Applied Magnetic Resonance

, 37:65

Incorporation of 2,3-Disubstituted-1,4-Naphthoquinones into the A1 Binding Site of Photosystem I Studied by EPR and ENDOR Spectroscopy

Authors

    • Department of ChemistryBrock University
  • Yulia Pushkar
    • Department of PhysicsPurdue University
  • Irina Karyagina
    • Max Planck Institute for Biophysical Chemistry
  • Branden Fonovic
    • Department of ChemistryBrock University
  • Travis Dudding
    • Department of ChemistryBrock University
  • Jens Niklas
    • Max-Planck-Institut für Bioanorganische Chemie
  • Wolfgang Lubitz
    • Max-Planck-Institut für Bioanorganische Chemie
  • John H. Golbeck
    • Department of Biochemistry and Molecular Biology and Department of ChemistryThe Pennsylvania State University
Article

DOI: 10.1007/s00723-009-0047-x

Cite this article as:
van der Est, A., Pushkar, Y., Karyagina, I. et al. Appl Magn Reson (2010) 37: 65. doi:10.1007/s00723-009-0047-x

Abstract

Transient electron paramagnetic resonance and pulsed electron-nuclear double resonance (ENDOR) spectra of the state \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) in photosystem I containing a series of non-native naphthoquinones (NQs) are presented. Previous studies have shown that quinones bind to the A1 site with only one of their carbonyl groups H-bonded to the protein and that the asymmetric H-bond produces an odd alternant distribution of the spin density within the quinone. It is known that the native phylloquinone binds with its methyl group meta and its phytyl tail ortho to the H-bonded carbonyl. Monosubstituted NQs with short alkyl chains have been found to bind preferentially with their alkyl side groups meta to the H-bonded carbonyl. The selectivity of the binding site toward methyl and short chain substituents is studied by incorporating disubstituted NQs that have a methyl group at the 2-position and a short chain at the 3-position of the quinone ring. The hyperfine couplings (hfcs) of the methyl group protons are sensitive to the spin density distribution on the quinone and are used to deduce the position of the methyl group relative to the H-bonded carbonyl. The measured methyl proton hfcs indicate that the disubstituted quinones bind exclusively with their methyl group in the meta position relative to the H-bonded carbonyl and no evidence for binding with the methyl group in the ortho position is found. The disubstituted quinones have also been chosen to study the effect of electron withdrawing substituents on the spin density distribution. When the short chain contains electronegative atoms such as sulfur or chlorine, the methyl proton hfcs of the quinone in the A1 binding site are found to be significantly larger than those of 2-methyl-1,4-naphthoquinone and phylloquinone in the same environment. Solution ENDOR measurements of the quinone radical anions in isopropanol and density functional theory (DFT) calculations in vacuo show that this increase in the hfcs is mostly intrinsic to the quinones due to the electron-withdrawing ability of the short chain and is not a result of differences in the binding to the protein. The DFT calculations suggest that the main reason for the increased methyl proton hfcs is delocalization of the singly occupied molecular orbital onto the side chain, which leads to an increase of the spin density on the neighboring carbon, which carries methyl group.

1 Introduction

In oxygenic photosynthesis, light is used to oxidize water, reduce nicotinamide adenine dinucleotide phosphate NADP+ and drive the synthesis of adenosine triphosphate (ATP). The heart of this process is a light-driven proton-coupled electron transfer reaction in the thylakoid membrane in which electrons are extracted from water on the lumenal side of the membrane and transferred to NADP+ in the stroma. Because the overall electrochemical potential of this redox reaction cannot be generated easily from a single photon in the visible region of the electromagnetic spectrum, the reduction of NADP and the oxidation of water are driven by two separate light-induced electron transfer reactions that occur in Photosystem I (PS I) and Photosystem II (PS II), respectively (see Refs. [1] and [2] for detailed reviews of the structure and function PS I and PS II, respectively). In both photosystems, light is used to transfer electrons from a chlorophyll donor along a chain of acceptors across the membrane to a quinone acceptor. In PS II, multiple turnovers of this reaction generate the highly positive oxidation potential and the four equivalents needed to oxidize water in the Mn-containing water-splitting complex. At the same time, the plastoquinone pool in the thylakoid membrane is reduced. In PS I, each turnover oxidizes plastocyanin or cytochrome c6 and reduces ferredoxin or flavodoxin, generating the low redox potential needed to reduce NADP+. The arrangement and nature of the cofactors involved in the initial electron transfer in PS I and PS II are very similar but they have vastly different redox potentials in accordance with the functional differences between the two photosystems.

