Electron Transfer in Cyanobacterial Photosystem I

I. PHYSIOLOGICAL AND SPECTROSCOPIC CHARACTERIZATION OF SITE-DIRECTED MUTANTS IN A PUTATIVE ELECTRON TRANSFER PATHWAY FROM A0 THROUGH A1 TO FX*

Wu Xu {ddagger}, Parag Chitnis {ddagger}, Alfia Valieva §, Art van der Est §, Yulia N. Pushkar ¶, Maciej Krzystyniak ¶, Christian Teutloff ¶, Stephan G. Zech ¶, Robert Bittl ¶, Dietmar Stehlik ¶, Boris Zybailov ||, Gaozhong Shen || and John H. Golbeck || **

From the {ddagger}Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, the §Department of Chemistry, Brock University, St. Catharines, Ontario L2S 3A1, Canada, the Fachbereich Physik, Freie Universität, D14195 Berlin, Germany, and the ||Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802

Received for publication, March 24, 2003 , and in revised form, April 23, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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 {pi}-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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Photosynthetic reaction centers (RCs)1 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 (1).

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 (2). 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 (3). 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 (4). 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. 57 for a summary), and a Trp in van der Waals contact with the phylloquinone (5, 6, 8, 9), most probably {pi}-stacked (5, 6, 911). Both the {pi}-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 (3). 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 (24), 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 (1216) (see also Ref. 10 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.



View larger version (29K):
[in this window]
[in a new window]
 
FIG. 1.
Environment of the two phylloquinones in PS I from the 2.5-Å resolution x-ray structure (3) (Protein Data Bank entry 1JB0 [PDB] ). The binding sites are viewed from a direction roughly parallel to the membrane plane. The locations of those amino acids discussed in the text are indicated. The figure was constructed using the program MOLMOL (23).

 

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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 pCRT{Delta}B 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.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 2.
Physical map of DNA fragments for site-directed mutagenesis of the psaA and psaB genes in Synechocystis sp. PCC 6803. A, restriction map of the Synechocystis sp. PCC 6803 genomic region containing the psaA-psaB operon in wild-type and two recipient strains pWX3 and pCRT{Delta}B. B, restriction map of pIBC and pGEM3C+ plasmids showing DNA fragments cloned into pBluescript II KS vector and pGEM7z and insertion of the chloramphenicol resistance (Cam-r) gene after the terminator of psaA-psaB operon. Mutation sites of Ser, Arg, and Trp are marked by arrows.

 

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 TransformerTM 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 pCRT{Delta}B. 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 x 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 (A730).

Quantitation of chlorophyll content, preparation of thylakoid membranes, and isolation of PS I particles were carried out according to previously published procedures (17). 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 c6 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. 12. 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 (18). The loaded Q-value for this dielectric ring resonator was about Q = 3000, equivalent to a rise time of {tau}r = Q/(2{pi} x {nu}mw) {approx} 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 P700+A1 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 ({pi}(microwave) – {pi}(radio frequency) – {pi}/2(microwave) {pi}(microwave) – echo) was used, with pulse lengths of 128 ns for the two microwave {pi} pulses and 64 ns for a microwave {pi}/2 pulse and 8 µs for the radiofrequency {pi} 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. 19 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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 {pi}-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 (1) 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 {pi}{pi} 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 {pi}-{pi} 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 {pi}{pi} 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 {pi}{pi} 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 m2 s1) than at low light intensities (about 5 µmol m2 s1). 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.


View this table:
[in this window]
[in a new window]
 
TABLE I
Growth rate and chlorophyll content of wild-type and mutant strains

 

When the wild-type and mutant strains were grown photomixotrophically with glucose in the medium, all mutant strains displayed growth rates similar to the wild-type (Table I). Under these conditions, heterotrophy is the major energy acquisition mode in Synechocystis sp. PCC 6803, and respiratory electron transport rates in the mutant strains are deemed sufficient to sustain a normal heterotrophic growth. When the cells were cultured under high light intensities, the W697FPsaA and W677APsaB mutant strains showed high light sensitivity as indicated by their longer doubling times. This effect was either absent or less pronounced in the S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB mutant strains. Thus, within the error bounds, the PsaA side mutants are more sensitive to high light conditions than the PsaB side mutants.

