Recruitment of a foreign quinone into the A1 site of photosystem I. In vivo replacement of plastoquinone-9 by media-supplemented naphthoquinones in phylloquinone biosynthetic pathway mutants of Synechocystis sp. PCC 6803.

Interruption of the phylloquinone (PhQ) biosynthetic pathway by interposon mutagenesis of the menA and menB genes in Synechocystis sp. PCC 6803 results in plastoquinone-9 (PQ-9) occupying the A(1) site and functioning in electron transfer from A(0) to the FeS clusters in photosystem (PS) I (Johnson, T. W., Shen, G., Zybailov, B., Kolling, D., Reategui, R., Beauparlant, S., Vassiliev, I. R., Bryant, D. A., Jones, A. D., Golbeck, J. H., and Chitnis, P. R. (2000) J. Biol. Chem. 275, 8523-8530. We report here the isolation of menB26, a strain of the menB mutant that grows in high light by virtue of a higher PS I to PS II ratio. PhQ can be reincorporated into the A(1) site of the menB26 mutant strain by supplementing the growth medium with authentic PhQ. The reincorporation of PhQ also occurs in cells that have been treated with protein synthesis inhibitors, consistent with a displacement of PQ-9 from the A(1) site by mass action. The doubling time of the menB26 mutant cells, but not the menA mutant cells, approaches the wild type when the growth medium is supplemented with naphthoquinone (NQ) derivatives such as 2-CO(2)H-1,4-NQ and 2-CH(3)-1,4-NQ. Since PhQ replaces PQ-9 in the supplemented menB26 mutant cells, but not in the menA mutant cells, the phytyl tail accompanies the incorporation of these quinones into the A(1) site. Studies with menB26 mutant cells and perdeuterated 2-CH(3)-1,4-NQ shows that phytylation occurs at position 3 of the NQ ring because the deuterated 2-methyl group remains intact. Therefore, the specificity of the phytyltransferase enzyme is selective with respect to the group present at ring positions 2 and 3. Supplementing the growth medium of menB26 mutant cells with 1,4-NQ also leads to its incorporation into the A(1) site, but typically without either the phytyl tail or the methyl group. These findings open the possibility of biologically incorporating novel quinones into the A(1) site by supplementing the growth medium of menB26 mutant cells.

Substituted quinones are usually employed as cofactors in electron transport chains, as demonstrated in bacterial reaction centers (1,2), and in the two photosystems of plants and cyanobacteria (3)(4)(5). These quinones comprise a relatively polar ring, which consists of either a benzoquinone (BQ) 1 or a naphthoquinone (NQ) "head group" and a non-polar isoprenoid "tail" of various chain lengths and degrees of saturation. Benzoquinones such as plastoquinone-9 (PQ-9) and ubiquinone-10 function either as fixed or exchangeable electron/proton carriers during photosynthetic and respiratory electron transport. In photosystem II (PS II), PQ-9 functions as a bound one-electron cofactor in the Q A site and as an exchangeable two-electron/ two-proton cofactor in the Q B site. The reduced PQH 2 -9 is displaced from the Q B site, diffuses laterally through the membrane, and becomes oxidized and deprotonated by the cytochrome b 6 f complex. Photosynthetic reaction centers (RCs) of purple bacteria use either ubiquinone-10 (e.g. Rhodobacter sphaeroides) in a similar double role or menaquinone-9 in the Q A site (e.g. Rhodosprillulum viridis). In photosystem I (PS I), phylloquinone (PhQ), a substituted 1,4-NQ with a 20-carbon, largely saturated phytyl tail, functions as a bound one-electron cofactor in the A 1 site. PS I contains two PhQ molecules/P700, but neither of the two quinones functions in a manner equivalent to Q B in the bacterial RC and PS II. Instead, the electron is transferred from the active quinone(s) to soluble ferredoxin via a chain of three bound iron-sulfur centers. Quinones are therefore extremely versatile; they can function as the interface between electron transfer involving organic cofactors and electron transfer involving iron-sulfur clusters (as in PS I), or between pure electron transfer and coupled electron/proton transfer involving a second organic cofactor (as in PS II). Each quinone displays equilibrium binding and redox properties that can be very different for each site of interaction (6), and these properties are conferred largely by the protein environment.
To understand the structural determinants that allow quinones to function with a low redox potential in the A 1 site of PS I, we embarked on a project aimed at biological replacement of * The work was supported by National Science Foundation Grants MCB 0078262 (to P. R. C.) and MCB 9723661 (to J. H. G.) and by Deutsche Forschungsgemeinschaft Grant SFB 498, TP A3, SPP "High Field EPR," and Fonds der Chemischen Industrie (to R. B.). This paper is fourth in the series, "Recruitment of a Foreign Quinone into the A 1 Site of Photosystem I." This is Journal Paper J-19488 of the Iowa Agriculture and Home Economics Experiment Station (Ames, IA) Project 3416 and is supported by Hatch Act and State of Iowa Funds. 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.
‡ ‡ To whom correspondence should be addressed. Tel.: 515-294-1657; Fax: 515-294-0453; E-mail: chitnis@iastate.edu. the native PhQ. Previously, the method used to remove PhQ was to extract lyophilized PS I complexes with solvents, which can remove one (7,8) or both (8 -10) PhQ molecules. Quinone substitution into the A 1 site has been demonstrated for these PS I complexes (11,12). However, up to 85% of the 100 chlorophylls and all of the carotenoids are lost, and it is uncertain whether or not the PS I complex has undergone structural changes as a result of solvent extraction.