Of particular interest are the quinone acceptors and the role of the protein environment in determining their properties (see Refs. [3] and [4] for recent reviews of the quinones in PS I and PS II). In PS II, the two plastoquinones have different redox potentials and very different binding properties; QA is tightly bound and has a reduction midpoint potential of approximately −80 mV [5], while QB, which is a mobile electron and proton carrier, has a slightly more positive midpoint potential. In stark contrast to the behavior of the plastoquinones in PS II, both phylloquinones in PS I do not exchange as part of their function and they have reduction potentials in the neighborhood of −700 mV or lower [6]. This huge difference in redox potential between the quinones in PS I and PS II is primarily a result of protein–cofactor interactions, although the structural differences between plastoquinone (a benzoquinone) and phylloquinone (a naphthoquinone) do account for some of the difference [7, 8]. The role of the protein–cofactor interactions in determining the quinone potential in PS I was demonstrated by experiments in which the biosynthetic pathway to phylloquinone was interrupted by inactivating either the menA or menB genes in the cyanobacterium Synechocystis sp. PCC 6803 [9]. In the absence of phylloquinone, it was found that plastoquinone-9 is incorporated into PS I [9] and is able to function with only slightly lower efficiency than phylloquinone [10]. An analysis of the electron transfer kinetics [11] suggests that the reduction midpoint potential of plastoquinone-9 in the PS I binding site is shifted at least 500 mV more negative compared to the values in the QA and QB binding sites in PS II.

These results reveal some intriguing properties of the quinones and the quinone binding pocket in PS I. First, the large shift in the quinone potential when it is bound to PS I is necessary because the electrons would otherwise be trapped by the quinones, which are generally good electron acceptors. Hence, it is of interest to understand which features of the binding site lead to the unusually negative potential for the quinone. One can also speculate as to why PS I contains quinones at all since dispensing with them would seem to be an easier and more efficient way of achieving the same goal. Second, the fact that in the absence of phylloquinone, plastoquinone-9 is seen to bind to PS I, shows that the binding site tailored to selectively bind phylloquinone, yet has a certain affinity for other quinones, e.g., plastoquinone-9. Thus, it is also of interest to understand which features of the quinones and binding site lead to this selectivity.

The selectivity of the binding site also leads to a very elegant way of introducing foreign quinones into the PS I binding site. It has been shown in a number of studies [1218] that foreign quinones and structurally related compounds can be introduced into the quinone binding site in PS I by extracting the native quinone using organic solvents and incubating the extracted PS I with the foreign quinone. However, this technique has the rather serious drawback that the extraction procedure partially denatures the protein and removes the β-carotene and many of the chlorophyll molecules (see Ref. [6] for a discussion). Thus, conclusions about the nature of the quinone binding drawn from studies on such samples may not accurately reflect the behavior of native PS I. However, it has been found that if PS I from the menA and menB mutants is incubated with an excess of various naphthoquinones, the plastoquinone-9 in the A1 binding site can be replaced [19, 20]. Since no organic solvents or strong detergents are required in the preparation of such samples, no alteration of the binding site compared to native PS I is expected and they are ideal for studying the interactions of the quinone in PS I with its surroundings. Indeed, a recent Q-band pulse ENDOR study showed that the 1H ENDOR spectra of PS I from the menB mutant substituted with phylloquinone in vitro and wild-type PS I are identical [21].

Transient electron paramagnetic resonance (TREPR) spectroscopy has been used extensively to study the binding and function of the phylloquinone acceptors in PS I (see Refs. [22, 23] for two recent reviews). In such experiments, the radical pair \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \), where P700 is the electron donor and A1 is the phylloquinone acceptor in the PsaA-branch (Fig. 1), is easily detected in native PS I. The corresponding radical pair involving the PsaB-branch quinone is not normally observed by TREPR [22] but its spectrum obtained under strongly reducing conditions has been reported [24]. When naphthoquinone derivatives are incorporated into PS I the corresponding spectra of \( P_{700}^{ \cdot + } NQ_{{}}^{ \cdot - } \) are observed. Because the TREPR spectra are sensitive to the g-tensor, hyperfine couplings and orientation of the quinone, they provide a convenient assay for the incorporation of foreign quinones into PS I [10, 12, 25, 26] and can be used to study the influence of the protein environment on the spin density distribution [2729]. One interesting feature of the TREPR spectra of \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) in native PS I is the presence of several shoulders that result from hyperfine coupling (hfc) to the protons of the methyl group at the 2-position of the phylloquinone headgroup. The assignment of these spectral features to the methyl hfc has been demonstrated most elegantly by the fact that they are absent in a mutant, lacking the methyltransferase that adds the methyl group during the biosynthesis of phylloquinone [26].
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Fig. 1

Structure of the phylloquinone binding pocket on the PsaA-side of PS I. Hydrogen bonds are shown as dotted lines. The structural model is based on the 2.5 Å X-ray structure (Protein Data Bank entry 1JB0) [53]

