds� PS I complexes of Synechocystis sp. PCC 6803. The sampleswere resuspended at a concentration of 1 mg of Chl a ml�1 in 100 mM
glycine (pH 10) containing 50 mM sodium dithionite. The spectra wererecorded in the dark at 15 K. Spectrometer settings were as follows:microwave power, 20 milliwatts; microwave frequency, 9.478 GHz; re-ceiver gain, 6.3 � 104; modulation amplitude, 10 G at 100 kHz; centerfield, 3480 G; scan width, 1740 G. The spectra shown are the average ofthree scans.
FIG. 6. Illuminated-minus-dark difference Q-band continuouswave EPR spectra of A1
� in WT, zds�, and pds� PS I complexesat 205 K. The samples were suspended to a concentration of 1 mg of Chla ml�1 in Buffer A (see “Materials and Methods”) to which were added100 mM glycine buffer (pH 10) and 50 mM sodium dithionite. Thepreillumination and post-illumination spectra were recorded as de-scribed under “Materials and Methods.” The instrument settings wereas follows: microwave power, 1 milliwatt; microwave frequency, 34.056GHz; modulation frequency, 100 kHz; modulation amplitude, 1 G; timeconstant, 10 ms; conversion time, 10 ms; 50 scans were averaged.
Carotenoids Sense PsaA- and PsaB-side Electron Transport 20035
strains. We concluded that the ability to photoreduce phyllo-semiquinone and the electronic structure of phyllosemiquinonein the WT and mutant PS I reaction centers was insensitive tothe absence and/or the replacement of �-carotenefam with�-carotenefam or phytoene.
Electron Transfer from A1� to FX—Fig. 7 shows kinetic
traces of the absorbance changes at 380 and 480 nm after theflash-induced formation of the phyllosemiquinone anion A1
�
for the WT, pds�, and zds� PS I complexes. The transientswere best fit with double exponential functions. Similar kineticprofiles were also determined at different wavelengths from300–540 nm in whole cells and PS I complexes, and the kineticdata were subjected to global fit analysis. The results aresummarized in Table II. For the WT PS I complexes, the fasterdecay component has a t1⁄2 � 8–9 ns (20–25% of total decayamplitude estimated by comparing the relative amplitudes ofthe UV signals at 380–390 nm of Fig. 8, see below), whereas theslower component has a t1⁄2 � 152–180 ns (�75–80% of totaldecay). These time constants and relative amplitudes weresimilar to previous determinations on different cyanobacterialPS I preparations (9), although considerable variability in therelative amplitudes of the fast and slow phases has been foundwith PS I complexes from the eukaryote, spinach, C. rein-hardtii, and Chlorella. In spinach, depending on preparationmethods, the ratio of the amplitudes of the fast to the slowcomponent varied considerably from 2:1 to 1:2, and in wholecells of C. reinhardtii and Chlorella, the difference spectrashowed 1:2 and 1:1 relative amplitudes, respectively. For thepds� and zds� strains, the relative amplitudes of the fast andslow phases were similar to the WT. However, the time con-stants in the PS I mutant complexes were somewhat longer,particularly for the slow phase (1.5 times) compared with WT(see Table II).
To verify that the fast and slow phases indeed arise from A1�
oxidation, the global exponential fits for these phases werecompared in the UV and in the near-UV where the phyllo-semiquinone minus phylloquinone difference spectrum is ex-pected to contribute most strongly. Fig. 8 shows the A1
�(FeS) �A1(FeS)� difference spectra from 300 to 540 nm obtained byglobal exponential fit analysis of the kinetic data for the WT,pds�, and zds� PS I complexes. The slow phase in the UV andnear-UV light difference spectra of the WT shows peaks at 375,405, and 430 nm, with a shoulder at 340 nm and a minimum at315 nm. In the fast phase difference spectrum, there is a peakat 400 and 425 nm, but the shorter wavelength peak, theshoulder, and the minimum are all shifted by 10–15 nm to thered with respect to the slow phase. In the case of the mutants,however, the fast and slow phase difference spectra lookedmore similar to each other except for the presence of a 340 nmshoulder in the slow phase difference spectra and a small 385nm feature present in the fast phase difference spectra. Thecontribution of A1
�(FeS) � A1(FeS)� is expected to be rela-
tively small in this region. With the possible exception of acontribution from FeS[X]
� FeS[A/B] �FeS[X]FeS[A/B]� to the
spectrum of the slow phase (e.g. at 340 nm), the similarity ofthe fast and slow phase difference spectra to each other and tothe Chromatium menasemiquinone-menaquinone (Vit K2
�� �Vit K2) difference spectra (40) supports the attribution of thefast and slow phases largely to the oxidation of phyllosemiqui-none. The PS I phyllosemiquinone-phylloquinone (Vit K1
�� �Vit K1) difference spectra, however, appear to be shifted toshorter wavelength by about 30 nm, compared with the differ-ence spectra reported by Romijn and Amesz (40).