To assess the physiological impact of the mutations on photosynthetic activity, oxygen uptake and oxygen evolution were measured on thylakoid membranes of the wild-type and mutant strains (Table II). PS II electron throughput was measured by oxygen evolution using benzoquinone as the electron acceptor; the activities of the wild-type and the mutants were found to be similar. PS I electron throughput was measured either by oxygen uptake using 3,6-diaminodurene as electron donor, and methyl viologen as electron acceptor or by NADP+ reduction using cytochrome c6 as donor and ferredoxin as acceptor. All mutant strains showed lower levels of PS I-mediated electron transfer activity compared with the wild-type. The electron throughput of W697FPsaA and W677FPsaB were the most severely impacted of all of the mutant strains; the electron throughput of the S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB mutant strains were also consistently lower than the wild-type. Regardless of whether the mutation was in the Trp, Ser, or Arg residues, the electron throughput activities of the PsaA side mutants were consistently lower than the PsaB side mutants. There was also no significant difference in the relative loss of activity when measured by the oxygen uptake assay or by NADP+ reduction. In general, the decrease in PS I-mediated electron transfer activity of the thylakoid membranes (Table II) correlated with the increase in the doubling time of the mutant strains (Table I).


View this table:
[in this window]
[in a new window]
 
TABLE II
Biochemical characterization of thylakoid membranes in the PS I mutants

 

When grown under normal light intensities, the mutant strains contained less chlorophyll than the wild-type (Table I). In particular, the replacement of the {pi}-stacked Trp with Phe in both the QK-A and QK-B binding sites has a more pronounced effect on PS I stability than does the alteration of a H-bond to the {pi}-stacked Trp residues or the loss of the H-bond by the replacement of the Arg residues with Ala residues. Note also that whether the mutation was in the Trp, Ser, or Arg residues, the chlorophyll content in all of the PsaA side mutant strains is lower than in the PsaB side mutant strains. On closer inspection, the lower chlorophyll content of the mutant strains roughly parallels the decrease in their growth rates (Table I) and the decrease in PS I-mediated electron throughput (Table II). This is especially evident in the W697FPsaA and W677FPsaB mutant strains. Because PS I is the major chlorophyll-containing complex under iron-replete growth conditions, the lower growth rates may be a function of a lower concentration of PS I in the mutant cells. The data implies that the electron transfer activity in the membranes may be more related to the amount of PS I in the mutant cells as to any direct effect on primary electron transfer. The lower levels of PS I could be a result of down-regulation in response to low electron transfer efficiency.

Accumulation of PS I Proteins in Thylakoid Membranes of the Mutant Strains—Western blotting assays using polyclonal antibodies against PsaB, PsaC, or PsaL were performed to estimate the steady state level of the PS I subunits in the thylakoid membranes of the mutant cells (Fig. 3). These antibodies typically give linear signal intensities over a wide range of protein concentrations and therefore are suitable for semi-quantitative estimation. The quantitation of PS I in the mutant membranes was based on the amount of wild-type membranes required to produce the same signal intensity. On the basis of the same chlorophyll content, the levels of PS I in the W697FPsaA and W677FPsaB membranes were reduced to about 80% of the wild-type levels. However, no obvious differences from the wild-type levels could be observed for the accumulation of PS I in the S692CPsaA and S672CPsaB membranes (the Arg mutants were not studied).



View larger version (24K):
[in this window]
[in a new window]
 
FIG. 3.
Western blotting analysis of PS I subunits accumulation in the thylakoid membranes of the Trp -> Phe (panel A) and Ser -> Cys (panel B) mutant strains. The proteins were resolved by Tricine/urea/SDS-PAGE and blotted onto nitrocellulose membranes. For quantitative comparison, thylakoid membranes of the wild type were loaded with three different concentrations (0.5, 1.5, and 5.0 µg of Chl) per lane; 5 µg of chlorophyll was loaded in each lane for thylakoids of mutants. Thylakoid membranes of the recipient strain pWX3 were loaded as the PS I-less control. The antibodies against Synechocystis sp. PCC 6803 PsaB and PsaL, and Synechococcus sp. PCC 7002 PsaC were used and the immunodetection was visualized by enhanced chemiluminescence.

 

To examine the polypeptide composition of the purified PS I complexes, the subunits were resolved by SDS-PAGE and visualized by staining with Coomassie Blue. The PS I subunits PsaA through PsaF, and PsaI through PsaM could be identified in PS I complexes isolated from the wild-type and all of the mutants (data not shown). The relative amounts of the different subunits in the PS I complexes were nearly identical in the wild-type and mutants. These results suggest that the Trp, Arg, or Ser mutations in PsaA and PsaB have no effect on the polypeptide composition of PS I, even though the absolute number of PS I complexes per cell may be reduced in the mutants. All further spectroscopic characterizations of the mutants were carried out on purified PS I complexes.