Pulsed electron-nuclear double resonance (ENDOR) studies of \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) [10, 21, 3033] and pulsed and continuous-wave (CW) ENDOR of photoaccumulated \( A_{1}^{ \cdot - } \) [21, 32, 34, 35] show that the methyl proton hfcs of phylloquinone in PS I are unusually large, which implies that a large spin density resides on the ring carbon adjacent to the methyl group (ring position 2). As can be seen in Fig. 1, which shows the X-ray structure of the neutral phylloquinone and its binding pocket in PS I, the phylloquinone QK-A is hydrogen-bonded to Leu A722 via carbonyl oxygen O4, while carbonyl oxygen O1 is not H-bonded. The large spin density at the carbon adjacent to the methyl proton is a result of this asymmetry of the H-bonding of the quinone, which distorts the spin density distribution [20, 27, 36].

The dependence of the methyl proton hfcs on the position of the methyl group relative to the H-bond can be used to determine how foreign quinones bind to the quinone binding site [26, 37]. Studies of this type have yielded some interesting features of the A1 binding site in PS I. First, out-of-phase echo experiments, which provide very precise values for the electron–electron spin–spin coupling, show that the distance between \( P_{700}^{ \cdot + } \) and \( A_{1}^{ \cdot - } \) is identical for PS I with the native quinone and foreign quinones in the binding site [10, 38]. Second, the polarization patterns indicate that for substituted naphthoquinones (NQs) the orientation of the carbonyl bonds relative to the dipolar coupling vector is the same as in native PS I [37]. Together, these two observations imply that the H-bonding of the NQs to the protein is similar to that of the native phylloquinone. However, they do not give enough information to specify which of the two carbonyl oxygens, O1 or O4, is H-bonded to Leu A722 (see Fig. 1 for the numbering of the atoms in the quinone headgroup). This ambiguity can be resolved using the hfcs of substituents at the 2 and 3 positions on the ring [20, 26, 37] because the asymmetric H-bonding leads to a high spin density on the ring carbon meta to the H-bonded carbonyl and low spin density on the carbon ortho to the H-bonded carbonyl. This difference in spin density is reflected in the strength of the proton hfcs of substituents at the two ring positions. Hence, a methyl or methylene group bound ortho to the H-bonded carbonyl should show significantly smaller proton hfcs than one bound meta to the H-bonded carbonyl. Using this line of reasoning it was argued [26, 37] that 2-methyl-1,4-naphthoquinone (MeNQ) binds with its methyl group meta to the H-bonded carbonyl, while 2-phytyl-1,4-naphthoquinone (PhNQ) binds with its phytyl tail ortho to the H-bonded carbonyl, i.e., in the same position as the corresponding substituent in native phylloquinone, which has both a methyl group and a phytyl tail. Interestingly, it was found that 2-ethyl- and 2-n-butyl-1,4-naphthoquinone (EthNQ and ButNQ, respectively) bind such that their single alkyl chain is meta to the H-bonded carbonyl. This means the alkyl chain assumes the position that would be otherwise occupied by the methyl group of phylloquinone and not that of the phytyl chain as one might assume [37]. Thus, it appears that having a substituent meta to the H-bonded carbonyl promotes stronger binding of quinones to PS I. However, since PhNQ is found to bind with its side chain ortho to the H-bonded carbonyl [26], it is apparently more favorable for the substituent to reside in the ortho position for long side chains. At present it is not known how long the chain must be before binding with the substituent ortho to the H-bonded carbonyl is preferred.

Here, we investigate the specificity of the binding site toward the positioning of methyl and short chain substituents, by incorporating 2,3-disubstituted NQs into PS I. We have selected two naphthoquinones, both of which have a methyl group and a short chain. The chemical structures of the quinones and their abbreviated names are shown in Fig. 2 (note that for consistency with phylloquinone and monosubstituted NQs, in the text we refer to the position of the methyl group as 2 and that of the short chain as 3 even though in some instances this numbering is reversed in the International Union of Pure and Applied Chemistry name). Since monosubstituted MeNQ, EthNQ, and ButNQ bind with their alkyl substituent meta to the H-bonded carbonyl, it is not clear, a priori, whether the disubstituted quinones will bind preferentially with their substituents in a specific position relative to the H-bonded carbonyl. Hence, these quinones provide a test of ability of the binding site to distinguish between a methyl group and a short chain on the quinone headgroup. We use highly sensitive ENDOR spectroscopy to measure the methyl proton hfcs from which the position of the two substituents relative to the H-bonded carbonyl can be deduced. The results show that the binding site selectively incorporates the disubstituted NQs exclusively with the methyl group meta to the H-bonded carbonyl as found for native phylloquinone. There is no evidence for any admixture of molecular orientations with the methyl group ortho to the H-bonded carbonyl. We will discuss possible reasons for this high degree of selectivity, although at present it remains unexplained. Additionally, we investigate the influence of electronegative atoms in the side chains on the overall spin density distribution on the quinone headgroup. The results indicate that if the short chain is electron withdrawing, the singly occupied molecular orbital (SOMO) delocalizes onto the side chain, thereby creating an alternating spin density around the ring that increases the spin density at the adjacent carbons of the naphthoquinone headgroup.
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Fig. 2