Fig. 8 shows the absorption difference spectra of the fast andslow phases of the A1
�(FeS) � A1(FeS)� difference spectra ofthe WT, zds�, and pds� PS I cores to 540 nm. Some of the mostmarked features of the visible difference spectrum were locatedat �440 nm, where the contribution of the phyllosemiquinoneminus phylloquinone difference spectrum was expected to besmall and relatively flat (40). The A1
�(FeS) � A1(FeS)� for theWT showed a marked second derivative-shaped signal centeredat �450 nm for both the fast and slow phases. Another deriv-ative-shaped signal was centered at �506 nm for the fast phaseand �500 nm for the slow phase. The spectral differencesbetween the fast and slow phases in this region have not beenreported previously, as the earlier work did not extend beyond500 nm (44). All of these features have been attributed toelectrochromic band shifts of carotenoids coupled to A1
� reduc-tion (11, 12). The construction of the pds� and zds� strainsprovided a test of these assignments. As shown in Fig. 8, theloss of �-carotenefam results in the complete disappearance ofthe 500 and 506 nm features but only a small change in the 450nm-centered feature. Clearly the former was due entirely to�-carotenefam, whereas the latter was not. The absence of the500 and 506 nm features in the pds� and zds� spectra wascaused either by the replacement of �-carotenefam by moresaturated carotenoids, phytoene or �-carotene, respectively,which are at least 50 nm blue-shifted compared with �-caro-tenefam, or the absence of carotenoid in the carotenoid-bindingsites that were nearest to A1. The second derivative-shapedfeature centered at 450 nm was likely due to an electrochromicband shift of a chlorophyll or chlorophylls located close to A1
�.The minimum of this signal appears at 448 and 452 nm for thefast and slow phases, respectively.
Also part of the fit for the global decomposition of the tran-sient kinetics was a component that remained following therelaxations in the submicrosecond range. As the only speciesremaining are P700� and FeS[A/B]
�, this component corre-sponds to the difference spectrum P700� FeS[A/B]
� � P700FeS[A/B]. Because the contribution of the FeS[A/B]
� � FeS[A/B]
was so much smaller than that of P700� � P700, this spectrumcorresponded primarily to the latter. This difference spectrum,shown in Fig. 9, shows a large bleaching at 435 nm and aderivative-shaped spectrum centered at about 518 nm. The
TABLE IIt1⁄2 and relative contributions of the fast and slow phases of A1
� oxidation
PS I cores
WT zds� pds�
Average 380–390 nm 480 nm Average 380–390 nm 480 nm Average 380–390 nm 480 nm
latter feature disappeared in the P700� FeS[A/B]� � P700
FeS[A/B] difference spectrum in the pds� and zds� strains,indicating that this feature was most likely due to a �-caro-tenefam that is located close to P700.