Photoaccumulated CW EPR Spectrum at Q-band—The photoaccumulated EPR spectrum of A1 in PS I complexes from the wild-type, W697FPsaA, W677FPsaB, S692CPsaA, and S672CPsaB mutants are depicted in Fig. 4. At 34 GHz (Q-band), the g-anisotropy of A1 dominates the EPR spectrum in wild-type PS I, allowing the gxx = 2.0062 and gzz = 2.0021 components of the tensor to be resolved as the positive and negative features on the left- and right-hand extremes of the spectrum, respectively. The four partially resolved hyperfine lines from the –CH3 fragment obscure the gyy component in the middle of the spectrum and result from the high spin density on the ring carbon of the phyllosemiquinone anion radical to which the methyl group is attached (see structure in Fig. 1). A small amount of A0 is also present in the wild-type spectrum, and contributes to the exaggerated highfield (gzz) trough. The photoaccumulated CW EPR spectrum of the W697FPsaA mutant is similar to the wild-type, but shows slightly more prominent hyperfine lines and the spectrum is contaminated with a greater contribution from A0. These features are not found in the spectrum of the W677FPsaB mutant, which is similar to the wild-type, showing some indication of hyperfine couplings and a relatively small contribution from A0. The spectrum of the S692CPsaA mutant shows a linewidth equivalent to that of the wild-type, but there is a (conspicuous) absence of prominent hyperfine lines and a slightly greater contribution from A0. In contrast, the photoaccumulated EPR spectrum of the S672CPsaB mutant was found to be identical with the wild-type, showing some indication of hyperfine coupling and a relatively small contribution from A0. The data on the Trp and Ser mutants are therefore internally consistent, showing that alterations on the PsaB side lead to an EPR spectrum identical to the wild-type, but that alterations on the PsaA side lead to a spectrum different from the wild-type.



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 4.
Photoaccumulated CW EPR spectra of PS I complexes isolated from the wild-type, W697FPsaA, W677FPsaB, S692CPsaA, and S672CPsaB mutants. The experimental conditions are described under "Materials and Methods." Because the microwave frequency was not identical in all scans, the spectra are plotted against g value.

 

Spin Polarization Patterns of P700+A1 at X-band, Q-band, and W-band–-The spin polarization patterns of P700+A1 are sensitive to the orientation of A1 (see Ref. 11 for a recent review). Thus, they provide a good method for probing the effect of the mutations on the environment of the quinone. Fig. 5 shows the direct detection transient X-band, Q-band, and W-band spectra of P700+A1 in the W697FPsaA and W677FPsaB mutants together with the corresponding wild-type spectra taken in frozen solution at low temperature. With the exception of the W-band spectra, the same comparison for S692CPsaA and S672CPsaB is shown in Fig. 6 and corresponding spectra of the R694APsaA and R674APsaB mutants are shown in Fig. 7. The overall polarization patterns for all of the mutants are very similar to wild-type. However, subtle differences in the partially resolved methyl hyperfine structure are observed mainly for the W697FPsaA and S692CPsaA mutants. The effect of the hyperfine couplings is most pronounced in the X-band spectra whereas the g-anisotropy plays a more important role in the Q-band and W-band spectra, which makes the latter more sensitive to the quinone orientation. With the exception of the W697FPsaA mutant, the Q-band spectra are virtually identical with the wild-type. We conclude that these mutations do not induce any significant change in either the orientation of the observed quinone or in its g-anisotropy. In the Q-band spectrum of the W697FPsaA mutant, there may be a small difference with the wild-type in the low field part of the spectrum. To increase the spectral resolution and to determine the significance of this effect (mainly the difference in the g-tensor) we performed studies of the W697FPsaA and W677FPsaB mutants at W-band (95 GHz). As shown in Fig. 5 (E and F), the PsaB mutant is identical with the wild-type, whereas the PsaA mutant may have a slightly more anisotropic g-tensor: the gxx component shifted to a higher value. However, even with the increased resolution available at W-band, the difference between the mutant and the wild-type is small and is within the S/N, indicating that there is no significant change in the orientation of the quinone.



