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Electron Transfer in Cyanobacterial Photosystem I

2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês

10.1074/jbc.m302962200

1083-351X

Wu Xu, Parag R. Chitnis, А. И. Валиева, Art van der Est, Yulia Pushkar, M. Krzystyniak, Christian Teutloff, Stephan G. Zech, Robert Bittl, D. Stehlik, Boris Zybailov, Gaozhong Shen, John H. Golbeck,

Spectroscopy and Quantum Chemical Studies

The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp697PsaA and Trp677PsaB are π-stacked with the head group of the phylloquinones and are H-bonded to Ser692PsaA and Ser672PsaB, whereas Arg694PsaA and Arg674PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and FX. The mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P700+A1– radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697FPsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P700+A1– and the CW EPR spectra of photoaccumulated A1–. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P700 and A1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR. The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp697PsaA and Trp677PsaB are π-stacked with the head group of the phylloquinones and are H-bonded to Ser692PsaA and Ser672PsaB, whereas Arg694PsaA and Arg674PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and FX. The mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P700+A1– radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697FPsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P700+A1– and the CW EPR spectra of photoaccumulated A1–. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P700 and A1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR. Photosynthetic reaction centers (RCs) 1The abbreviations used are: RC, reaction centers; PS, photosystem; CW, continuous wave, EPR, electron paramagnetic resonance; ENDOR, electron nuclear double resonance; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethylethyl]glycine; Chl, chlorophyll; phylloquinone, 2-methyl-3-phytyl-1,4-naphthoquinone or 2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-1,4-naphthalenedione. are classified into two general types depending on the identity and function of the terminal electron acceptors. Those RCs that incorporate iron-sulfur clusters are classified as "Type I," and those that incorporate a mobile (secondary) quinone are classified as "Type II." Type I RCs include Photosystem I (PS I) of cyanobacteria and plants and those in heliobacteria and green sulfur bacteria. Type II RCs include Photosystem II of cyanobacteria and plants and those in green non-sulfur bacteria and purple bacteria. Despite the difference in the identity of the terminal electron acceptors, Type I and Type II RCs share a common motif in terms of polypeptide arrangement and cofactor composition (1Schubert W.D. Klukas O. Saenger W. Witt H.T. Fromme P. Krauss N. J. Mol. Biol. 1998; 280: 297-314Crossref PubMed Scopus (194) Google Scholar). The primary cofactors are bound to proteins that are present as dimers in the membrane. This results in a set of electron transfer cofactors that are arranged (pseudo)symmetrically (2Fromme P. Singhal G.S.R.G. Sopory S.K. Irrgang K.D. Govindjee Concepts in Photobiology: Photosynthesis and Photomorphogenesis. Narosa Publishing House, New Delhi, India1999: 181-220Crossref Google Scholar). In PS I, these cofactors include a special pair of chlorophyll a/a′ molecules as the primary donor, two bridging chlorophyll a molecules, and two chlorophyll a molecules, at least one of which functions as the primary acceptor (3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauß N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2081) Google Scholar). In the purple bacterial reaction center, the cofactors include a special pair of bacteriochlorophyll a molecules as the primary donor, two bridging bacteriochlorophyll a molecules, and two pheophytin molecules, one of which functions as the primary acceptor (4Deisenhofer J. Epp O. Miki K. Huber R. Michel H. J. Mol. Biol. 1984; 180: 385-398Crossref PubMed Scopus (1535) Google Scholar). PS I and the purple bacterial reaction center contain two quinones; in the latter, one quinone is rather immobile (QA) and the other is mobile (QB), whereas in PS I both quinones (QK-A and QK-B) are, to the best of our knowledge, immobile in their normal function. In Type II reaction centers, a single turnover results in the reduction of QB, to a semiquinone and a second turnover results in the further reduction (and protonation) of QB to