The molecular structures of several mono- and disubstituted naphthoquinones. From top to bottom: 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone); 2-methyl-1,4-naphthoquinone (MeNQ, vitamin K3); 2-ethyl-1,4-naphthoquinone (EthNQ); 2-chloromethyl-3-methyl-1,4-naphthoquinone (CMMeNQ); 2-ethylthio-3-methyl-1,4-naphthoquinone (ETMeNQ). The bottom four quinones were used for incorporation into the A1 site in PS I from the menB mutant strain of Synechocystis sp. PCC 6803

2 Materials and Methods

2.1 Preparation of PS I with Artificial Doubly Substituted Quinones

The quinones were purchased from Sigma-Aldrich Canada Ltd. and used without further purification. A 100-fold molar excess of quinone (10 μl of a 0.034 M solution of quinone in ethanol) was added to PS I trimers (150 μl in Tris buffer, pH 8.3, containing 0.2% Triton X-100) isolated from menB mutant cells of Synechocystis sp. PCC 6803. Incubation was carried out at room temperature (2–4 h) with intensive stirring. The PS I particles were washed two times with 150 μl buffer solution to remove excess quinone; the final volume of sample was 150 μl. Prior to the TREPR measurements, 10 μl of 1 M ascorbate solution was added and the sample was dark-adapted and frozen in the dark.

2.2 REPR Spectroscopy

X-band TREPR experiments were carried out using a Bruker ER046 XK-T microwave bridge equipped with a Flexline dielectric resonator and an Oxford liquid helium gas-flow cryostat. The loaded Q-value for this dielectric ring resonator was about Q = 3000, equivalent to a rise time of τr = Q/(2πvmw) ≈ 50 ns. Q-band (34 GHz) TREPR spectra of the samples were also measured with the same setup except that a Bruker ER 056 QMV microwave bridge equipped with a home-built cylindrical resonator was used. The samples were illuminated using a Spectra Physics Nd-YAG/MOPO laser system operating at 10 Hz at either the second harmonic (532 nm) or near the long wavelength absorption edge of PS I at approximately 700 nm. Spectra obtained with these two excitation wavelengths were found to be identical. All measurements were carried out at T = 80 K. Spin-polarized EPR spectra were extracted from the complete time/field datasets in a 500 ns wide time window centered at 250 ns after the laser flash.

2.3 Pulsed ENDOR Studies of \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \)

Pulsed ENDOR experiments on the radical pair \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) were performed on a Bruker ESP 380E X-band Fourier-transform (FT) EPR spectrometer with an ESP360D-P ENDOR accessory, an ER4118X-MD-5W1-EN ENDOR resonator, and an ENI A500 radio frequency amplifier. The Davies-ENDOR pulse sequence [(π (microwave) − π (radio frequency) − π/2 (microwave) − π (microwave) − echo] was used, with pulse lengths of 128 ns for the two microwave π pulses and 64 ns for a microwave π/2 pulse and 8 μs for the radio frequency π pulse. The delay time between the laser flash and the first microwave pulse was 800 ns. The ENDOR experiments were carried out at 80 K. The field position was chosen to provide as close to equal and opposite intensity for the two halves of the spectrum as possible. See Ref. [30] 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.

2.3.1 CW EPR and ENDOR Measurements

Semiquinone radical anions for liquid solution EPR and ENDOR measurements were prepared in perdeuterated isopropanol as described in Ref. [39]. The initial quinone concentrations were 0.5 mM. CW EPR and ENDOR spectra of the samples were taken at −10°C on a Bruker ESP 300E X-band EPR spectrometer with ENDOR accessories similar to the ones described in Ref. [39].

2.3.2 DFT Calculations

Geometry optimizations of MeNQ, CMMeNQ, and ETMeNQ (Fig. 2) radical anions in vacuo were carried out using the Gaussian 03 [40] suite of programs. For calculation of the methyl hfcs, free rotation of the methyl group was modeled as discussed in Ref. [41] by varying the dihedral angle (φ) of the methyl group relative to the aromatic system in 15° increments to generate a total of seven conformers for each quinone. For CMMENQ the chloromethyl group was also rotated generating a total of 16 conformers for this molecule. The hfcs were computed in additional single-point calculations of these conformers. The isotropic methyl proton hfcs were then calculated as the average of the hfcs of the conformers. For all cases the UB3LYP [42, 43] functional was used in combination with the 6-311+G(2d,2p) basis set [4448]. It is noted that while the EPR-II basis set is the preferred, and most widely used, basis set for calculating EPR parameters, it is unparameterized for S and Cl and hence could not be used here.