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 5.
X-band (top), Q-band (middle), and W-band (bottom) spin-polarized transient EPR spectra of PS I complexes from the W697FPsaA and W677FPsaB mutants (solid lines) compared with wild-type (dashed lines)at80K. Positive signals correspond to absorption (A) and negative signals correspond to emission (E). The spectra are assigned to P700+A1 and have been extracted from the full time/field data sets by integrating the signal intensity in a time window from 152 to 1520 ns following the laser flash. Note that the field axes are different for the X-, Q-, and W-band spectra. The experimental conditions are given under "Materials and Methods."

 


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 6.
X-band (top) and Q-band (bottom) spin-polarized transient EPR spectra of PS I complexes from the S692CPsaA and S672CPsaB mutants (solid lines) compared with the wild type (dashed lines) at 80 K. Conditions are as described in the legend to Fig. 5.

 


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 7.
X-band (top) and Q-band (bottom) spin-polarized transient EPR spectra of PS I complexes from the R694APsaA and R674APsaB mutants (solid lines) compared with the wild type (dashed lines) at 135 K. The spectra are assigned to P700+A1 and are the integrated signal intensity in a time window of 0.8–1.6 µs following the laser flash. All other conditions are as described in the legend to Fig. 5.

 

A more careful inspection of the X-band spectra shows that while the spectra of the PsaB mutants (Figs. 5B and 6B) are almost indistinguishable from the wild-type, the methyl hyper-fine splitting pattern is slightly more pronounced for the W697FPsaA mutant (Fig. 5A) and less pronounced for the S692CPsaA mutant (Fig. 6A). This is seen most clearly on the low field shoulder of the central absorptive peak and shows that the PsaA mutations affect the methyl hyperfine coupling. There is also a slight shift of the low field emission in the Q-band spectrum of the W697FPsaA mutant compared with wild-type, which is consistent with an increase in the methyl hyperfine coupling.

Pulsed ENDOR of P700+A1To quantify the effects on the hyperfine couplings of the Trp, Ser, and Arg mutants, we performed pulsed ENDOR experiments on P700+A1. Fig. 8 compares the pulsed ENDOR spectra of P700+A1 in the W697FPsaA, W677FPsaB, S692CPsaA, and S672CPsaB mutants (top) as well as the R694APsaA and R674APsaB mutants (bottom). The ENDOR spectra are taken at a field position for which the spectra appear approximately symmetric with respect to the proton Larmor frequency. ENDOR lines occur in absorption (positive features) and emission (negative features) depending on the electron spin polarization of the radical pair (for a detailed analysis see Ref. 19). Two clearly resolved powder patterns of an axially symmetric tensor are observed in the wings of all spectra in the range 8.6–10.6 and 18.9–20.9 MHz. These features are assigned to the ring methyl group of phylloquinone. A qualitative comparison shows that the spectra of all PsaB mutants are virtually identical to each other. In addition, the spectra of the PsaB mutants agree well with spectra recorded for PS I from Synechococcus elongatus (19). The spectra of the PsaA mutants display systematic changes. In Fig. 8, top, both edges of the methyl hyperfine powder patterns, representing the A|| and A{perp} hyperfine tensor components, shift within experimental accuracy by the same amount of about 5% for both PsaA mutants, compared with the PsaB mutants, but the direction of the shift is opposite in the two cases with larger couplings observed for the W697FPsaA mutant and smaller couplings in the S692CPsaA mutant. Because the A|| and A{perp} components change together, the tensor asymmetry does not change but rather the isotropic hyperfine coupling constant, which is determined by the spin density at the carbon ring position to which the methyl group is attached. Using the hyperfine tensor components of S. elongatus (which also apply to all PsaB mutants) of A|| = 12.3 ± 0.1 MHz and A{perp} = 8.8 ± 0.1 MHz (19), the shifts in the PsaA mutant corresponds to A|| = 11.8 ± 0.1 MHz and A{perp} = 8.3 ± 0.1 MHz for S692CPsaA, and A|| = 12.9 ± 0.1 MHz and A{perp} = 9.4 ± 0.1 MHz for W697FPsaA. In addition to these changes in the methyl hyperfine couplings, the S692CPsaA mutant also shows spectral changes compared with the wild-type in the 11.5–12.5 and 17.0–18.0 MHz range (Fig. 8). The hyperfine couplings corresponding to the features in these regions of the spectrum are also reduced by about the same amount as the methyl couplings, however, assignments to specific protons is not yet certain. In Fig. 8, bottom, again the ENDOR spectrum of the R674APsaB mutant is identical to those of the PsaB mutants above (and of wild type) (19). In contrast, the spectrum of the R694APsaA mutant shows characteristic shifts of relevant spectral features, actually very similar to those observed for the S692CPsaA mutant in Fig. 8, top. Thus, consistent with the pact that changes in the transient EPR spectra are only observed for the PsaA mutants, the R694APsaA mutation alters the ENDOR spectrum, while the R674APsaB mutation does not. Note that the different result for the two mutants confirms nicely the specificity of each of the mutants. There are no obvious structural hints why the spectral changes for the S692CPsaA and R694APsaA mutants should be so similar. On the other hand, the relative changes are quite small (about 5%) but have the same sign. As a final note we remark that all of the observed changes in the hyperfine parameters for the PsaA and not the PsaB mutants are most precisely evaluated from the ENDOR data. Nevertheless, they can also be recognized in and are consistent with the changes observed in the EPR spectra of photoaccumulated A1 (Fig. 4) and the spin polarization patterns of P700+A1 (Figs. 5 and 6).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 8.
Pulsed ENDOR of the P700+A1 state in PS I point mutants. Top, S672CPsaB (solid line), S692CPsaA (dashed line), W677FPsaB (dotted line), W697FPsaA (dashed-dotted line). Bottom, R694APsaA (solid line), R674APsaB (dashed line). See "Materials and Methods" for details of the pulse sequences and other experimental conditions.