To better resolve the spectra arising from carotenoid electro-chromism coupled to A1
�(FeS) � A1(FeS)� and to P700�
FeS[A/B]� � P700 FeS[A/B], we calculated the double difference
spectra, [A1�(FeS) � A1(FeS)�]WT � [A1
�(FeS) �A1(FeS)�]mutant and [P700� FeS[A/B]
� � P700 FeS[A/B]]WT �[P700� FeS[A/B]
� � P700 FeS[A/B]]mutant for the fast and slowphases of A1
�(FeS) � A1(FeS)� and for P700� FeS[A/B]� � P700
FeS[A/B] (Fig. 10). The spectra were normalized to the 430 nmsignal of P700� FeS[A/B]
� � P700 FeS[A/B] measured at 20 �s.In principle, all absorbance changes other than those arisingfrom the carotenoids alone should disappear. It should be keptin mind, however, that spectral subtractions in the region ofstrong chlorophyll absorption corresponded to differences be-
tween two large signals, thus creating a greater uncertainty inthe difference spectra in this region. The A1 double differencefor the WT � zds� and for the WT � pds� both show verysimilar three peaked differences, consistent with their arisingfrom electrochromic red shifts of carotenoids. Both the posi-tions of the inflections (corresponding to the carotenoid absorb-ance maxima) and their displacement from each other (30–35nm) were consistent with their identification with the �-caro-tenefam. A comparison of the fast and slow phases in both casesindicated that the slow phase was displaced to the blue relativeto the fast phase by �6 nm. The difference between the twophases implied that the carotenoids that detected the fast andslow phases were located in different environments. This dif-ference in the locations of the carotenoid band shifts was alsoobserved in WT cells (Fig. 11), indicating that it is an intrinsicproperty of the reaction center and not an artifact of detergentisolation. The reasons for this displacement will be discussedbelow.
Fig. 10 also shows the P700 double difference spectrum forthe WT and each of the mutants. Although there are somedifferences in the 400–450 nm range, these double differencespectra also indicate the clear involvement of �-carotenefam inelectrochromism tied to P700 oxidation. By taking the P700double difference of zds� minus pds�, band shifts are apparentthat likely arise from the two longer wavelength peaks of
FIG. 9. Comparison of the zds� and the pds� decay-associatedspectra with that of WT for P700� FeS[A/B]
� � P700 FeS[A/B] in PSI complexes. This spectrum was detected at 20 us following the actinicflash.
FIG. 8. Decay-associated spectra from a global analysis of therelaxation kinetics of the fast (f) and slow phases ( ) of the A1
�
(FeS) � A1(FeS)� in PS I complexes from the WT (top), zds�
(middle), and pds� strains (bottom).
FIG. 10. Fast (f) and slow phase ( ) double difference spectraof the [A1
�-carotenefam. These are responsible for the differences ob-served in the WT minus zds� and the WT minus pds� differ-ence spectrum between 400 and 450 nm. Thus, the carotenoidor carotenoids that sense the oxidation of P700 are �-carotenefam in the WT and are replaced by �-carotenefam in thezds� strain. The double difference zds� minus pds� in the A1
case leaves smaller residuals than those observed for P700, pos-sibly indicating a lower probability of the �-carotenefam replacing�-carotenefam in the neighborhood of the phylloquinones.
Charge Recombination from FA/FB� to P700�—The slowing
of the slow phase of oxidation of A1� in the mutants compared
with WT (Table II) could reflect a change in the reductionpotential of A1/A1
� (Equations 1 and 2). Any mutation-inducedchanges in the reduction potential of A1/A1
� could be quanti-fied by measuring the rate of charge recombination (Vobs, Equa-tion 3) between P700� and FA/FB
�, assuming that the rate ofacceptor side equilibration was rapid relative to the rate ofcharge recombination and that the intrinsic rate of chargerecombination (kin � 95 �s (41)) of P700�A1
�3 P700A1 did notchange in the mutants. The rate of charge recombinationshould then be proportional to the equilibrated concentration ofA1
� (Equations 1 and 2) determined by equilibrium constantKAF (Equation 4). An increase in the reduction potential ofA1/A1
� would consequently accelerate charge recombination.The rate of charge recombination between P700� and FA/FB
�
was measured by saturating flash excitation followed by detec-tion at 435 nm, a minimum of the P700� � P700 differencespectrum (Fig. 9). Fig. 12 shows an increase in the rate ofcharge recombination from a t1⁄2 of 35 ms (� � 51 ms) in the WTto a t1⁄2 of 19 ms (� � 27 ms) in the zds� strain (Fig. 12). Asimilar experiment performed on PS I complexes of WT and thepds� strain gave a t1⁄2 of 33 and 24 ms, respectively (not shown).The change in recombination rate corresponded to an increasein reduction potential of A1/A1
� of 16 and 8 mV, respectively,for the zds� and pds� complexes.