a hydroquinone. The stability of QB– requires that there is no recombination pathway; hence, there is also no direct forward pathway to QB. The hydroquinone in the QB site is loosely bound and is replaced by an oxidized quinone, thereby recharging the site for a new round of light-induced turnover. In PS I, a single turnover results in the reduction of the quinone to a semiquinone; the electron is then passed to FX, an interpolypeptide iron-sulfur cluster that serves to vector the electron to ferredoxin (with the participation of two additional iron-sulfur clusters, termed FA and FB, bound within the stromal subunit PsaC). As a result, there is only the need for a one-electron reduction of a single quinone to the semiquinone state. In PS I there is no a priori reason that electron transfer must be either uni- or bidirectional because there is no obvious need to accumulate two electrons in a QB-type quinone as in the bacterial reaction center. Indeed, the simpler requirement that only a single electron needs to be passed to the iron-sulfur cluster FX implies that there may be no preferred pathway for the electron. Thus, a bidirectional pathway of electron transfer is possible. Alternately, the 2.5-Å electron density map of PS I indicates that there are subtle differences between the two branches, starting with the primary donor P700, which the x-ray structure reveals to be a Chl a/Chl a′ heterodimer. In addition, there are differences in the distances, orientations, and environments of the cofactors along both branches. Because rates of electron transfer are sensitive to such factors, the probability that both branches are exactly equivalent is miniscule. Thus, a unidirectional pathway of electron transfer is equally possible. Spectroscopic indicators do not provide any conclusive evidence for directional electron transfer in native PS I because the two branches cannot be distinguished, unlike the case with bacterial reaction centers, in which the two pheophytins on the L and M sides have different optical spectra. Mutagenesis provides a technique by which the two branches can be rendered distinguishable by introduction of a specific modification into only one branch of cofactors. However, there are a number of important criteria that must be met if this method is to be used successfully. First, the changes induced by the mutations should be sufficiently subtle that the cell should still be able to grow. Second, the changes should be localized to the immediate vicinity of the electron transfer cofactors. This is necessary because otherwise, any correlation between spectroscopic changes and electron transfer along a given branch may be lost. Third, the mutations should not disturb the overall structure and function of the primary charge separation. For example, a mutation that inadvertently alters the directionality of electron transfer cannot be used to infer this behavior in wild-type PS I. Last, the mutations should produce changes that are readily characterized using spectroscopic techniques. These considerations led us to approach the problem of distinguishing between the two potential pathways by introducing site-directed mutations in and around the QK-A and QK-B binding sites on the PsaA and PsaB reaction center proteins in Synechocystis sp. PCC 6803. EPR and ENDOR spectroscopy of A1– and P700+A1– have yielded considerable details about the local environment of the quinone as well as information about electron transfer kinetics. These studies indicate asymmetric H-bonding to the phylloquinone, with a dominant H-bond to the oxygen meta to the methyl group (see Refs. 5Kamlowski A. Altenberg-Greulich B. van der Est A. Zech S.G. Bittl R. Fromme P. Lubitz W. Stehlik D. J. Phys. Chem. B. 1998; 102: 8278-8287Crossref Scopus (40) Google Scholar, 6Zech S.G. Hofbauer W. Kamlowski A. Fromme P. Stehlik D. Lubitz W. Bittl R. J. Phys. Chem. B. 2000; 104: 9728-9739Crossref Scopus (75) Google Scholar, 7Rigby S.E. Evans M.C. Heathcote P. Biochim. Biophys. Acta. 2001; 1507: 247-259Crossref PubMed Scopus (36) Google Scholar for a summary), and a Trp in van der Waals contact with the phylloquinone (5Kamlowski A. Altenberg-Greulich B. van der Est A. Zech S.G. Bittl R. Fromme P. Lubitz W. Stehlik D. J. Phys. Chem. B. 1998; 102: 8278-8287Crossref Scopus (40) Google Scholar, 6Zech S.G. Hofbauer W. Kamlowski A. Fromme P. Stehlik D. Lubitz W. Bittl R. J. Phys. Chem. B. 2000; 104: 9728-9739Crossref Scopus (75) Google Scholar, 8Hanley J. Deligiannakis Y. MacMillan F. Bottin H. Rutherford A.W. Biochemistry. 