 

Clearly, only mutations in the PsaA protein lead to a change in the hyperfine couplings whereas PsaB mutations do not, which shows that only the quinone on the PsaA branch is observed in the transient EPR and ENDOR experiments. Keeping in mind that for the low temperature spin polarization patterns and pulsed ENDOR experiments, only the fraction of RCs in which reversible electron transfer through A1 takes place is observed, we conclude that for this fraction electron transfer occurs along the PsaA branch. It is conceivable that the behavior at low temperature or under the strongly reducing conditions used for the photoaccumulation experiments might be different from the behavior under physiological conditions. Therefore it is important to combine these experiments with measurements at room temperature and below, in which forward electron transfer past P700+A1 is observed.

Room Temperature Electron Spin-polarized EPR Spectra at X-band—Electron spin-polarized EPR spectra from the wild-type and the mutants measured at room temperature are depicted in Fig. 9. The temperature dependence of the spectra was also measured and no significant differences in the polarization patterns were observed between 250 K and room temperature (see Ref. 24). The kinetically separated spectra are extracted from the complete time/field data sets using the fitting procedure described in Refs. 20 and 21. As the electron is transferred to the iron-sulfur clusters, the initial emission/absorption/emission (E/A/E) polarization pattern of P+A1 changes to the primarily emissive P+ contribution to the P+FeS spectrum. In Fig. 9, the spectra of the W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB mutants are shown to be very similar, however, some small but significant changes are observed as a result of the mutations. (Note that the weaker P+FeS spectrum for the S692CPsaA mutant (middle left) is an artifact because of difficulties in extracting the spectrum when the lifetime of the electron transfer from A1 to FX and the lifetime of the spin polarization are approximately equal.) The spectral differences are most obvious in the case of the W697FPsaA mutant (Fig. 9, left column, top), which shows a more pronounced hyperfine structure than found for the wild type. In contrast no difference is detectable within experimental error between the spectra of the corresponding PsaB side mutant and wild type (Fig. 9, right column, top). This is the same result as observed in the low temperature transient EPR spectra (Fig. 5) and in the pulsed ENDOR (Fig. 8). The spectra of the S692CPsaA mutant (middle left) and the R694APsaA mutant (bottom left) also show some indication of the weaker hyperfine structure seen at low temperature; however, the lower signal to noise ratio at room temperature does not allow the effect to be detected clearly. The correspondence between the low temperature and room temperature spectra of the Trp mutants (and the other mutants) is significant because it shows that conclusions based on the low temperature spectra are relevant for PS I under physiological conditions. The data shown here are from isolated PS I complexes and it is known that for some species, the isolation procedures have an influence on the biphasic electron transfer kinetics from A1 to FX. To ensure that such effects are not occurring in these samples, we performed transient EPR experiments on whole cells of wild-type Synechocystis sp. PCC 6803 and the spectra (not shown) are indistinguishable from the spectra of isolated wild-type PS I complexes.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 9.
Room temperature transient EPR spectra of PS I with point mutations in PsaA (left) and PsaB (right). Positive signals correspond to absorption (A) and negative signals correspond to emission (E). The spectra are assigned to P700+A1 (E/A/E pattern) and P700+FeS (emissive spectrum) and have been extracted from the time/field data sets by fitting the individual transients. The parameters used in the fitting procedure are given in the accompanying paper (24). The solid curves are from the mutants and the dashed curves are the corresponding spectra from wild-type PS I. Top left, W667FPsaA; top right, W677FPsaB; middle left, S692CPsaA; middle right, S672CPsaB; bottom left, R694APsaA; bottom right, R674APsaB.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Effect of the Mutations—The interpretation of the spectra is predicated on the assumption that the mutations cause only subtle changes in the properties of the cofactors without altering the electron transfer pathway. Ideally, the physiological properties of the mutants would be identical to the wild type. The data in Tables I and II show that although the mutants do not rigorously fulfill this criterion, the differences are quite small, especially for the Ser and Arg mutants. Indeed, the lower growth rate and the lower rates of steady-state PS I-based activity in the membranes correlate with a lower than normal PS I content in the mutant cells. Furthermore, there appears to be no correlation between growth rate and the kinetic data, i.e. the rate of oxidation of A1 (see Ref. 24). Because the mechanism by which the PS I content in the membranes is regulated is not known, it is difficult to infer whether the lower amount of PS I is a result of poor assembly because of structural changes or of down-regulation of PS I because of impairment of its function. However, the physiological data show that any such perturbations induced by the mutations are minor.