In a series of three papers (13)(14)(15), we reported the generation and characterization of PhQ-less mutants in the cyanobacterium Synechocystis sp. PCC 6803. The chosen method was to employ interposon mutagenesis of the menA and menB genes, which code for phytyltransferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, in the biosynthetic pathway of PhQ (Fig. 1). The goal was to prevent PhQ biosynthesis with the intent of creating an empty A 1 site. We found instead that the A 1 site is occupied by a BQ derivative, identified as PQ-9, a molecule that is otherwise exclusively associated with PS II. Detailed EPR and optical spectroscopic analyses show that the PQ-9 radical is at the same distance from P700 ϩ and has the same orientation as the PhQ radical in the wild type. Surprisingly, PQ-9 was found to function in oxidation-reduction reactions as an intermediate electron transfer carrier between A 0 and F X . The redox potential of PQ-9 in the A 1 site was estimated to be more oxidizing than PhQ, rendering the electron transfer step from Q Ϫ to F X thermodynamically unfavorable. Forward electron transfer nevertheless occurs because the large favorable free energy change from A 0 Ϫ to flavodoxin remains unchanged.
In principle, it should be possible to introduce novel, phytylated quinones into the A 1 site in vivo by utilizing the phytyltransferase and/or methyltransferase enzymes in the existing biosynthetic pathway of the menA and/or menB mutant cells (Fig. 1). If successful, biological incorporation of various quinones into the A 1 site would allow better understanding of how the protein modulates the redox potential of a bound, organic cofactor in membrane-bound bioenergetic systems. In this paper, we put biological incorporation to a test by supplementing the growth medium of the menA and menB mutant cells with authentic PhQ and PhQ precursors.

MATERIALS AND METHODS
Cyanobacterial Strains and Growth-A glucose-tolerant strain of Synechocystis sp. PCC 6803 was used as the wild-type strain. The menA mutant strain lacks a functional phytyltransferase and has been described previously (13). The menB mutant strain used in this study (menB26) was selected as a spontaneous light-tolerant mutant from the menB18 strain reported previously (13). The wild-type and mutant cells were grown in BG-11 medium with kanamycin and spectinomycin added to the media of the menA and menB26 mutant strains, respectively (16). Agar plates for the growth of the stock cells were kept at low light intensity (2-10 E m Ϫ2 s Ϫ1 ). Liquid cultures of the wild-type and mutant strains were grown autotrophically under normal light conditions (40 -60 E m Ϫ2 s Ϫ1 ). Cells grown in liquid cultures were bubbled with sterile filtered air and were monitored by measuring the absorbance at 730 nm (1-cm pathlength) with a Shimadzu spectrophotometer (13). Cells from liquid cultures in the late exponential phase of growth (A 730 ϭ 0.8 -1.2) were harvested by centrifugation at 5000 ϫ g for 15 min.
Growth Rates of the Wild-type and Mutant Cells with NQ Supplements-Cyanobacterial cultures in late exponential phase were pelleted by centrifugation, washed twice with BG-11 medium, and suspended in BG-11 medium containing ϳ10 g/ml spectinomycin. For estimating growth rates, the cultures were grown in six-well plates with 8 ml of medium in each well. All cultures were adjusted to the same initial cell density (A 730 ϭ 0.1). Final concentrations of naphthoquinones, glucose, and 3-(3,4-dichlorophenyl)-1,1-dimethylorea were 5 M, 5 mM, and 10 M, respectively. The cells were shaken on an orbital shaker at 110 rpm. Growth was monitored by measuring absorbance of cultures at 730 nm. Chlorophyll was extracted from whole cells with 100% methanol, and the concentration was determined as described in Ref. 17.
Isolation of Thylakoid Membranes and PS I Complexes-Thylakoid membranes were prepared from cells as described previously (18). The membranes were pelleted by centrifugation at 50,000 ϫ g for 90 min and resuspended in SMN (0.4 M sucrose, 10 mM MOPS at pH 7.0, 10 mM NaCl) buffer. Chlorophyll was extracted from thylakoid membranes and PS I trimers with 80% acetone and determined as described in Ref. 17. For the isolation of PS I complexes, thylakoid membranes were incubated in SMN buffer with 20 mM CaCl 2 for 0.5-1.0 h at room temperature in the dark to enhance the trimerization of PS I. To this mixture, n-dodecyl-␤-D-maltoside was added to a final concentration of 1.5% (w/v) and incubated in the dark on ice with occasional gentle stirring for 0.5-1.5 h. The non-solubilized material was removed by centrifugation at 10,000 ϫ g for 15 min. The trimeric and monomeric PS I complexes and PS II were separated by centrifugation in 10 -30% (w/v) sucrose gradients with 0.04% n-dodecyl-␤-D-maltoside in 10 mM MOPS, pH 7.0.
FIG. 1. Proposed pathway of phylloquinone biosynthesis in Synechocystis sp. PCC 6803. The proposed entry point for 2-CO 2 H-1,4-NQ and 2-CH 3 -1,4-NQ into the phylloquinone biosynthetic pathway is likely to be just prior to the phytyltransferase. Details of the initial steps are not depicted.
Measurement of Photosystem I Activity-Steady-state rates of electron transfer in isolated PS I complexes were measured using cytochrome c 6 as electron donor and flavodoxin as electron acceptor as described in Refs. 19 and 20. Analysis of PhQ Using HPLC and Mass Spectrometry-Quinones were extracted from PS I complexes and analyzed similar to that described previously (13). Chromatograms were run on the basis of 0.026 mg of chlorophyll content of the PS I complexes. Sample injections (35 l) were made on a 4.6 mm ϫ 25-cm Ultrasphere C 18 column (4.6 mm ϫ 250 mm) with 5-m packing (Beckman) using gradient elution (solvent A ϭ methanol; solvent B ϭ isopropanol; 100% A from 0 to 10 min to 3% A, 97% B at 30 min, hold until 40 min) at 1.0 ml/min. A 40 mM solution of PhQ was prepared in absolute ethanol and kept at Ϫ20°C as a standard for calibration. PhQ and derivatives were identified by a combination of spectrophotometry and mass spectrometry. When available, known standard solutions were used to confirm identifications. Demethylphylloquinone elutes at 17.7 min, which is 2.3 min prior to PhQ due to the lack of a hydrophobic methyl group. It was identified by its mass and by its similarity to the PhQ spectrum. 2-OH-Phylloquinone elutes 11.0 min prior to PhQ because of the presence of a hydrophilic hydroxy group. It was only identified by mass; thus, its assignment is tentative. PQ-9 elutes at 20.1 min and was identified by a combination of spectrophotometry and mass spectroscopy. Two pigments, chlorophyll a at 18 min and ␤-carotene at 29 min, served as internal standards and allowed quantitative comparison of the wild-type to the PhQless mutants.