1997; 36: 11543-11549Crossref PubMed Scopus (51) Google Scholar, 9Itoh S. Iwaki M. Ikegami I. Biochim. Biophys. Acta. 2001; 1507: 115-138Crossref PubMed Scopus (72) Google Scholar), most probably π-stacked (5Kamlowski A. Altenberg-Greulich B. van der Est A. Zech S.G. Bittl R. Fromme P. Lubitz W. Stehlik D. J. Phys. Chem. B. 1998; 102: 8278-8287Crossref Scopus (40) Google Scholar, 6Zech S.G. Hofbauer W. Kamlowski A. Fromme P. Stehlik D. Lubitz W. Bittl R. J. Phys. Chem. B. 2000; 104: 9728-9739Crossref Scopus (75) Google Scholar, 9Itoh S. Iwaki M. Ikegami I. Biochim. Biophys. Acta. 2001; 1507: 115-138Crossref PubMed Scopus (72) Google Scholar, 10Brettel K. Leibl W. Biochim. Biophys. Acta. 2001; 1507: 100-114Crossref PubMed Scopus (294) Google Scholar, 11van der Est A. Biochim. Biophys. Acta. 2001; 1507: 212-225Crossref PubMed Scopus (56) Google Scholar). Both the π-stacked Trp and one H-bond that occurs between the backbone amide from Leu722PsaA and Leu706PsaB, and the carbonyl of phylloquinone meta to the methyl group, along with the absence of a significant H-bond to the other carbonyl ortho to the methyl group, have been confirmed in the 2.5-Å PS I structure (3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauß N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2081) Google Scholar). Combined with optical studies of the electron transfer kinetics, a very detailed picture of the influence of the mutations on structure and function can be constructed, which is a prerequisite to uncovering the electron transfer pathway in the wild-type. In this paper, we present physiological and EPR/ENDOR spectroscopic characterization of the following mutants: W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB. These amino acids were chosen because they may constitute a potential electron transfer pathway between A0 and FX (Fig. 1). In a companion paper (24Xu W. Chitnis P.R. Valieva A. van der Est A. Brettel K. Guergova-Kuras M. Pushkar J. Zech S.G. Stehlik D. Shen G. Zybailov B. Golbeck J.H. J. Biol. Chem. 2003; 278: 27876-27887Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), the optical and EPR measurements of the electron transfer kinetics will be presented. The results presented here are aimed at gaining an understanding of the physiological and structural changes induced by the mutations. The importance of a detailed characterization is highlighted by the fact that a number of recent papers addressing the issue of directionality in various mutants and PS I preparations using a variety of spectroscopic techniques (12Yang F. Shen G. Schluchter W.M. Zybailov B. Ganago A.O. Vassiliev I.R. Bryant D.A. Golbeck J.H. J. Phys. Chem. 1998; 102: 8288-8299Crossref Scopus (65) Google Scholar, 13Joliot P. Joliot A. Biochemistry. 1999; 38: 11130-11136Crossref PubMed Scopus (163) Google Scholar, 14Guergova-Kuras M. Boudreaux B. Joliot A. Joliot P. Redding K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4437-4442Crossref PubMed Scopus (271) Google Scholar, 15Purton S. Stevens D.R. Muhiuddin I.P. Evans M.C. Carter S. Rigby S.E. Heathcote P. Biochemistry. 2001; 40: 2167-2175Crossref PubMed Scopus (54) Google Scholar, 16Hastings G. Sivakumar V. Biochemistry. 2001; 40: 3681-3689Crossref PubMed Scopus (23) Google Scholar) (see also Ref. 10Brettel K. Leibl W. Biochim. Biophys. Acta. 2001; 1507: 100-114Crossref PubMed Scopus (294) Google Scholar for a summary) have reported conflicting evidence compatible with both unidirectional and bidirectional electron transfer. In most of these studies, the directionality is inferred from either optical or EPR/ENDOR spectroscopic properties of modified PS I complexes but little attention has been paid to direct comparison of optical and EPR/ENDOR data on the same samples and to ensuring that the modifications do not alter the structure or function of the quinone. A careful characterization of the environment of the mutation is a necessary prerequisite to drawing conclusions about the function of the wild type from the function of the mutants. We show here that the mutations described above lead to only subtle changes in the quinone environment and have no perceptible effect on P700. Thus, they are ideally suited to the study of the directionality of electron transfer. Moreover, the specific changes in the spectra can be explained satisfactorily in terms of expected changes in the electronic environment of the quinone. Together, the combined results from six different mutants and several spectroscopic techniques clearly indicate that the