�-Carotenefam Is Needed for PS II Assembly—Carotenoidsplay multiple roles in photosynthesis. They are accessory pig-ments used for light-harvesting and for photoprotection in chlo-rophyll-protein complexes by quenching of chlorophyll triplet
states (42). We show here that the replacement of �-carotenefam
with shorter conjugated chain carotenoids in Synechocystis sp.PCC 6803 results in the loss of PS II complexes in the thylakoidmembranes. This observation is consistent with the observa-tions of Trebst and Depka (43) in which inhibition of phytoenedesaturase by norflurazon and fluridone results in the loss ofthe D1 polypeptide. Either the inability to quench chlorophylltriplet states accelerates the rate of D1 photodamage and deg-radation or the lack of an appropriately shaped carotenoidresults in an inability to correctly assemble PS II reactioncenters. In either case, accumulation of PS II in the thylakoidmembranes is prevented.
Spectroscopic Characterization—The replacement or ab-sence of �-carotene and its hydroxylated derivatives results ina 2-nm blue shift of the Qy absorption maximum of the Chls inthe PS I complexes From the 2.5 Å x-ray crystallographicstructure of PS I in T. elongatus, close associations are ob-served between the carotenoids and the chlorophylls of thereaction center (2, 3). Because the ringed carotenoids (�-caro-tene, zeaxanthin, �-cryptoxanthin, and isocryptoxanthin) arereplaced by ones with shorter �-conjugation (�-carotene or phy-toene) or no carotenoid at all, structural adjustments are to beexpected. Such structural changes could result in alterations inlocal dielectric, in excitonic interaction between pigments, or ina loss of carotenoid-associated chlorophyll-binding sites. Thatthe last two of these contribute to the above-mentioned absorb-ance changes will be documented in a forthcoming publication.
Forward Electron Transfer from A1� to FX—Brettel et al.
(44) showed in P700�A1� charge recombination experiments
with prereduced FA and FB at 10 K (t1⁄2 50 �s) that the A1� �
A1 difference spectrum bears a close resemblance to the absorb-ance difference spectrum for Vit K1
. � Vit K1 (40). This resem-blance is consistent with the presence of phylloquinone in PS Icomplexes (45) and provided strong evidence for the identifica-tion of A1
� with phyllosemiquinone. Brettel et al. (44) were thefirst to measure the UV and visible region absorbance changesassociated with the forward oxidation of A1
�. The A1�(FeS) �
A1(FeS)� absorbance difference spectra were measured in PS Icomplexes from the cyanobacteria Synechocystis sp. PCC 6803(8), Synechococcus sp. (11), and spinach (46). These spectrahave now been examined with improved signal/noise and overa more extended wavelength range in PS I complexes fromSynechocystis sp. PCC 6803 (the present work) and in wholecells of C. reinhardtii (47). These spectra bear a close resem-blance in the UV to the difference spectra generated byP700�A1
� charge recombination (44).The forward oxidation of A1
� has been shown to be biphasicwith half-times (relative amplitudes) of 25 (65%) and 150 ns
FIG. 11. Decay-associated spectra in Synechocystis sp. 6803whole cells of the 8 ns (f, fast phase) and 180 ns ( , slow phase)components from a global analysis of kinetic data in WT. Thecells were suspended in 20 mM HEPES (pH 7.2), 20% (w/v) Ficoll, and5 �M carbonyl cyanide (4-(trifluoromethoxy)phenyl)hydrazone.
FIG. 12. Charge recombination kinetics of electron transferfrom FeS[A/B]
� to P700� monitored at 435 nm (minimum ofP700� � P700) after a saturating actinic flash in WT and zds� PSI complexes. The complexes were suspended in 25 mM Tris-HCl (pH8.3), 10 �M sodium ascorbate, 4 �M DCPIP, and 0.03% �-DM. Thechlorophyll a concentration for both samples was 14 �g/ml.