Q-band EPR Spectroscopy of Photoaccumulated PS I Complexes-CW EPR spectrometry at 34 GHz was carried out using the same instrumentation and methods described previously (14).
Time-resolved EPR Spectroscopy at X-and Q-band-Transient EPR spectra and pulsed EPR spectra were performed according to methods described previously (14,21).
Optical Kinetic Spectroscopy in the Near-infrared Region-Optical absorbance changes in the near-infrared region were measured using a laboratory-built spectrophotometer as described in Ref. 15. To assure resolution of kinetics in the microsecond time domain, a high frequency roll-off amplifier described in the original specifications was not employed, and the signal was fed directly into the plug-in (11A33 differential comparator, 100-MHz bandwidth) of the Tektronix DSA601 oscilloscope. The sample cuvette contained the PS I complexes isolated from the menA or menB mutants at 50 g/ml Chl in 25 mM Tris-HCl, pH 8.3, 10 mM sodium ascorbate, 4 M DCPIP, and 0.04% n-dodecyl-␤-D-maltoside.
PhQ Pulse Experiment-Cells were grown under normal light conditions to an optical density (OD) of 0.550 -0.675 at 730 nm in 3-liter flasks with BG-11 medium supplemented with 5 mM ferric ammonium citrate and 5 mM glucose. The cells were pelleted under sterile conditions and resuspended in fresh medium with ferric ammonium citrate lacking glucose and grown for an additional day. At t ϭ Ϫ15 min, three antibiotics were added (132 g/ml chloramphenicol, 30 g/ml kanamycin, 75 g/ml erythromycin) to one-half of the flasks. At time ϭ 0 min, PhQ was added to a final concentration of 10 M. At set time intervals, 400 ml of liquid culture was pelleted and resuspended in SMN buffer, with 5 mM EDTA and 5 M phenylmethylsulfonyl fluoride. Thylakoid membrane extractions were performed on a minipreparative scale. The vials were shaken eight times at 3400 rpm for 30 s on a mini-bead beater with an 8 vial adaptor (Biospec, Bartlesville OK). PS I complexes were isolated (13) and washed twice with 10 mM MOPS buffer, pH 7.0, containing 0.05% n-dodecyl-␤-D-maltoside to remove sucrose and frozen at Ϫ80°C. HPLC analysis of the pigments was performed on an equal chlorophyll basis.

Isolation of a High Light Tolerant Variant of the menB Mutant-
The original menB strain (menB18) has a low content of PS I per cell and is sensitive to high light due to the low PS I to PS II ratio (13). To select for a light-tolerant strain, colonies of the menB18 strain were replica-plated on BG-11 plates with 5 mM glucose and grown at "normal" light intensity (40 E m Ϫ2 s Ϫ1 ) while another set was grown at "high" light intensity (Ͼ140 E m Ϫ2 s Ϫ1 ). The menB26 strain was one of several isolates that could grow at a high light intensity. This strain showed no changes in electron transfer rates for a given amount of PSI and PSII compared with the menB18 strain (data not shown). The PS I complexes of the menB26 and menB18 strains showed identical optical and EPR characteris-tics as well as identical pigment compositions (data not shown). The amino acid sequences of PsaA and PsaB from the A 1 site to the C terminus were identical in menB18, menB26, and the wild type (data not shown). The molecular basis for the suppression of the original menB phenotype is under investigation; however, the PS I content in the menB26 strain is higher than in the menB18 strain. Since the growth of the menB26 revertant was robust under a variety of growth conditions, it was used in the growth supplementation studies described below.
Growth of Cells with Naphthoquinone Supplements-Growth rates of the wild-type and the menA and menB26 mutant strains were monitored in cultures grown in BG-11, medium which contains minerals including bicarbonate (Table  I). Under normal light conditions (40 E m Ϫ2 s Ϫ1 ), photoautotrophic doubling times for the wild type (26 h) were nearly 3 times faster than the mutant strains (menA, 86 h; menB26, 77 h). The menA mutant strain reached stationary growth at ϳ0.4 OD similar to the original menB18 mutant strain, whereas the menB26 mutant strain and the wild type reached stationary phase at ϳ0.8 and ϳ1.0 OD, respectively. Photomixotrophic growth that uses both respiration and photosynthesis for energy metabolism was achieved by adding 5 mM glucose. Under these conditions, the menA mutant strain grew slower than the wild-type and the menB26 mutant strains. Photoheterotrophic growth conditions included addition of glucose for respiration and 10 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea to suppress PS II activity. Under these conditions, the wildtype and mutant strains had similar doubling times and reached stationary growth at a similar culture density (2.75-3.5 OD). Therefore, respiratory energy metabolism and cyclic photophosphorylation function normally in the mutants without PhQ.