quinone detected by CW EPR, transient EPR and ENDOR is located on the PsaA branch of cofactors. Generation of the PsaA and PsaB Point Mutants—For site-directed mutagenesis of the psaA and psaB genes, two recipient strains were constructed with deletion of a portion of the psaA gene or deletion of the whole psaB gene. As shown in Fig. 2A, the Synechocystis sp. PCC 6803 pWX3 recipient strain was constructed through deletion of the EagI-EcoRI fragment that contains the 1130-bp 3′ part of the psaA gene and the whole psaB gene, and replacement with a spectinomycin-resistance cartridge gene. The Synechocycstis sp. PCC 6803 pCRTΔB recipient strain was obtained through deletion of the HindIII-EcoRI fragment that contains the 3′ half of the psaB gene and replacement with a 1.3-kilobase pair kanamycin-resistance cartridge gene. As shown in Fig. 2B, two plasmids pGEM-3C+ and pIBC were constructed as the templates for PCR-based site-specific mutagenesis. To generate mutations in the Qk-B binding site, the plasmid pGEM-3C+ was constructed through cloning of a 1588-bp segment of the psaB 3′ region and the 760-bp region downstream of the psaB into the pGEM7z vector. A chloramphenicol resistance gene was inserted at the EcoRI site just downstream of the psaB gene. To generate mutations in the Qk-A binding site, the pIBC plasmid was constructed through cloning of a DNA fragment that contained most of the psaA gene, the psaB gene, and a 760-bp downstream region of the psaB gene into a pBluescript II KS vector. A chloramphenicol-resistant cassette gene was inserted after the 3′ terminator of the psaB gene. PCR mutagenesis was carried out using the Transformer™ site-directed mutagenesis kit (Clontech Laboratories, Inc). The constructs with specific mutations in the psaA gene were generated through PCR mutagenesis using the pIBC plasmid DNA as the template and appropriate primers for W697FPsaA, R694APsaA, and S692CPsaA. The constructs with specific mutations in the psaB gene were generated through PCR mutagenesis using the pGEM-3C+ plasmid DNA as the template and appropriate primers for W677FPsaB, R674APsaB, and S672CPsaB. All mutation sites were confirmed by DNA sequencing. The plasmids with the desired psaA mutations derived from pIBC were used to transform the Synechocystis sp. PCC 6803 recipient strain pWX3. The plasmids with the desired psaB mutations derived from pGEM-3C+ were used to transform the Synechocystis sp. PCC 6803 recipient strain pCRTΔB. Transformants with chloramphenicol resistance were selected under low light intensities. To verify the full segregation of the transformants, DNA fragments containing the mutation sites were amplified through PCR from the genomic DNA of the mutant strains and sequenced to confirm the desired nucleotide change. Physiological and Biochemical Characterization—The Synechocystis sp. PCC 6803 wild-type and mutant strains were cultured in BG-11 medium with 5 mm glucose under lower light intensities. To start a growth experiment, cells of actively growing cultures were centrifuged at 4000 × g and the pellet was suspended in BG-11 medium. The centrifugation-resuspension procedure was repeated three times to separate the glucose from the cells. Cultures were shaken constantly at 120 rpm under different light intensities at 30 °C with (photomixotrophic) or without (photoautotrophic) glucose. Growth rates of the liquid cultures were monitored by the absorbance at 730 nm (A 730). Quantitation of chlorophyll content, preparation of thylakoid membranes, and isolation of PS I particles were carried out according to previously published procedures (17Shen 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). Light-driven PS I-mediated electron transport from 3,6-diaminodurene to methyl viologen was monitored using a Clark-type oxygen electrode (Hansatech, Norfolk, United Kingdom). PS I activity was also determined with a NADP+ photoreduction assay using cytochrome c 6 and ferredoxin as electron donor and acceptor, respectively. PS II activity was measured as light-driven oxygen evolution, in which electrons are transferred from water to p-benzoquinone. The polypeptide composition of PS I complexes in the wild-type and mutant strains was examined by analytical SDS-PAGE and Western immunoblotting. Immunoblots were probed with antisera against PsaB and PsaL from Synechocystis sp. PCC 6803, and PsaC from Synechococcus sp. PCC 7002. For chemiluminescence immunodetection, the ECL Western blotting detection method was used (Amersham Biosciences). Q-band CW EPR of