The ENDOR and EPR data, on the other hand, give details about the local environment of the cofactors. The small but clearly visible shifts in the ENDOR spectra and corresponding subtle changes in the EPR spectra show that the mutations in the QK-A binding site lead to changes in the spin density distribution on the phylloquinone while at the same time the spin polarization patterns, especially at Q- and W-band, show that none of the mutations affect its position and orientation. Two protein-cofactor interactions are well established as having a significant influence of spin density distribution: (i) asymmetric H-bonds to the two quinone carbonyl groups and (ii) {pi}-stacking between an aromatic residue and the quinone ring (see Refs. 5 and 6, and references therein). The changes induced by the mutations can be interpreted in terms of these two interactions, however, this interpretation remains preliminary because other factors such as local charges or induced dipoles can influence the spectral results. In wild-type PS I, the single H-bond to Leu722PsaA (see Fig. 1, top) introduces an asymmetric spin density distribution such that it is increased on the ring carbon atom to which the methyl group is attached. The {pi}-stacking arrangement with Trp697PsaA, on the other hand, reduces the effect of the asymmetric H-bonding. In the W697FPsaA mutant it is likely that the {pi}-stacking is weakened so that the distortion of the spin density because of the H-bond to Leu722PsaA is increased. Consistent with this, the methyl hyperfine couplings are increased in the W697FPsaA mutant. The S692CPsaA mutant, on the other hand, can be expected to change the environment of the carbonyl oxygen ortho to the methyl group of the quinone. Although Ser and Cys differ only by the substitution of the hydroxyl group in serine by a thiol group in cysteine, the van der Waals radius of the sulfur is larger than that of oxygen. The effect of this change on the environment is difficult to predict. The weaker methyl hyperfine coupling in the S692CPsaA mutant is consistent with a change in the environment for the carbonyl oxygen ortho to the methyl group possibly as a result of an increase in the electron withdrawing ability of the surroundings.

Consequences for Electron Transfer—All data reported here from the point mutants suggest that the quinone measured by EPR and ENDOR is located in the PsaA branch. The quinone in the PsaA branch was also implicated in EPR studies of a PsaE/PsaF double deletion mutant (12), which showed complete loss of the photoaccumulated A1 spectrum and appearance of an A0 spectrum. The appearance of A0 is thought to require double reduction and protonation of the quinone. Because PsaE and PsaF are located on the PsaA side of the PS I complex, it was suggested that removal of these subunits facilitates protonation of the quinone via a water channel. The ability to photoaccumulate A1 rests largely on a favorable combination of kinetic rates, particularly on forward electron transfer between the sulfite ion, and P700+ outcompeting the inherent backreaction between P700+ and A1 in a statistically meaningful number of turnovers. This condition is clearly met for the PsaA side quinone; however, it is not clear whether or not this condition can be met for the PsaB side quinone, which, if active may have different forward and backward kinetics. If electron transfer along the PsaB branch occurred to any significant extent, it would require that either (i) no accumulation of QK-B occurs or (ii) the point mutations have no effect on QK-B whereas removal of the PsaE and PsaF has the same effect on both quinones. Clearly, the latter assumption is physically unreasonable.