When the cyanobacterial cells attained a culture density of ϳ0.15 OD, several substituted naphthoquinones were added to the growth medium to a final concentration of 10 M. The growth rate of wild-type cells slowed in the presence of nearly all supplemented NQ derivatives (Table I). The addition of 1,4-NQ, 2-CO 2 H-1,4-NQ, or 2-CH 3 -1,4-NQ, which are identical or closely related to the biosynthetic precursors of PhQ, to the growth medium led to a highly reproducible increase in the 6803 with supplemented naphthoquinones Light intensity was 40 E, normal light conditions. Error is in the fit of the linear slope and was between 5 and 15%, with an n ϭ 3 or more. Concentrations of naphthoquinones, DCMU, and glucose were 5 M, 10 M, and 5 mM, respectively. Initial cell density was 0.10 OD at 730 nm. The "none" column is the basal growth rate in BG-11 medium. DCMU, a Cells doubled once and then died. Numbers without a statistical error were derived from less than three observations. doubling times by ϳ25%. A similar increase in the doubling time was seen when 2-carboxy-NQ derivatives such as 2-CO 2 H-3,5-NQ and 2-CO 2 H-3,7-NQ or naphthoquinones with alkyl chain tail such as menaquinone-4 (MQ-4) and 2-OH-3-prenyl-1,4-NQ were added to the wild-type cultures. Doubling times in the wild-type cells were not affected by the addition of either 1,2-NQ or 2-OH-1,4-NQ to the growth medium. Thus, NQ derivatives that are analogs or direct products of the naphthoate synthase enzyme appear to slow the growth of the wild-type cells. All three cyanobacterial strains ceased to grow shortly after addition of 5-OH-1,4-NQ, 2-CH 3 -5-OH-1,4-NQ, and 2,3-diCl-1,4-NQ, which are known herbicides (22)(23)(24).
In general, the menA mutant strain was stressed (or remained unaffected) by the addition of NQ derivatives (Table I).
In contrast, naphthoquinones that structurally resemble the product of naphthoate synthase (1,4-NQ, 2-OH-1,4-NQ, 2-CO 2 H-1,4-NQ, and 2-CH 3 -1,4-NQ) accelerated the growth rates of the menB26 mutant cells (Table I). Similarly, MQ-4, a PhQ analog with a C-20 unsaturated alkyl group, improved the growth rate by a factor of 1.7 (Table I). Another compound with a shorter alkyl tail, 2-OH-3-prenyl-1,4-NQ, and naphthoquinones in which the head group does not structurally resemble 1,4-NQ (e.g. 1,2-NQ) have marginal effects on growth rates of the menB26 mutant cells. Other 2-carboxy naphthoquinones (2-CO 2 H-3,5-NQ and 2-CO 2 H-3,7-NQ) had virtually no effect on the growth rate of the menB26 mutant cells. Thus, the placement of the carbonyl oxygens on the NQ ring is important and may be related to the ability of the quinone to become phytylated. Those naphthoquinones that decreased the doubling times of the menB26 mutant cells were used in further studies.

HPLC-MS and HPLC-UV Analysis of Pigment Extracts from PS I Complexes-
The identity of the quinone in isolated PS I complexes was examined using HPLC coupled to a chemical ionization time-of-flight mass spectrometer. By co-injecting standards and by interpreting the spectra at selected masses, chlorophyll a (m/z 892), PhQ (m/z 450), and PQ-9 (m/z 748) were identified with retention times of 19.0, 19.9, and 29.1 min, respectively. MQ-4 elutes earlier (14.2 min) than PhQ in this solvent system. Virtually identical chromatograms were obtained for pigment extracts of the wild-type and the menA and menB26 mutant strains, except that the PhQ peak at 19.9 min was missing in the mutants (Table II). Quantification by MS is somewhat difficult, given that each molecule has different ionization characteristics. It is possible to determine the number of counts at a particular molecular weight, thereby calculating the area under the peak. However, without quantitative controls and standards, the mass spectroscopy data are only semiquantitative. To extract quantitative information, we measured the absorption spectra of the HPLC peaks with a UV-visible range diode array detector. Because the extinction coefficients are well known for most of the quinones of interest, it is possible to quantify the results. The retention times in the HPLC studies were consistent and similar to the LC-MS studies, with slight variations that can be attributed to slightly different lengths of tubing connecting the HPLC to the mass spectrometer or UV spectrophotometer. The absorption spectra of peaks identified as PhQ, MQ-4, and PQ-9 are consistent with spectra of known standards (25). The spectrum of 2-CH 3 -1,4-NQ is similar to PhQ with an absorption maximum at 248 nm and a shoulder at 270 nm.
The amount of PQ-9 in wild-type PS I complexes appears to be small (typically Ͻ1% of the total quinone); nevertheless, the PhQ:PQ-9 ratio of pigment extracts of wild-type PS I complexes varies from preparation to preparation. When assayed by HPLC-UV spectroscopy, several preparations showed no detectable PQ-9 in wild-type PS I complexes, whereas others (Table II) showed a PhQ:PQ-9 ratio as high as 70:1. The latter corresponds to less than 2 PQ-9 molecules/100 PS I complexes. This high degree of variability suggests that any detected PQ-9 is carryover from the membranes in the isolation of the PS I complexes.
Supplementing growth medium of menB26 mutant cells with certain NQ derivatives led to the complete replacement of PQ-9 in the PS I complexes. The addition of authentic PhQ to menB26 mutant cells revealed that the cells could harvest this highly nonpolar quinone from the liquid media and incorporate it into PS I complexes in an amount similar to the wild type (Table II). The menB26 mutant cells are also capable of harvesting MQ-4 (m/z 444), an analog of PhQ with a C-20 partially unsaturated tail; however, the efficiency of MQ-4 incorporation as indicated by the MQ-4:PQ-9 ratio is somewhat lower than that for PhQ incorporation (Table II). The difference in the degree of PQ-9 displacement can be attributed to the C-20 unsaturated tail of MQ-4 as opposed to the largely saturated phytyl tail in PhQ. This represents a potentially important finding because the unsaturated MQ-4 tail resembles the unsaturated PQ-9 tail except for length, and suggests that a variety of chain lengths and degrees of unsaturation can be accommodated in the A 1 site. Since MQ-4 is able to either out compete or displace PQ-9 with an unsaturated C-20 carbon tail, the specificity for the site must be conferred largely by the head group with a significant contribution by the tail.