3 Results and Discussion

3.1 Q-band TREPR Spectra

The structures of the quinones incorporated into PS I are shown in Fig. 2. Before examining their methyl proton hfcs using ENDOR spectroscopy and discussing their binding, we characterize the incorporation of the quinones into the A1 site using their TREPR spectra. Figure 3 shows spin-polarized Q-band (34 GHz) TREPR spectra of native PS I (top) and PS I from the menB mutant incubated with the four non-native quinones shown in Fig. 2. As can be seen, all of the samples give a strong \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) spectrum but there are clear differences between them. This suggests that the non-native quinones have all been successfully incorporated in the A1 binding site. Spin polarization patterns such as those shown in Fig. 3 are known to be particularly sensitive to the orientation of the quinone g-tensor relative to the dipolar coupling vector that joins \( P_{700}^{ \cdot + } \) and \( A_{1}^{ \cdot - } \) (see Ref. [49] for a review). For native PS I it is known that the x-axis of the phylloquinone g-tensor, which is parallel to the carbonyl bonds, is aligned along the dipolar coupling vector. Thus, the spectra in Fig. 3 can be used to determine whether the carbonyl bonds of the non-native quinones are also parallel to the dipolar coupling vector or whether there are deviations from this arrangement. The experimental spectra in Fig. 3 (solid lines) are compared to simulated spectra (dashed lines) calculated using the known parameters [50, 51] for native PS I. For the non-native methyl NQs, the methyl proton hfcs (Table 1) obtained from the pulsed ENDOR spectra, which will be discussed below, have been used. For EthNQ, the methylene couplings have been taken from Ref. [37]. The inhomogeneous line widths of the quinones have been adjusted to account for the presence or absence of other substituents that have unresolved hfcs. Because the principal g-values of the non-native quinones were not determined independently, the values from native PS I were used for all of the calculated spectra. Any difference between the g-values of phylloquinone and the non-native quinones is expected to be sufficiently small that it will have only a minor influence on the spectra. More importantly, the geometric parameters for all of the simulated spectra are the same as for native PS I. Since the experimental spectra are reproduced well by the calculations, we conclude that in all cases the carbonyl bonds of the respective quinone are aligned along the dipolar coupling vector and that all non-native quinones take the same position as the native phylloquinone in the A1 binding site.
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Fig. 3

Q-band spin-polarized TREPR spectra 80 K of the \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) radical pair state in PS I particles at 80 K. Solid lines experimental spectra with native phylloquinone (top), MeNQ, EthNQ, CMMeNQ and ETMeNQ in the A1 binding site of PS I from the menB mutant strain of Synechocystis sp. PCC 6803. Dashed lines calculated spectra using the parameters reported in Refs. [50, 51]. For the non-native methyl NQs, the principal values of the corresponding methyl hfc tensors in Table 1 have been used. For EthNQ, the hfc values have been taken from Ref. [37]. The inhomogeneous line widths for the quinones have been adjusted to 0.18 mT (phylloquinone), 0.16 mT (MeNQ), 0.16 mT (EthNQ), 0.20 mT (CMMeNQ) and 0.16 mT (ETMeNQ) to take the broadening caused by the respective groups adjacent to the methyl group into account

Table 1

Methyl hyperfine coupling constants of several naphthoquinone derivatives in different environments

Quinonea

Methyl hyperfine coupling (MHz)

 

a (PS I)b

a|| (PS I)b

aiso (PS I)c

aiso (solution)d

aiso (in vacuo)e

Phylloquinone

9.0

12.1

10.0

7.39f

6.56

MeNQ

9.0

12.0

10.0

7.88

5.89

CMMeNQ

10.9

14.5

12.1

8.90

8.75

ETMeNQ

11.6

15.1

12.8

8.59

8.34

aThe structures of the quinones are shown in Fig. 2

bFrom the pulsed ENDOR spectra shown in Fig. 6

cCalculated from \( a_{\text{iso}} = {\tfrac{1}{3}}(a_{||} + 2a_{ \bot } ) \)

dIsopropanol solution at −10°C

eDFT calculation

fFrom Ref. [39]

3.2 X-band TREPR Spectra

Figure 4 shows corresponding spin-polarized X-band TREPR spectra of the same samples. At X-band, the lower resolution of the g-tensor components means that the hfcs have a much stronger influence. As can be seen, the spectra of the non-native quinones all show more structure than that of native PS I (Fig. 4, top). Figure 5 shows corresponding simulated spectra to illustrate the effect of changes in the hfc. The top spectrum in Fig. 5 is calculated using the parameters for native PS I tabulated in Refs. [50, 51]. In the middle spectrum the effect of strong methylene proton hfcs is illustrated by replacing the methyl proton hfc with coupling to the –CH2 protons using the values estimated for EthNQ given in Ref. [37]. The bottom spectrum in Fig. 5 illustrates the effect of a 25% increase in the principal values of the methyl hfc tensor compared to native PS I.
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Fig. 4