Photoaccumulated PS I Complexes—Photoaccumulation experiments were performed using a Bruker ER300E spectrometer and an ER 5106-QT resonator equipped with an opening for in-cavity illumination similar to that described in Ref. 12Yang F. Shen G. Schluchter W.M. Zybailov B. Ganago A.O. Vassiliev I.R. Bryant D.A. Golbeck J.H. J. Phys. Chem. 1998; 102: 8288-8299Crossref Scopus (65) Google Scholar. Low temperatures were maintained with an ER4118CV liquid nitrogen cryostat and an ER4121 temperature controller. The microwave frequency was measured with a Hewlett-Packard 5352B frequency counter, and the magnetic field was measured with a Bruker ER035M NMR gaussmeter. The pH of sample was adjusted to 10.0 with 1.0 m glycine buffer, and sodium dithionite was added to a final concentration of 50 μm. After incubation for 10 min in the dark, the sample was placed into the resonator and the temperature was adjusted to 205 K. The sample was illuminated at 630 nm for 40 min using a 20 mW He-Ne laser. A dark background spectrum was subtracted from the experimental spectrum. EPR spectral simulations were carried out on a dual 1 GHz Power Macintosh G4 computer using a Windows 3.1 emulator (SoftWindows, FWB Software Inc., Redwood Shores, CA) and SimFonia software (Bruker Analytik GMBH). Variable Temperature X-band, Q-band, and W-band Transient EPR—The low temperature X-band (9 GHz) transient EPR experiments were carried out on a laboratory built spectrometer using a Bruker ER046 XK-T microwave bridge equipped with an ER-4118X-MD-5W1 dielectric ring resonator and an Oxford CF935 helium gas-flow cryostat (18van 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 Q = 3000, equivalent to a rise time of τr = Q/(2π × νmw) ≈ 50 ns. Q-band (35 GHz) transient EPR spectra of the samples were also 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 and 50 μm phenazine methosulfate as external redox agents and were frozen in the dark. The samples were illuminated using a Spectra Physics Nd-YAG/MOPO laser system operating at 10 Hz. The low temperature X-band experiments on the R694APsaA and R674APsaB mutants were carried out using the set-up described below for room temperature measurements except that the sample was placed in a quartz tube and a liquid nitrogen cryostat was used to control the temperature. W-band (95 GHz) transient EPR spectra were measured using a Bruker E680 spectrometer. Illumination was accomplished with a frequency-doubled Nd-YAG laser using an optical fiber fed into the sample capillary and ending directly above the PS I sample. Room Temperature Transient EPR measurements—Room temperature X-band experiments were performed using a modified Bruker ESP 200 spectrometer equipped with a home-built, broadband amplifier (bandwidth >500 MHz). A flat cell and a rectangular resonator were used and the samples were illuminated using a Q-switched, frequency-doubled Continuum Surelite Nd-YAG laser at 532 nm with a repetition rate of 10 Hz. To mediate cyclic electron transfer, 1 mm sodium ascorbate and 50 μm phenazine methosulfate were added. Pulsed ENDOR Studies of the P 700+ A 1– State—Pulsed ENDOR experiments on the radical pair P700+A1– were performed using a Bruker ESP 380E X-band FT-EPR spectrometer using a ESP360D-P ENDOR accessory, an ER4118X-MD-5W1-EN ENDOR resonator, and an ENI A500 radiofrequency amplifier. The Davies-ENDOR pulse sequence (π(microwave) – π(radio frequency) – π/2(microwave) – π(microwave) – echo) was used, with pulse lengths of 128 ns for the two microwave π pulses and 64 ns for a microwave π/2 pulse and 8 μs for the radiofrequency π pulse. The delay time between the laser flash and the first microwave pulse was 800 ns. The ENDOR experiments were carried out at 80 K, and the field position was chosen to provide the most symmetric spectra. See Ref. 19Fursmann C. Teutloff C. Bittl R. J. Phys. Chem. B. 2002; 106: 9679-9686Crossref Scopus (22) Google Scholar for a detailed discussion of the field-dependence of the ENDOR spectra. The light source for the experiments was a Q-switched and frequency-doubled Nd-YAG laser (Spectra Physics GCR 130) operating at a wavelength of 532 nm with a pulse width of 8 ns (full width at half-height) and a repetition rate of 10 Hz. Rationale for the Choice of the Mutant Strains— Fig. 1, top, shows a view (parallel to the membrane plane) of the QK-A region of the PsaA subunit and Fig. 1, bottom, shows the corresponding view of the QK-B region of the PsaB subunit. Examination of this region of the structure reveals a network of contacts between