Supplementing the menB26 mutant cells with the direct product of naphthoate synthase, 2-CO 2 H-1,4-NQ, results in the presence of PhQ in the PS I complexes at a ratio of ϳ16:1 PhQ:PQ-9 (Table II). When the medium is supplemented with 2-CH 3 -1,4-NQ, PhQ is found in the PS I complexes (Table II) at a ratio of ϳ5:1 PhQ:PQ-9. Thus, 2-CH 3 -1,4-NQ is probably not incorporated in the biosynthetic pathway as efficiently as 2-CO 2 H-1,4-NQ. This may be because 2-CH 3 -1,4-NQ lacks the carboxy group, which may be important for the function of the phytyltransferase, or it may be because the solubility of 2-CH 3 -1,4-NQ is lower than that of 2-CO 2 H-1,4-NQ.
When menB26 mutant cells are grown with 1,4-NQ, the LC-MS analysis of pigments extracted from isolated PS I complexes usually showed only the presence of PQ-9 and no phytylated or methylated NQ. However, one sample showed the presence of PhQ (m/z ϭ 450) as well as a species that eluted with a retention time of 17.7 min and with a mass (m/z) of 436, probably representing unmethylated 2-phytyl-1,4-NQ. The ratio of unmethylated to methylated was ϳ2, suggesting that the rate of methylation may be slower than the rate of phytylation. In this sample, the ratio of the total phytylated 1,4-naphthoquinones (m/z ϭ 450 plus m/z ϭ 436, representing methylated and unmethylated species) to PQ-9 was 12:1, which is lower than when the cells were grown with 2-CO 2 -1,4-NQ (Table II). The reason for the batch-to-batch variability in the 1,4-NQ samples is unclear and is under active investigation; nevertheless, this result shows that phytylation of unsubstituted 1,4-NQ can occur under appropriate conditions. Although 2-OH-1,4-NQ supports an increase in the growth rate of the menB26 mutant, no peaks at m/z 450, corresponding to PhQ, or m/z 436, corresponding to unmethylated PhQ, were found. Rather, a new peak at 11 min, corresponding to a m/z of 453, was observed. We tentatively assign this peak to 2-OH-3-phytyl-1,4-NQ containing a 2-OH instead of 2-CH 3 group. The ratio of 2-OH-3-phytyl-1,4-NQ:PQ-9 in PS I complexes is ϳ1:10. Therefore, PQ-9 is not significantly replaced by the 2-OH derivative of PhQ in the mutant PS I complexes.
Since the phytyltransferase has already been shown to function when a hydrogen or a carboxy group is present at ring position 2, it is not surprising that it also functions when a hydroxy group is present. However, the low amount of 2-OH-3-phytyl-1,4-NQ in the PS I complexes may indicate that the specificity of the phytyltransferase for the 2-OH-1,4-NQ substrate is low.
CW EPR Spectroscopy at Q-band of Photoaccumulated A 1 -The presence of phytylated naphthoquinones in solvent extracts of PS I complexes shows that certain quinones are capable of passing through the cell membrane and entering into the remaining PhQ biosynthetic pathway to become phytylated by the product of the menA gene and methylated by the product of the menG gene. However, whether the quinone is functional in the A 1 site is best determined using methods such as CW and transient EPR spectroscopy (see below) of isolated PS I complexes. Fig. 2 shows the Q-band (34-GHz) CW EPR spectra of photoaccumulated PS I complexes isolated from the wild-type and the menB26 mutant strain. At high magnetic fields, the g-anisotropy dominates the spectrum of A 1 Ϫ in wildtype PS I, allowing the g xx ϭ 2.0062 and g zz ϭ 2.0021 components of the tensor to be resolved. The four prominent hyperfine lines obscure the g yy component of the tensor, and result from the high spin density at the carbon position 2 of the phyllosemiquinone anion radical with the methyl group attached. In the menB26 mutant, the g-tensor of the PQ-9 anion radical has principal values of g xx ϭ 2.0067, g yy ϭ 2.0051, g zz ϭ 2.0022 and is therefore more anisotropic than the phyllosemiquinone anion radical in the wild-type (14). The methyl groups at positions 2 and 3 of PQ-9 are unresolved but could be observed by electron-nuclear double resonance (ENDOR) spectroscopy as inequivalent (14); in the CW spectrum the two methyl groups simply add to the inhomogeneous linewidth. (The spectra of the wild-type and the menB26 are both contaminated with a contribution from A 0 Ϫ .) When the growth medium of the menB26 mutant cells is supplemented with authentic PhQ, 2-CO 2 H-1,4-NQ, or MQ-4, the photoaccumulated EPR spectrum of A 1 Ϫ resembles the wild-type spectrum (Fig. 2). The recovery of the smaller ganisotropy and the reappearance the prominent hyperfine couplings are consistent with a near-quantitative replacement of the PQ-9 with PhQ or MQ-4 in the A 1 site. Given that the head group of MQ-4 is identical to PhQ, this is not a surprising result. Similarly, when the growth medium of the menB26 mutant cells is supplemented with 2-CH 3 -1,4-NQ, the g-anisotropy of PhQ is partially restored and the prominent hyperfine couplings are visible, but they are not as well resolved. Given the lower ratio of PhQ/PQ-9 in these PS I complexes (Table II), this spectrum indicates that the majority population of A 1 sites contain PhQ with a minority population that contains PQ-9. When the growth medium of the menB26 mutant cells is supplemented with 1,4-NQ, the results are highly variable. In the sample depicted here, the hyperfine couplings are either missing or obscured, and the quinone in the A 1 site appears to be an admixture of PQ-9 and a species with the g-anisotropy of a naphthoquinone (see below). In other samples, the spectrum is exclusively that of PQ-9, and is therefore nearly identical to the menB26 mutant.