X-band spin-polarized TREPR spectra of the radical pair \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) in PS I particles at 80 K. Comparison of spectra of PS I, containing phylloquinone (top, native PS I), MeNQ, EthNQ, CMMeNQ and ETMeNQ in the A1 binding site of PS I from the menB mutant strain of Synechocystis sp. PCC 6803

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Fig. 5

Calculated X-band spin-polarized TREPR spectra, demonstrating the effect of changes in the proton hfc. Top the spectrum of \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) in native PS I calculated using the parameters reported in Refs. [50, 51]. Middle spectrum of EthNQ calculated using the same parameters as for native PS I except that the methyl hfc has been replaced with the methylene hfc values reported for EthNQ in Ref. [37]. Bottom the effect of an increase of the methyl proton hfc. The spectrum is calculated with the same parameters as the top spectrum except that the principal values of the methyl hfc tensor have been increased by 25%

In the spectrum of the MeNQ-containing sample in Fig. 4, the more pronounced structure is due to a smaller inhomogeneous line width as a result of the absence of the phytyl tail. In native PS I, the spin density on carbon 3 (see Fig. 1) of the phylloquinone headgroup is low and hence the hfc to the methylene protons is not resolved and only contributes to the inhomogeneous line width in the TREPR spectrum. In MeNQ, the methylene protons are replaced by a ring proton, which is expected to have a smaller hfc because it lies in a node of the π*-orbital, carrying the unpaired electron. Hence, the inhomogenous line width is expected to be smaller for MeNQ and the structure caused by the hfc of the methyl protons is more pronounced in the TREPR spectrum. Comparison of the top two spectra in Fig. 4 suggests that the methyl hfc is approximately the same for the native PS I sample and the MeNQ sample, which implies that the spin density adjacent to the methyl group is similar for both quinones. This, in turn, implies that the two quinones are bound in the same manner with the carbonyl group meta to the methyl group H-bonded to the protein.

The spectrum of the EthNQ sample also shows very pronounced structure but with a different pattern than for the MeNQ sample. The spectrum can be reproduced by introducing a strong methylene coupling as illustrated by the middle spectrum in Fig. 5 and it is essentially identical to the spectrum reported previously for EthNQ incorporated into solvent-extracted PS I [37]. As demonstrated in Ref. [37], the hyperfine structure in the spectrum is lost if the methylene protons of the ethyl group are replaced with deuterons and the observed pattern is consistent with the quinone, being bound such that the ethyl group occupies the same position as the methyl group in MeNQ and phylloquinone. This is a somewhat surprising result since one might expect that it would be energetically more favorable for EthNQ to bind with the ethyl group in the position normally occupied by the phytyl tail of phylloquinone (see above).

In the case of the disubstituted quinones (bottom two spectra in Fig. 4), the same pattern is observed as for MeNQ but the hyperfine structure is even more pronounced, particularly in the case of ETMeNQ. As illustrated by the bottom spectrum in Fig. 5, the observed pattern is consistent with a larger methyl proton hfc than found in native PS I. The large methyl hfc implies that the –CH3 group is meta to the H-bonded carbonyl in both cases.

3.3 Pulsed ENDOR Spectra

Although the hyperfine structure is clearly visible in the X-band TREPR spectra, it is difficult to estimate the values of the coupling constants from them. Moreover, an important question that cannot be answered easily from the TREPR spectra is whether there are two different methyl hfc tensors arising from the quinones, being bound in two different orientations, or whether there is only a single methyl hyperfine tensor with the quinones bound in only one orientation, i.e., with the methyl group meta to the H-bonded carbonyl. Pulsed ENDOR spectra of the radical pair \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) have been used to provide accurate values of the principal components of the methyl hfc tensor [21, 30, 32], to investigate the effect of quinone binding site point mutations on the spin density distribution [52] and to illustrate the recruitment of a foreign quinone into PS I [10]. Thus, they provide an important complement to the TREPR spectra.