At low temperature, the photoaccumulated A1 spectra, the spin polarization patterns, and the pulsed ENDOR spectra all show an effect when mutations are made to PsaA but the corresponding spectra from the PsaB mutants are identical to those from wild type. Thus, we conclude that only the phylloquinone bound to PsaA (QK-A) contributes to these spectra, whereas the phylloquinone bound to PsaB (QK-B) does not. Because the observed changes in the properties of the quinone are small, it is reasonable to extrapolate from this result to the conclusion that the electron cycle between P700 and A1 measured by EPR at low temperature occurs along the PsaA branch. This is consistent with the conclusion reached by Heathcote et al. (15) and Boudreaux et al. (22) from point mutation studies in the eukaryotic organism, Chlamydomonas reinhardtii. However, it must be kept in mind that cyclic electron transfer involving P700 and A1 occurs in only a fraction of the RCs in frozen solution, whereas stable charge separation to the ironsulfur clusters takes place in the remainder. Therefore, we also need to consider the pathway taken by the fraction that is involved in non-cyclic electron transfer. An important feature of the cyclic and non-cyclic fractions is that their relative amplitudes do not change under prolonged illumination. This behavior rules out the possibility that they result from two competing pathways because if all RCs had even a low probability for the non-cyclic pathway, prolonged illumination would lead to complete non-cyclic charge separation.

At room temperature the changes in the spectra from the Trp and Ser mutants (Fig. 9) mirror those at low temperature, i.e. mutations in PsaB have no effect on the patterns whereas those in the PsaA result in visible changes. Therefore we conclude that the EPR spectra measured at room temperature are also dominated by radical pairs generated by electron transfer along the PsaA branch. Optical experiments reveal a component of electron transfer from A1 to Fx with a lifetime of ~10 ns and it has been proposed that this corresponds to electron transfer in the PsaB branch (see Ref. 10 for a summary) (13, 14). Therefore, it is important to compare time-resolved optical and EPR results for the same mutant samples and try to interpret the results in a unified manner. In the accompanying paper (24) we present such data and discuss the kinetics of electron transfer in greater detail.


    CONCLUSIONS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
This work represents part I of a comprehensive study of residues that surround the phylloquinone, which are postulated to be involved in an electron transfer pathway between A0 and FX. A spectroscopic characterization of the W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB mutants in Synechocystis sp. PCC 6803 includes ENDOR studies, which shows that the W697FPsaA mutation leads to a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a decrease in this coupling by about the same amount. These changes correlate with spectral alterations observed by CW EPR spectroscopy of photoaccumulated PS I complexes as well as those studied by transient EPR spectroscopy. Thus, electron transfer detected by EPR spectroscopy in this cyanobacterium occurs on cofactors associated with the PsaA side. The transient EPR studies performed at low temperature correlate with those at room temperature, indicating that conclusions reached at low temperatures are valid at physiological temperatures. Again, the wild-type and the Trp677PsaB and Ser672PsaB mutants yield the same spectra. This agrees with an assessment that those electron transfer steps detected by EPR spectroscopy in the eukaryotic organism C. reinhardtii also involve cofactors associated with the PsaA side (15, 22). It is more difficult to assess from EPR data whether any electron transfer occurs on the cofactors associated with the PsaB side, and the accompanying paper (24) will address the biphasic kinetics of electron transfer in the point mutants in detail.


    FOOTNOTES
 
* This work was supported by National Science Foundation Grants MCB-0117079 (to J. H. G.) and MCB-9723001 (to P. R. C.), Deutsche Forschungsgemeinschaft Grants SFB 498, TPA3, C5, and SPP "High Field EPR" (to D. S. and R. B.), grants from the Fonds der Chemischen Industrie (to R. B.), the Natural Sciences and Engineering Research Council, The Canada Foundation for Innovation, and The Ontario Innovation Trust (to A. v. d. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed. Fax: 814-863-7024; E-mail: JHG5{at}psu.edu.