Time-resolved EPR Spectroscopy at X-and Q-bands-The spin polarization pattern of transient EPR spectra of the functional radical pair state P700 ϩ Q Ϫ is known to be highly sensitive to the relative orientation of the cofactors involved. Due to the spin polarization pattern, the g-anisotropy of the particular quinone present in the A 1 site is more readily resolved. Similar to the photoaccumulated CW spectra, the increased hyperfine splitting of the methyl protons of native PhQ in the A 1 site as compared with the semiquinone anion radical in solution is partially resolved. These latter two characteristic features of native PhQ in PS I have been shown to change drastically in biosynthetic pathway mutants such as menA and menB, consistent with the identification of the quinone in the A 1 site as PQ-9 recruited during bacterial growth (14). All three mentioned characteristic features are applied here to monitor the extent to which feeding of menB mutants with 1,4-NQ derivatives results in recovery of the wild type (or a modified) transient EPR spectrum. To protect the sample from slow deterioration under prolonged laser flash excitation, all experiments were performed in frozen solution (80 K). It is known that, under these conditions, only a fraction of the PS I complexes (about 1/3) contributes to the signal because this fraction is able to perform cyclic electron transfer to A 1 required to observe the transient P700 ϩ Q Ϫ state (26). Fig. 3 shows transient spectra at X-and Q-band for those cases where growth supplementation result in full recovery of the wild-type spectrum. In each panel, the quinone-supplemented menB26 mutant spectrum (solid line) is shown together with the wild-type spectrum (dashed line). For comparison, the spectrum with PQ-9 in the A 1 site of the menB26 mutant is also included (Fig. 3, bottom spectrum). Note the distinct differences to the wild-type spectrum in the latter case. In contrast, the wild-type spectrum, including the methyl hyperfine splitting, is fully recovered with the supplemented quinones 2-CO 2 H-1,4-NQ, 2-CH 3 -1,4-NQ, and MQ-4. These transient EPR data are in agreement with the results of the biochemical and CW EPR experiments described previously.
Since 2-CO 2 H-1,4-NQ is the precursor in the biosynthetic pathway for the phytylation and methylation steps and is missing in the menB26 mutant, recovery of the wild-type spectrum corresponds to expectation. On the other hand, 2-CH 3 -1,4-NQ as such could, in principle, replace PQ-9 in the A 1 site and this would lead also to recovery of the wild-type spectrum if the methyl group takes up the same position as for wild-type PhQ. For that, 2-CH 3 -1,4-NQ must be at least phytylated in order to exhibit the wild-type spectrum as demonstrated in Fig. 3. Indeed, a chain substituent is required for proper orientation of the quinone in the A 1 site (11). However, the result raises an interesting question since there are two possibilities for enzymatic modification. 1) Phytylation may occur at the ring position adjacent to the methyl group of 2-CH 3 -1,4-NQ. The methyl group that is already present then may or may not be affected by the final methylation step of the biosynthetic pathway (2). Phytylation may occur at the site of the methyl group, followed by a methylation step at the adjacent ring position. This will also result in a PhQ restored into the A 1 site. In both cases, the wild-type spectrum would be observed as demonstrated in Fig.  3. It is possible to distinguish between these two possibilities with the use of deuterated d-2-CH 3 -1,4-NQ. The characteristic methyl hyperfine splitting will only be recovered if the deuterated methyl group is replaced by a protonated one in a methylation step during biosynthesis, but it will be lost if the methyl group remains deuterated (i.e. the methylation step of the biosynthetic pathway remains inoperative with 2-CH 3 -1,4-NQ supplemented in the menB26 growth medium). In the following, we provide experimental evidence in support of the latter. Fig. 4 presents results for two additional quinones (d-2-CH 3 -1,4-NQ and 1,4-NQ) supplemented to the growth medium. Despite numerous batches tested for each type of sample in a persistent effort to reproduce conditions for PQ-9-free samples, the (best) samples chosen still contain a noticeable percentage of PS I complexes with PQ-9 remaining in the A 1 site. Nevertheless, the mixed spectra in Fig. 4 are readily interpretable when an appropriate spectral contribution due to PS I complexes with PQ-9 in the A 1 site are subtracted (see respective difference spectra in Fig. 4). In contrast to the results reported in Fig. 3, the transient spectra with these two supplemented quinones clearly differ from the wild-type spectrum. In the case of d-2-CH 3 -1,4-NQ, the difference spectrum (Fig. 4) clearly lacks the methyl hyperfine splitting but its orientation is consistent with that of PhQ in the A 1 site, which should not be changed by a deuterated methyl group. Thus, we are left with a clear-cut experimental conclusion. The deuterated methyl group provided by d-2-CH 3 -1,4-NQ survives the phytylation and methylation steps of the biosynthetic pathway.