residues extending from Met688PsaA (Met668PsaB), which is the axial ligand to Chl eC-A3 (eC-B3), through QK-A (QK-B) to Gly572PsaB (Gly585PsaA) in the respective FX binding loop, which reaches over from the other subunit of the heterodimer. The backbone of Met688PsaA (Met668PsaB) is H-bonded to Ser692PsaA (Ser672PsaB), which in turn is H-bonded to Trp697PsaA (Trp677PsaB). This Trp is π-stacked with the phylloquinone, which is H-bonded to Leu722PsaA (Leu706PsaB). In turn, the backbone oxygen of Leu722PsaA (Leu706PsaB) is H-bonded to the FX binding loop through Arg694PsaA (Arg674PsaB), which originates from the start of the respective stromal surface helix jk (1Schubert W.D. Klukas O. Saenger W. Witt H.T. Fromme P. Krauss N. J. Mol. Biol. 1998; 280: 297-314Crossref PubMed Scopus (194) Google Scholar) and acts as a bridge between the return loop containing Leu722PsaA (Leu706PsaB) and the FX binding loop provided by the other heterodimeric subunit. Note that for this purpose the FX binding loop of PsaB (PsaA) crosses over into the region occupied below the stromal membrane surface by the other subunit PsaA (PsaB) and establishes the intersubunit H-bond between Arg694PsaA (Arg674PsaB) and the backbone oxygen of Gly572PsaB (Gly585PsaA). Additional intraloop contacts stabilize the loop configuration as part of the FX binding site. This continuous set of covalent bonds, ionic contacts, H-bonds, and π–π contacts may constitute a highly favorable electron transfer pathway from A0 through A1 to FX. When inferring functional properties of the wild-type from studies of mutants, it is important to base the conclusions on as many mutants as possible. This is particularly true if it is not known with certainty whether structural changes are induced by the mutations. Immediate candidates for mutation are Trp697PsaA and Trp677PsaB, which are in π-π contact with QK-A and QK-B and are therefore likely to influence the redox properties of the quinones. Because we wanted to restrict the influence of the mutations to the vicinity of the amino acids involved, we chose to make a conservative replacement, changing the Trp to a Phe. As can be seen in Fig. 1, top, a side chain oxygen of Ser692PsaA forms a H-bond with the imidazole nitrogen of Trp697PsaA, which is in π–π contact with QK-A. The hydrogen atom, which is shared between the Ser and Trp residues, is also close to the carbonyl oxygen ortho to the methyl group on the quinone ring. Thus, the quinone may participate to some extent in the H-bonding, although the distances and angles argue against a significant interaction. Rather, the function of the Ser residue is likely to stabilize the Trp in π–π contact with the quinone. Hence, Ser692PsaA and Ser672PsaB were considered further good candidates for mutagenesis. Again, by making a conservative mutation of the Ser to a Cys, we expect only subtle changes in the H-bonding to the neighboring Trp. Fig. 1 also highlights the two symmetry-related residues, Arg694PsaA and Arg674PsaB, which link the A1 and FX binding sites as described above. Thus, Arg694PsaA and Arg674PsaB residues were also considered candidates for mutagenesis. The PsaA- or PsaB-specific mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were therefore constructed, because they would be most likely to show an effect on the acceptors involved in the electron transfer pathways. Physiological Characterization of the Mutant Strains—The growth rates of the wild-type and mutant strains were compared at different light intensities under photoautotrophic and photomixotrophic growth conditions (Table I). All mutant strains grew photoautotrophically, yet all displayed reduced growth rates compared with the wild-type. The growth rates of all of the mutant strains were significantly slower at high light intensities (about 250 μmol m–2 s–1) than at low light intensities (about 5 μmol m–2 s–1). In contrast, the growth rate of the wild-type strain was marginally faster at high light intensities than at normal light intensities. Regardless of light intensity, the growth rates of W697FPsaA and W677FPsaB were the most severely impacted of all of the mutant strains; the growth rates of the S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB mutant strains were nearly equivalent. The growth rate of the PsaA side mutant W697FPsaA was slower than the PsaB side mutant W677APsaB, especially under high light intensities.Table IGrowth rate and chlorophyll content of wild-type and mutant strainsStrainsChlorophyll contentaChlorophyll concentration was measur