Carotenoids Sense PsaA- and PsaB-side Electron Transport20038
(35%) in spinach PS I complexes (46) and 7 (33%) and 190 ns(66%) in Synechocystis sp. PCC 6803 (8). Similar biphasic ki-netics have been observed in Chlorella sorokiniana (12) and inC. reinhardtii (14). The A1
�(FeS) � A1(FeS)� difference spectrafor the two phases are shown in Fig. 8 for WT Synechocystis sp.PCC 6803 and the two knock-out strains pds� and zds�. Theyboth bear an overall resemblance in the UV to Chromatium VitK2
. � Vit K2 and to the in vitro Vit K. � Vit K1 differencespectrum (11, 40). The spectra observed here peak at 375–380nm instead of the 390 nm observed in the in vitro spectrum andare shifted by about 30 nm to shorter wavelength comparedwith the Chromatium difference spectrum. There is also addi-tional fine structure in the biological spectra, as observed byBrettel (8, 11). In the case of the WT, the slow and fast phasesof A1
� oxidation show similar spectral components, the relativeamplitudes of which are different. A component at 385 nmappears to dominate in the fast phase spectrum, and one at 378nm dominates in that of the slow phase. Also a shoulder at 340nm in the slow phase appears to be missing in the fast phase.The fast and slow phase UV spectra are more similar in thecase of the pds� and zds� strains. In the two mutants, the 385nm component is smaller than the 378 and 405 nm componentsresulting in a closer resemblance in the spectra of the twophases. These observations argue in favor of the fast and slowphases arising from the same chemical species, possibly insomewhat different environments, the differences for which aremore marked in the WT difference spectrum. It is also possiblethat there is an additional small contribution from FX
� toFA/FB electron transfer that contributes to the slow phase in allthree strains. The shoulder at 340 nm in the slow phase mightarise from such a contribution.
The Vit K1. � Vit K1 in vitro difference spectrum shows only
small and flat absorbance changes above 440 nm (40). In thisrange there are marked features (450–470 nm and 500–520 nm)in the A1
�(FeS) � A1(FeS)� difference spectra of the fast andslow phases that most likely come from electrochromic bandshifts of pigments located in close proximity to A1, reflecting theanionic charge present in the A1
� state. The pigments responsi-ble for these electrochromic band shifts were suggested to becarotenoids (11, 12). A comparison of the A1
�(FeS) � A1(FeS)�
from the WT and from the two carotenoid mutants indicatesslight modifications in the 450–470 nm range and large differ-ences in the 500–520 nm range. In both the pds� and the zds�
strains, the band shifts in the 500–520 nm range have com-pletely disappeared in the difference spectra of both phases.However, the large second derivative-shaped signal in the 430–470 nm range has remained largely intact, including the dis-placement of the fast phase minimum at 448 to 452 nm in theslow phase. A derivative-shaped shoulder present at 460 nm inthe WT fast phase has disappeared in the mutants. By taking thedouble difference [A1
�(FeS) � A1(FeS)�]WT � [A1�(FeS) �
A1(FeS)�]mutant, one obtains the spectra shown in Fig. 10. Hereone can see that there are three electrochromic bands that un-dergo a red shift in both the fast and slow phases. The 30–35 nmspacing between these shifts is consistent with their arising from�-carotenefam. It is clear that the band shifts are not located inthe same positions for the fast and slow phases, with the bandshifts displaced by �6 nm to higher wavelength for the fast phaseas compared with the slow phase. This observation supports theidea that the A1 species in each of the two phases is present in adifferent environment, giving rise to electrochromic band shifts ofa different set of carotenoid pigments. That the second deriva-tive-shaped feature in the 430–470 nm range remains prominentin the mutant difference spectra, despite the absence of carote-noids absorbing in this range, argues that the remaining bandshift most likely arises from one or more chlorophyll molecules.