1 The 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. Back


    ACKNOWLEDGMENTS
 
We thank Wolfgang Lubitz for kindly providing access to the pulsed ENDOR spectrometer at the Max Planck Institute für bioanorganische Chemie, Mülheim, Germany, and Maurice van Gastel for assistance with the ENDOR experiments on the R694APsaA and R674APsaB mutants.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Schubert, W. D., Klukas, O., Saenger, W., Witt, H. T., Fromme, P., and Krauss, N. (1998) J. Mol. Biol. 280, 297–314[CrossRef][Medline] [Order article via Infotrieve]
  2. Fromme, P. (1999) in Concepts in Photobiology: Photosynthesis and Photomorphogenesis (Singhal, G. S., R. G., Sopory, S. K., Irrgang, K. D., and Govindjee, eds) pp. 181–220, Narosa Publishing House, New Delhi, India
  3. Jordan, P., Fromme, P., Witt, H. T., Klukas, O., Saenger, W., and Krauß, N. (2001) Nature 411, 909–917[CrossRef][Medline] [Order article via Infotrieve]
  4. Deisenhofer, J., Epp, O., Miki, K., Huber, R., and Michel, H. (1984) J. Mol. Biol. 180, 385–398[Medline] [Order article via Infotrieve]
  5. Kamlowski, A., Altenberg-Greulich, B., van der Est, A., Zech, S. G., Bittl, R., Fromme, P., Lubitz, W., and Stehlik, D. (1998) J. Phys. Chem. B 102, 8278–8287[CrossRef]
  6. Zech, S. G., Hofbauer, W., Kamlowski, A., Fromme, P., Stehlik, D., Lubitz, W., and Bittl, R. (2000) J. Phys. Chem. B 104, 9728–9739[CrossRef]
  7. Rigby, S. E., Evans, M. C., and Heathcote, P. (2001) Biochim. Biophys. Acta 1507, 247–259[Medline] [Order article via Infotrieve]
  8. Hanley, J., Deligiannakis, Y., MacMillan, F., Bottin, H., and Rutherford, A. W. (1997) Biochemistry 36, 11543–11549[CrossRef][Medline] [Order article via Infotrieve]
  9. Itoh, S., Iwaki, M., and Ikegami, I. (2001) Biochim. Biophys. Acta 1507, 115–138[Medline] [Order article via Infotrieve]
  10. Brettel, K., and Leibl, W. (2001) Biochim. Biophys. Acta 1507, 100–114[CrossRef][Medline] [Order article via Infotrieve]
  11. van der Est, A. (2001) Biochim. Biophys. Acta 1507, 212–225[Medline] [Order article via Infotrieve]
  12. Yang, F., Shen, G., Schluchter, W. M., Zybailov, B., Ganago, A. O., Vassiliev, I. R., Bryant, D. A., and Golbeck, J. H. (1998) J. Phys. Chem. 102, 8288–8299
  13. Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130–11136[CrossRef][Medline] [Order article via Infotrieve]
  14. Guergova-Kuras, M., Boudreaux, B., Joliot, A., Joliot, P., and Redding, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4437–4442[Abstract/Free Full Text]
  15. Purton, S., Stevens, D. R., Muhiuddin, I. P., Evans, M. C., Carter, S., Rigby, S. E., and Heathcote, P. (2001) Biochemistry 40, 2167–2175[CrossRef][Medline] [Order article via Infotrieve]
  16. Hastings, G., and Sivakumar, V. (2001) Biochemistry 40, 3681–3689[CrossRef][Medline] [Order article via Infotrieve]
  17. Shen, G., Zhao, J., Reimer, S. K., Antonkine, M. L., Cai, Q., Weiland, S. M., Golbeck, J. H., and Bryant, D. A. (2002) J. Biol. Chem. 277, 20343–20354[Abstract/Free Full Text]
  18. van der Est, A., Hager-Braun, C., Leibl, W., Hauska, G., and Stehlik, D. (1998) Biochim. Biophys. Acta 1409, 87–98[Medline] [Order article via Infotrieve]
  19. Fursmann, C., Teutloff, C., and Bittl, R. (2002) J. Phys. Chem. B 106, 9679–9686
  20. van der Est, A., Bock, C., Golbeck, J., Brettel, K., Sétif, P., and Stehlik, D. (1994) Biochemistry 33, 11789–11797[Medline] [Order article via Infotrieve]
  21. Kandrashkin, Y. E., and van der Est, A. (2002) RIKEN Rev. 44, 124–127
  22. Boudreaux, B., MacMillan, F., Teutloff, C., Agalarov, R., Gu, F., Grimaldi, S., Bittl, R., Brettel, K., and Redding, K. (2001) J. Biol. Chem. 276, 37299–37306[Abstract/Free Full Text]
  23. Koradi, R., Billeter, M., and Wuthrich, K. (1996) J. Mol. Graph. 14, 29–32; 51–55
  24. Xu, 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., and Golbeck, J. H. (2003) J. Biol. Chem. 278, 27876–27887