Pulsed ENDOR spectra of \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) in native PS I and PS I containing MeNQ, CMMeNQ, and ETMeNQ are shown in Fig. 6. All of the spectra show a pattern of lines arranged symmetrically around the proton Larmor frequency. In the top trace in Fig. 6, there are two axial powder patterns, one in absorption and one in emission, on the low- and high-frequency regions of the ENDOR spectrum of native PS I, respectively. These patterns are known to arise from the methyl hfc tensor of the A1A phylloquinone [21, 30, 32]. The vertical dashed lines indicate the positions of the parallel and perpendicular components of the axially symmetric methyl proton hyperfine tensor responsible for the powder patterns; these allow easier comparison with the other spectra. The hyperfine tensor components, a|| and a, are indicated above the spectra. The values of the coupling constants obtained from the spectra are given in Table 1.
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Fig. 6

Pulsed ENDOR of the \( P_{700}^{ \cdot + } A_{1}^{ \cdot - } \) radical pair state in PS I particles at 80 K. Comparison of spectra with native phylloquinone (top), MeNQ, EthNQ, CMMeNQ and ETMeNQ in the A1 binding site of PS I from the menB mutant strain of Synechocystis sp. PCC 6803. See “Materials and Methods” for details of the pulse sequences and other experimental conditions. The a|| and a components of the methyl group protons tensor of the native phylloquinone are indicated by dashed lines

For MeNQ, the coupling is the same as for phylloquinone within experimental error, in agreement with a previous ENDOR study [21]. In contrast, the methyl coupling is noticeably larger for CMMeNQ and ETMeNQ (bottom two traces in Fig. 6). For all of the spectra, the smaller couplings near the center of the spectrum arise from the NQ ring protons and protons of the surrounding protein. Most importantly, there is no clear evidence for two distinct methyl coupling tensors as would be expected if the NQs were bound in two different orientations. In this regard, the corresponding pulsed ENDOR spectra of PS I with plastoquinone-9 in the A1 site reported in Ref. [10] provide a useful comparison. Because plastoquinone-9 has two methyl groups at the 5 and 6 positions of its headgroup, one of them is necessarily meta to the H-bonded carbonyl and the other is ortho to it, regardless of which carbonyl is H-bonded. The pulsed ENDOR spectra of PS I with plastoquinone-9 in the binding site reveal two methyl hfc tensors with a|| = 9.8 ± 0.2 MHz, a = 6.8 ± 0.2 MHz and a|| = 5.8 ± 0.2 MHz, a = 2.8 ± 0.2 MHz [10]. Based on these values, the bottom two spectra in Fig. 6 would show a second methyl powder pattern with hfcs about 40% smaller than the main pattern, if a significant fraction of the disubstituted quinone molecules were bound with the methyl group ortho to the H-bonded carbonyl. Moreover, in the case of CMMeNQ, strong methylene proton hfcs would also be expected, if the chloromethyl group was bound meta to the H-bonded oxygen. There is no clear evidence for either of these powder patterns suggesting that disubstituted quinones bind exclusively with their methyl group meta to the H-bonded carbonyl.

3.4 Solution ENDOR and DFT Calculations

The fact that the methyl proton hfcs of the disubstituted MeNQs, observed in the pulsed ENDOR and TREPR spectra, are larger than for phylloquinone and MeNQ could have two possible origins. First, it is possible that the presence of the second substituent simply leads to higher spin density on the carbon adjacent to the methyl group, irrespective of the environment of the quinone. Alternatively, the second substituent on the quinones might alter their interaction with the A1 binding site in such a way that the hyperfine coupling is increased. For example, the H-bonding to Leu A722 might be stronger for the disubstituted quinones, which would also lead to a higher spin density on the carbon adjacent to the methyl group. To distinguish these two possibilities we have measured solution ENDOR spectra of the quinones in isopropanol and also performed DFT calculations of the quinones in vacuo. The comparison of values of the methyl hfcs in these different environments allows the intrinsic effect of the substituents to be qualitatively distinguished from the effect of the environment. The isotropic methyl couplings in the different environments are shown in Table 1. The presence of ethylthio and chloromethyl substituents ortho to the methyl group consistently leads to larger values in all three environments. Thus, we conclude that the increased methyl hfc in CMMeNQ and ETMeNQ are primarily due to the presence of substituents. The values in Table 1 show that for all of the quinones the isotropic methyl hfc of the quinone in the A1 site is approximately 30–40% larger than in isopropanol. Thus, the influence of the H-bonding to Leu A722 on the spin density distribution appears to be approximately the same for both the mono- and disubstituted quinones.

To better understand why placing a chloromethyl or ethylthio substituent ortho to the methyl group increases the methyl group hfcs, the spin density was mapped onto the Singly Occupied Molecular Orbital (SOMO) obtained from the DFT calculations for MeNQ, CMMeNQ, and ETMeNQ (Fig. 7). The shading of the SOMO indicates the spin density, with darker shading corresponding to higher spin density. The fractional Mulliken spin populations for selected atoms or groups of atoms are also given in Table 2. From Fig. 7, it is apparent that the presence of a substituent containing a heavy, electronegative element (S or Cl) causes significant delocalization of the SOMO onto the substituent and distortion at the ring carbon adjacent to it. This is most apparent in the case of CMMeNQ (Fig. 7, middle). As a result of the delocalization, the spin density on side chain increases and changes in the spin density around the ring are induced. These changes are given in parentheses (Table 2) as the difference in the fractional Mulliken spin populations between CMMeNQ and MeNQ and between ETMeNQ and MeNQ. These differences reveal that the sign of the change alternates around the ring such that it is negative on C1, C3, and O4 and positive on O1, C2, and C4. Thus, the electron-withdrawing effect of the chloromethyl and ethylthio groups causes an increase of spin density on C2, which is next to the methyl group, leads to the larger methyl proton hfcs. This effect is analogous to the influence of an asymmetric H-bond, which also withdraws electron density from the quinone and leads to alternating increases and decreases in spin density around the ring.
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Fig. 7