There has been considerable controversy concerning whetherone or both of the phylloquinones are active for electron trans-fer in PS I to explain the biphasic kinetics in the electrontransfer step between A1
� and the FeS clusters. Brettel (8) andSetif and Brettel (46) had initially attributed the presence ofthe biphasic kinetics of A1
� oxidation to the equilibrium be-tween A1 and FX, the reduction potentials of which were pro-posed to be close. The fast phase would then arise from theestablishment of the equilibrium A1
� FXi A1FX�, whereas the
slow phase would be limited by the electron transfer from FX tothe FA/FB centers (8, 46). Joliot and Joliot (12) have argued thatas the electron transfer from A1
� to FX is electrogenic (13), thenif the fast phase were indeed the establishment of the equilib-rium concentrations of A1
� and FX�, its relative amplitude
should be sensitive to the transmembrane electric field. Theyshowed that, on the contrary, the relative amplitudes of thefast and slow phases were independent of the membrane po-tential. Leibl and co-workers (13, 48) have also concluded fromphotovoltage measurements that the rate of electron transferfrom FX
� to FA/FB (� �50 ns) is faster than that of the slowphase of A1
� oxidation. Both of these experimental observa-tions thus argue against the equilibrium model and in favor ofone where either the two quinones operate independently in atwo-pathway model or where only one of the quinones is func-tional, but where there is a heterogeneity in the centers givingrise to two different rate constants for A1
� to FX electrontransfer (12).
Site-directed mutations have been constructed in C. rein-hardtii and in Synechocystis sp. PCC 6803 in the region of eachof the two phylloquinones. Mutations PsaA-W693F and PsaB-W673F (10, 14) resulted in a substantial slowing, respectively,of the slow and fast phases of A1
� forward oxidation. In eachcase, the rates of the complementary fast and slow phases,respectively, were unchanged as were the relative amplitudesof the fast and slow phases. Similar results were obtained withthe PsaA- and PsaB-side mutations, PsaA-S692C and PsaB-S672C in Synechocystis sp. PCC 6803, although the effect onthe fast kinetic phase of the PsaB-side mutant was not aspronounced as in the PsaB-W673F (10, 47). These results arguein favor of the two-pathway model in which the fast phasewould arise from the PsaB side and the slow phase the PsaAside. Also supporting the two-pathway model are EPR-detectedkinetics of P700�A1
� charge recombination at 100 K followingillumination at 205 K in the presence of sodium dithionite. Twophases are observed for charge recombination, the slower ofwhich is lost upon illumination in the PsaA-W693H and PsaA-W693L mutants (49). Xu et al. (10), while confirming the roomtemperature optical results with both sets of mutants, wereunable to observe a slowing of the fast phase using transientEPR. They concluded that the PsaA branch is active but thatthere was no EPR-based evidence for electron transfer up thePsaB branch. Thus, according to their data, a unidirectionalscheme using the A pathway of electron transfer with centerheterogeneity could not be ruled out. The inability of Xu et al.(10) to observe the same relative amplitude of fast phase bytransient EPR as they observe in optical experiments, however,may be due to the inability of transient EPR to detect, evenindirectly, a kinetic phase of �10 ns, especially when the fastkinetic phase represents a minority of the electron flux to FX.
The present work adds support for the two-pathway modeland provides additional spectroscopic markers with which todistinguish the two electron transfer pathways. We haveshown here that the carotenoid pigments that act as electro-chromic detectors for the fast and slow phases are different andare shifted 6 nm with respect to each other. That these samedifference spectra are observed in PS I complexes and in PS I in
Carotenoids Sense PsaA- and PsaB-side Electron Transport 20039
whole cells, with similar rates for the fast and slow phases andwith similar relative amplitudes, would argue against the twophases originating from center heterogeneity. In the �-caro-tene-deficient pds� and zds� strains, that the A1
�(FeS) �A1(FeS)� difference spectra of the fast and slow phases becomepractically identical with little change in relative amplitudecompared with WT also argues that the spectral differencesbetween the fast and slow phases and the biphasic electrontransfer do not arise from a heterogeneity between PS I reac-tion centers. The spectral differences in the two phases in WTmost likely arise then from carotenoids with different confor-mations and/or configurations or carotenoids in different envi-ronments associated with each of the two phylloquinones.These spectral differences associated with each phase thusargue for the participation in electron transfer of both thePsaA-side and PsaB-side quinones.