FIG. 3. Spin-polarized transient EPR spectra of PS I from menB mutants (solid curves) compared with the wildtype spectrum (dashed) at two microwave frequencies (left, X-band at 9
GHz; right, Q-band at 35 GHz). Results are shown for growth medium supplementation with the indicated 1,4-naphthoquinones (from top: 2-CO 2 H-1,4-NQ, 2-CH 3 -1,4-NQ, MQ-4) and compared with the spectra of the menB mutant with PQ-9 in the A 1 site. The spectra were recorded at a temperature of 80 K. The field scales are adjusted to reference to the same free electron (g ϭ 2.0023) resonance. The spectral amplitude represents the integrated signal intensity in a time window 0.5-1.5 s following the laser flash (533 nm). For further experimental details, see Ref. 14 and references therein. tom) is distinct from the wild-type spectrum. In fact, it agrees surprisingly well with the spectra reported earlier (11,12,27) for deuterated d-1,4-NQ substituted into PhQ-less PS I particles. The different spin polarization pattern compared with PhQ in wild-type results from a different orientation of the NQ headgroup in the A 1 site (g yy for 1, 4-NQ instead of g xx for PhQ being approximately parallel to the connecting vector between the centers of the radical ions P700 ϩ and Q Ϫ ). The top spectrum of Fig. 5 (same difference spectrum reproduced from Fig. 4, bottom) agrees well with the bottom spectrum, re-measured at Q-band for deuterated 1,4-NQ reconstituted into PhQ-free PS I particles. (The bottom spectrum exhibits less inhomogeneous broadening in the Q Ϫ spectral region due to reduced hyperfine coupling for deuterated NQ.) Again, it is emphasized that the latter sample was obtained with a different strategy for 1,4-NQ substitution. First, PhQ-free PS I complexes were prepared by organic solvent extraction (8 -10), and then d-1,4-NQ was incorporated by incubation of the PhQ-free PS I complexes. For this protocol, the justified criticism has been raised that organic solvent extraction extracts other cofactors as well and may be accompanied by changes of the PS I polypeptides (see Introduction). In this respect, the menB26 mutants studied here have a decisive advantage, since modifications concern exclusively the PhQ biosynthetic pathway and hence can be assumed to leave the PS I assembly as such intact. In both cases the same kind of transient P700 ϩ Q Ϫ spectra are observed. This serves as convincing experimental evidence that growth conditions for 1,4-NQ feeding of the menB26 mutant can be established that result in incorporation of (unsubstituted) 1,4-NQ into the A 1 site. Although this conclusion is quite straightforward from the available results, it must be termed qualitative, since PQ-9 could not be fully replaced in the batches of PS I complexes investigated thus far. In any case, 1,4-NQ (and not PhQ) incorporation has clearly been realized, and this despite the still intact phytyl-and methyltransferases of the biosynthetic pathway. This surprising result will be addressed more closely under "Discussion." In contrast to the electron spin echo envelope modulation (ESEEM) of regular radical spin ensembles, the corresponding echo modulation of correlated radical pair spins has the special property to be selective in its frequency composition to just to the pair spin-spin coupling, the dipolar part of which yields the distance between the radicals. Moreover, the latter is phase shifted by 90°compared with ESEEM of a single radical spins and was thus termed "out-of-phase" ESEEM. In the case of the transient P700 ϩ Q Ϫ state, EPR signal measurement of the out-of-phase ESEEM been demonstrated to yield accurate determination of the distance between the centers of the donor P700 ϩ and of the acceptor Q Ϫ in the charge separated state (see Ref. 28 and references therein). The out-of-phase ESEEM presented in Fig. 6 demonstrate that, within experimental accuracy, the distances are identical in all samples studied. For the wild type, a distance of 25.3 Ϯ 0.3 Å has been evaluated, which does not change for any of the substituted quinones investigated in this study.
P700 ϩ Optical Recombination Kinetics-As we showed in our previous paper (15), the replacement of PhQ by PQ-9 results in altered kinetics of the backreaction from [F A /F B ] Ϫ to P700 ϩ . In PS I complexes isolated from the wild type, the reduction of P700 ϩ is multiphasic after a saturating flash (29). When measured in the absence of an external electron acceptor, the reduction of P700 ϩ by the reduced iron-sulfur clusters is biphasic, with typical lifetimes of 10 -30 ms and 80 -100 ms. There is a long-lived kinetic phase of P700 ϩ reduction by re- duced DCPIP that contributes ϳ5-20% to the total absorbance change. In PS I complexes isolated from the menB26 mutant, the reduction of P700 ϩ is also multiphasic after a saturating flash (Fig. 7). When measured in the absence of external electron acceptor, the reduction of P700 ϩ is biphasic, with lifetimes of 3 and 10 ms. The menA/B mutants also exhibit a minor long-lived kinetic phase attributed to electron donation of DCPIP to the oxidized P700 ϩ that contribute the remainder of the absorbance change. Thus, the kinetics of the backreaction in isolated PS I complexes can be used to determine whether PQ-9 or PhQ is present in the A 1 site.
When the growth medium of the menB26 mutant cells is supplemented with authentic PhQ (data not shown) or 2-CO 2 H-1,4-NQ (Fig. 7), the backreaction kinetics from [F A / F B ] Ϫ to P700 ϩ are nearly identical to the wild-type. This agrees with the 15.9:1PhQ:PQ-9 ratio measured in the 2-CO 2 H-1,4-NQ-grown sample (Table II). When the growth medium of the menB26 mutant cells is supplemented with MQ-4, the 3-and 10-ms kinetic phases are replaced by a 60-ms kinetic phase (the 10-ms kinetic phase was not resolved), consistent with electron transfer to the terminal iron-sulfur clusters (data not shown). When the growth medium of the menB26 mutant cells is supplemented with 2-CH 3 -1,4-NQ, there is a significant decrease in the contribution of the ϳ3-5-ms kinetic phase (the ϳ10-ms kinetic phase is not resolved), indicating that PhQ occupies at least a fraction of the A 1 sites. The substantial recovery of the long-lived 10 -30-and 80 -100-ms kinetic phases indicates that a large portion of the A 1 sites are occupied with PhQ. This qualitatively agrees with the transient EPR results described above, indicating incomplete replacement of PQ-9 in the 2-CH 3 -1,4-NQ-grown sample.
Quinones that did not lead to enhanced rates of growth show only the presence of PQ-9 in the A 1 site. Thus, mutants that were grown in the presence of 2-CO 2 H-3,5-NQ, 2-OH-1,4-NQ, and 2-OH-3-prenyl-1,4-NQ show the fast 3-and 10-ms kinetic phases (data not shown). However, the smaller contribution of these fast kinetics phases in the case of 2-OH-3-prenyl-1,4-NQ, a quinone with a single unit prenyl tail, suggests that there may be partial replacement of PQ-9. Thus, the kinetic data, coupled with the pigment extract analysis, are largely consistent with the CW and transient EPR results in identifying the nature of the replacement quinone and in confirming that only certain quinone metabolites function in forward electron transfer to the iron-sulfur clusters.