The spin density mapped onto the electron density isosurface of the SOMO of MeNQ, CMMeNQ and ETMeNQ. The electron density surfaces are plotted at an isovalue of 0.028. The spin density is indicated by the shading of the surface and is plotted from −1.2 × 10−3 to 1.2 × 10−3 e/Å3 with darker shading indicating a larger magnitude for the spin density. In order to main consistency across the structures, the same conformation of the methyl group was chosen for the plots in each case

Table 2

Fractional Mulliken spin populations calculated in vacuo

Atoma

MeNQ

ETMeNQb

CMMeNQc

O1

0.25

0.25 (0.00)

0.26 (0.01)

C1

0.11

0.09 (−0.02)

0.10 (−0.02)

C2

0.09

0.13 (0.04)

0.15 (0.06)

C3

0.11

0.10 (−0.01)

0.10 (−0.01)

C4

0.06

0.09 (0.03)

0.06 (0.00)

O4

0.24

0.22 (−0.02)

0.21 (−0.03)

Methyl C

−0.01

−0.01 (−0.01)

−0.01 (0.00)

Methyl H’s

0.01

0.01 (0.00)

0.01 (0.00)

Side chaind

−0.01

0.01 (0.02)

0.02 (0.03)

The fraction of the total spin population on selected atoms or groups of atoms is shown

aThe numbering of the atoms is as shown in Fig. 1

bThe values in parentheses are the differences in the spin density between ETMeNQ and MeNQ

cThe values in parentheses are the differences in the spin density between CMMeNQ and MeNQ

dThe side chain varies and is attached to C3

4 Conclusions

We have performed a TREPR and pulse ENDOR study of PS I from the menB mutant of Synechocystis sp. PCC 6803 with 2-monosubstituted and 2,3-disubstituted 1,4-naphthoquinones incorporated into the A1 binding site. Because the PS I samples used here are derived from the menB mutant, which does not contain phylloquinone, the foreign quinones can be introduced without using the harsh solvent extraction needed to remove the phylloquinone from the wild-type PS I. Thus, the fact that the TREPR spectra of EthNQ and MeNQ reproduce those reported previously for incorporation into solvent-extracted PS I [37] confirms that the solvent extraction does not significantly change the A1 binding site.

Together, the TREPR and ENDOR data indicate that the disubstituted methyl NQs bind to the A1 site in the same orientation as the native phylloquinone and MeNQ such that the carbonyl bonds are parallel to the dipolar coupling vector and the methyl group is meta to the H-bonded carbonyl. In the case of EthNQ in the A1 binding site, strong methylene hfcs are observed, suggesting that the ethyl group is likewise meta to the H-bonded carbonyl group. Thus, for all of the quinones, there is a strong tendency to bind with a substituent in this position. However, it is also known from previous experiments that PhNQ binds with its phytyl tail ortho to the H-bonded carbonyl. From this, it appears that above a certain chain length longer than four carbons, it becomes favorable for the quinone to bind with its single substituent ortho to the H-bonded carbonyl rather than meta.

The results presented here show that if there are two substituents present at the 2 and 3 positions of the naphthoquinone headgroup and one of them is a methyl group, the NQs bind preferentially with the methyl substituent meta to the H-bonded oxygen like native phylloquinone. However, the data do not reveal a clear reason for this selectivity. In general, steric effects are expected to play a significant role and it is reasonable to assume that any substituent larger than a methyl group would experience greater steric hindrance, if placed in the part of the binding pocket where the methyl group would normally reside. Thus, it is not surprising that the disubstituted naphthoquinones bind in the same orientation as phylloquinone. However, the fact that 2-alkyl NQs have been found to bind with their substituent meta to the H-bonded carbonyl group suggests that steric hindrance is not the only factor that determines the orientation of the quinone in the binding site.

Acknowledgments

This article is dedicated to the memory of Dietmar Stehlik, mentor, scientist and friend. This work was supported by grants from the Natural Science and Engineering Research Council Canada to A.v.d.E. and T.D. and from the US National Science Foundation to J.H.G. (MCB-0519743), as well as by the Deutsche Forschungsgemeinschaft (Sfb 663, TP A7).

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© Springer 2009