Examination of the T. elongatus PS I x-ray crystal structure(2) indeed shows that there are structural differences in thecarotenoids associated with the PsaA and PsaB sides of thereaction center in the immediate vicinity of the phylloquinones.Two �-carotenefam carotenoids with cis double bonds are foundnear the phylloquinone on the PsaA side, whereas only one isfound on the PsaB side. On the PsaA side, there is a 9,13-di-cis(see numbering in Fig. 1) located at a distance of 19 Å fromPhQA (edge-to-edge distance). There is also a 13-cis �-carotenefam carotenoid 25 Å away. On the PsaB side, the onlycis-�-carotenefam carotenoid found near phylloquinone boundby PsaB is 9-cis, located 22 Å away. Hashimoto and Koyama(50) have shown in n-hexane that the absorption spectrum of9-cis-�-carotene is 6 nm shifted to the blue compared with thecorresponding all-trans isomer. The 13-cis isomer, on the otherhand, is shifted by 11 nm to the blue, again compared with thecorresponding all-trans isomer. In general, the nearer the cisdouble bond is to the center of the carotenoid, the greater theextent of the blue shift (50). A 9,13-di-cis-�-carotene is expectedto be even more blue-shifted than its corresponding mono-cisisomers. It is most likely that the carotenoids nearest to thephylloquinones all experience electrochromic effects but thatthe cumulative effect of having two cis-carotenoids (having atotal of three cis double bonds) on the PsaA side shifts theoverall absorption spectrum of the carotenoids on the PsaA side6 nm to the blue compared with the carotenoids on the PsaBside with only one cis double bond between them.
The differences in the minima of the second derivative-shaped feature in the 430–470 nm range between the fast andslow phases also likely reflect a difference in the electrochromiceffects of the PsaA- and PsaB-side phyllosemiquinone anionson nearby chlorophylls. Consistent with this observation,Dobek and Brettel (51) have recently reported differences inthe spectra of the fast and slow phases of A1
� oxidation in the660–694 nm range that likely reflect electrochromic effects ondifferent reaction center chlorophylls. The absorbance spectraof most of the PS I component chlorophylls are not known,however, making it difficult to make structural assignments tothe detecting chlorophylls.
The zds� and pds� strains both show a marked slowing ofthe slow phase of A1
� oxidation (t1⁄2 � 250 � 18 ns) comparedwith WT (t1⁄2 �170 � 15 ns) (Table II). There may also be aslight slowing of the fast phase, but the alteration in rate, if itexists, is within the noise level of the measurement. The mu-tant strains also show an acceleration of the rate of chargerecombination relative to WT. These observations are under-standable in terms of a model proposed by Agalarov and Brettel(9) in which A1
� 3 FX electron transfer is exergonic on oneside of the reaction center and endergonic on the other side,most likely the PsaB and PsaA sides, respectively. We pro-
pose that the replacement of �-carotenefam by shorter chaincarotenoids or the loss of �-carotenefam in the mutant strainsresults in the raising of the reduction potential of the phyl-loquinones. As the Marcus curve is parabolic for the A1
� 3FX electron transfer, a given change in the free energy of thereaction will have a larger effect on the reaction rate of theendergonic than on the exergonic reaction (Fig. 13). This isindeed what we observe (Table II and Fig. 7).
The rate of charge recombination is governed by the productof the intrinsic rate (kin) of charge recombination betweenP700� and A1
� times the concentration of A1�. The latter is
determined by the relative reduction potentials of the A1/A1�
and (FA/FB)/(FA/FB)� redox couples. The more positive the re-duction potential of A1/A1
� the higher the concentration of A1�
following charge separation and the higher the rate of chargerecombination. Assuming the intrinsic rate of P700�A1
� re-combination to be the same in the mutants and WT, then thehigher rate of charge recombination in the mutants reflects anincrease in the reduction potential of A1/A1
�. Recombinationshould occur predominantly through the PsaA-side phyllo-quinone, the more positive of the two resident quinones. Themore rapid recombination in the mutants is thus consistentwith the slowed rate of the slow phase forward reaction, withboth arising from an increased reduction potential of thePsaA-side phylloquinone (Fig. 13). The 1.4–1.8-fold increasein rate of charge recombination corresponds to an increase inreduction potential of A1/A1
� of 8–16 mV. Although lessvisible, it is likely that the PsaB-side phylloquinone has alsoundergone a similar increase in reduction potential. Theexistence of endergonic and exergonic components of the ki-netics of oxidation of A1
� is most consistent with the bidirec-tional model.