In Vivo PhQ Exchange-To assess the stability of PhQ in the A 1 site over time, an in vivo pulse experiment was conducted using authentic PhQ. This in vivo study was performed to determine if protein synthesis is required for reincorporation of PhQ in the A 1 site, i.e. whether only newly synthesized PS I complexes take up exogenously supplied PhQ. The necessary precondition for the experiment, that PhQ supplied in the growth medium is able to enter the cell and displace PQ-9 in growing cells, has already been demonstrated (see above). The menB26 strain was grown to mid-log phase, at which time PhQ was added to all of the cultures at a final concentration of 10 M and antibiotics were added to one-half of the cultures. We then assayed the incorporation of PhQ in PS I complexes of normal growing cells and of cells in which protein synthesis had been inhibited. By the first measurement time point of 15 min, PhQ completely displaces PQ-9 in PS I complexes in both sets of cultures (Fig. 8). The initial incorporation of PhQ is very rapid, suggesting a significant difference in the binding affinity of the A 1 site for PhQ and PQ-9. Over the course of 3 days, the level of PhQ drops as it is re-replaced by PQ-9. This may occur because of PhQ catabolism and mass action that could result from over 10 times excess of PQ-9 over PhQ in the membranes. The cellular chlorophyll content does not change significantly, suggesting that the PS I complexes do not undergo substantial degradation during the course of the experiment. The implication is that PhQ turns over much more rapidly than the PS I complex in living cells. Both conditions, with and without protein synthesis inhibitors, show a similar trend in PhQ incorporation. Given that the antibiotic-containing cultures cannot synthesize new PS I complexes, PhQ must be entering the A 1 site by diffusion and expelling the resident PQ-9. DISCUSSION These studies show that a certain set of substituted and unsubstituted naphthoquinones supplied during growth of menB26 mutants results in recovery of native PhQ in the A 1 site. The complete restoration of PhQ in the A 1 site after growth medium supplementation with 2-CO 2 H-1,4-NQ indicates that the phytyltransferase (the product of the menA gene) and the methylase (the product of the menG gene) remain functional (see Fig. 1). In contrast, only PQ-9 is found in the A 1 site of the menA mutants after growth medium supplementation with PhQ biosynthetic precursors such as 2-CO 2 H-1,4-NQ or 2-CH 3 -1,4-NQ. Because the menA gene codes for phytyltransferase, the phytyl group must be important either for the de novo placement of PhQ in the A 1 site or in the displacement of PQ-9 in pre-formed PS I complexes.
The restoration of PhQ in the A 1 site also occurs. after growth medium supplementation with 2-CH 3 -1,4-NQ. Experiments with deuterated 2-CH 3 -1,4-NQ supplementation prove that the deuterated methyl group is unaffected by the phytyland methyltransferases. Hence, the phytyl tail is attached at the ring position next to the methyl group by displacing a H rather than a CO 2 group. The reader should note that 2-CO 2 H-1,4-NQ as well as 2-CH 3 -1,4-NQ break the C 2v symmetry of 1,4-NQ ring with a single substituent in the same position, which we take as reference 2-position. With respect to this common reference, we conclude that the phytyltransferase affects different ring positions depending on which substituent it encounters at the 2-position. With the charged carboxylate group substituted in the ring 2-position, the phytyl tail replaces this group; with the nonpolar methyl group in the 2-position, the phytyl tail will be attached in the ring position adjacent to the methyl group. As a result, the same PhQ is synthesized in both cases. Thus, relevant properties of the recognition mechanism of the phytyltransferase appear to become addressable in these experiments and suggest further efforts in the development of appropriate growth supplementation strategies.
Our results also imply that an asymmetrically substituted 1,4-NQ is required for efficient phytyltransferase activity. This follows from fact that, for the preparations studied thus far, growth media supplementation with symmetric 1,4-NQ resulted in large batch-to-batch variability. In only one instance, PhQ and non-methylated PhQ (2-phytyl-1,4-naphthoquinone) has been detected by LC-MS analysis, but it has not yet been found in any of the batches studied by the various EPR methods. In all instances, there is clear evidence for partial incorporation of 1,4-NQ into the A 1 site without either phytylation or methylation, even though both transferases are functional. Incorporation of 1,4-NQ is partial with respect to PQ-9 content and the batch-to-batch variability concerns mostly the 1,4-NQ: PQ-9 ratio in the A 1 site without any detectable contribution of either phytylated and methylated, or only phytylated, or only methylated 1,4-NQ in the A 1 site. These results do not exclude that reliable and reproducible biosynthesis of PhQ from 1,4-NQ supplementation is feasible using other suitable strategies, which are currently under investigation. Independent of the outcome, we have demonstrated here that preparation protocols can be arranged which result in incorporation of unsubstituted 1,4-NQ into the A 1 site. Transient EPR spectra of the P700 ϩ Q Ϫ state are particularly suited to draw this conclusion, because they are most sensitive to the orientation of the quinone and 1,4-NQ has been shown to enter the A 1 site with an orientation rotated by about 90°as compared with that of PhQ in wild type (11,12,27).
For most of the samples that have been grown in the presence of supplemented quinones, the observed Chl/Q total ratio (Table II) is close to the norm, suggesting the A 1 site is fully occupied. Hence, there appear to be no empty A 1 sites in PS I complexes from the wild-type or the menB mutants. A deviation arises only in the 1,4-NQ sample, where it appears that the quinone content has dropped, resulting in an increase in the Chl:Q total ratio. However, the HPLC conditions employed here did not allow the resolution of unmodified 1,4-NQ from the solvent front. Since 1,4-NQ was detected by spectroscopic techniques, even in this instance the A 1 site is likely fully occupied by a quinone.
Obviously, thus far only a very small selection of quinones and even 1,4-NQ derivatives have been used for the results presented in this paper. As a general rule, naphthoquinones that decreased the doubling times of the menB26 cells were found to be present in PS I complexes, usually as phytylated derivatives, and naphthoquinones that did not significantly change the growth rates of the menB26 cells were not found in PS I complexes as phytylated derivatives. In contrast, an extremely wide range of quinones and even non-quinone alternatives has been shown to be able to substitute for the native quinone in the first quinone acceptor site Q A of bacterial reaction centers (1,2,30). Likewise, a wide variety of quinones and even non-quinone head groups (i.e. without the phytyl tail) could be incorporated into the A 1 site of solvent-extracted PS I (31). All of these are potential candidates to be used in the type of quinone supplementation studies of phylloquinone biosynthetic pathway mutants introduced with this paper.