Fig. 2 shows the cluster of carotenoids located close to eachof the phylloquinones in the x-ray crystal structure of PS I.The increase in reduction potential of the phylloquinones inthe mutants suggests that the loss/replacement of the caro-tenoids close to the phylloquinone in the WT results in astabilization of the phyllosemiquinone relative to phylloqui-none. One of the striking characteristics of the phylloquino-
FIG. 13. Relative reduction potentials of photosystem I co-factors in the WT and mutant PS I complexes. Shown in the insetis a portion of the inverted region of Marcus curve.
Carotenoids Sense PsaA- and PsaB-side Electron Transport20040
nes of PS I is their very low reduction potential. These havebeen estimated to be �531 and �686 mV for the PsaA- andPsaB-side PhQ/PhQ� redox couples, respectively (52). Thecarotenoids are apparently one contributing factor to this lowpotential, and a role for these molecules in photosynthesiswas not appreciated previously.
The small increase in the A1/A1� reduction potential associ-
ated with the loss/replacement of the carotenoid could resultfrom a small conformational shift of the peptide backbone inthe vicinity of the phylloquinone, a factor that Ishikita andKnapp (52) have signaled as being a determining factor forquinone redox behavior in PS I. We have also found that theloss/replacement of carotenoid results in the depletion of thePsaF and PsaL subunits in the isolated complex along withsome long wavelength chlorophylls.2 van der Est and co-work-ers (53) have argued that the loss of PsaF in a psaF deletionmutant (accompanied by the loss of chlorophylls 1138 and1139) could influence the redox properties of PhQA by increas-ing access to bulk water. van der Est and co-workers (53)report, relative to WT PS I complexes, an increased rate ofPhQA
� oxidation by FX in the presence of Triton X-100 but aslowing of the rate by 10% in the presence of �-DM, the deter-gent used here. The slowing of the slow phase of the A1
�
oxidation kinetics that we observe here in the pds� and zds�
mutants is larger (50%) relative to WT (Table II and Fig. 7)than what was reported by van der Est and co-workers inSynechococcus sp. PCC 7002 (53) and observed here both invivo and in isolated PS I complexes from Synechocystis sp. PCC6803. Although the relative amplitudes of the effect on ratemay be due to species differences, it is likely that the loss/replacement of carotenoid, with consequences for both PsaFand chlorophyll binding, enhances the impact on the reductionpotential of PhQA/PhQA
�, beyond what is observed by deletionof PsaF alone. Although we interpreted above the difference inkinetic effects of carotenoid loss/replacement on the relativeenergetics of the PsaA and PsaB branches, it is also possiblethat the structural perturbations associated with the carote-noid loss/replacement have a larger impact on A1/A1
� reductionpotential on the PsaA side.
Acknowledgments—We thank Dexter Chisholm for assistance in theconstruction of the mutants and Boris Zybailov for help with EPRmeasurements.
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Carotenoids Sense PsaA- and PsaB-side Electron Transport 20041
H. Golbeck, Jamie Yehong Wang, Daniel Béal and Bruce A. DinerJames A. Bautista, Fabrice Rappaport, Mariana Guergova-Kuras, Rachel O. Cohen, John
CYANOBACTERIA6803: EVIDENCE FOR PsaA- AND PsaB-SIDE ELECTRON TRANSPORT IN
Sp. PCCSynechocystis-Carotene Desaturase Deletion Mutants of ζDesaturase and Biochemical and Biophysical Characterization of Photosystem I from Phytoene
doi: 10.1074/jbc.M500809200 originally published online March 9, 20052005, 280:20030-20041.J. Biol. Chem.
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