Photo-CIDNP 13C magic angle spinning NMR on bacterial reaction centres: exploring the electronic structure of the special pair and its surroundings.

Photochemically induced dynamic nuclear polarisation (photo-CIDNP) in intact bacterial reaction centres has been observed by 13C-solid state NMR under continuous illumination with white light. Strong intensity enhancement of 13C NMR signals of the aromatic rings allows probing the electronic ground state of the two BChl cofactors of the special pair at the molecular scale with atomic selectivity. Differences between the two BChl cofactors are discussed. Several aliphatic 13C atoms of cofactors, as well as 13C atoms of the imidazole ring of histidine residue(s), show nuclear-spin polarisation to the same extent as the aromatic nuclei of the cofactors. Mechanisms and applications of polarisation transfer are discussed.

Pigment organization and their interactions in reaction centers of photosystem II: optical spectroscopy at 6 K of reaction centers with modified pheophytin composition.

Photosystem II reaction centers (RC) with selectively exchanged pheophytin (Pheo) molecules as described in [Germano, M., Shkuropatov, A. Ya., Permentier, H., Khatypov, R. A., Shuvalov, V. A., Hoff, A. J., and van Gorkom, H. J. (2000) Photosynth. Res. 64, 189-198] were studied by low-temperature absorption, linear and circular dichroism, and triplet-minus-singlet absorption-difference spectroscopy. The ratio of extinction coefficients epsilon(Pheo)/epsilon(Chl) for Q(Y) absorption in the RC is approximately 0.40 at 6 K and approximately 0.45 at room temperature. The presence of 2 beta-carotenes, one parallel and one perpendicular to the membrane plane, is confirmed. Absorption at 670 nm is due to the perpendicular Q(Y) transitions of the two peripheral chlorophylls (Chl) and not to either Pheo. The "core" pigments, two Pheo and four Chl absorb in the 676-685 nm range. Delocalized excited states as predicted by the "multimer model" are seen in the active branch. The inactive Pheo and the nearby Chl, however, mainly contribute localized transitions at 676 and 680 nm, respectively, although large CD changes indicate that exciton interactions are present on both branches. Replacement of the active Pheo prevents triplet formation, causes an LD increase at 676 and 681 nm, a blue-shift of 680 nm absorbance, and a bleach of the 685 nm exciton band. The triplet state is mainly localized on the Chl corresponding to B(A) in purple bacteria. Both Pheo Q(Y) transitions are oriented out of the membrane plane. Their Q(X) transitions are parallel to that plane, so that the Pheos in PSII are structurally similar to their homologues in purple bacteria.

Ultrahigh field MAS NMR dipolar correlation spectroscopy of the histidine residues in light-harvesting complex II from photosynthetic bacteria reveals partial internal charge transfer in the B850/His complex.

Low-temperature 15N and 13C CP/MAS (cross-polarization/magic angle spinning) NMR has been used to analyze BChl-histidine interactions and the electronic structure of histidine residues in the light-harvesting complex II (LH2) of Rhodopseudomonas acidophila. The histidines were selectively labeled at both or one of the two nitrogen sites of the imidazole ring. The resonances of histidine nitrogens that are interacting with B850 BChl a have been assigned. Specific 15N labeling confirmed that it is the tau-nitrogen of histidines which is ligated to Mg2+ of B850 BChl molecules (beta-His30, alpha-His31). The pi-nitrogens of these Mg2+-bound histidines were found to be protonated and may be involved in hydrogen bond interactions. Comparison of the 2-D MAS NMR homonuclear (13C-13C) dipolar correlation spectrum of [13C6,15N3]-histidines in the LH2 complex with model systems in the solid state reveals two different classes of electronic structures from the histidines in the LH2. In terms of the 13C isotropic shifts, one corresponds to the neutral form of histidine and the other resembles a positively charged histidine species. 15N-13C double-CP/MAS NMR data provide evidence that the electronic structure of the histidines in the neutral BChl a/His complexes resembles the positive charge character form. While the Mg...15N isotropic shift confirms a partial positive charge transfer, its anisotropy is essentially of the lone pair type. This provides evidence that the hybridization structure corresponding to the neutral form of the imidazole is capable of "buffering" a significant amount of positive charge.

Heteronuclear 2D-correlations in a uniformly [13C, 15N] labeled membrane-protein complex at ultra-high magnetic fields.

One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C, 15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the alpha-helical segments of the protein, mostly found in the membrane interior.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680(.+) primary donor chlorophyll.

We report (13)C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo-CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance (13)C provide information on the electronic structure of the primary electron donor P(680) (chlorophyll a molecules absorbing around 680 nm) and on the p(z) spin density pattern in its oxidized form, P(680)(.+). Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P(680)(.+). PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P(680) by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Exploring the calcium-binding site in photosystem II membranes by solid-state (113)Cd NMR.

Calcium (Ca(2+)) is an essential cofactor for photosynthetic oxygen evolution. Although the involvement of Ca(2+) at the oxidizing side of photosystem II of plants has been known for a long time, its ligand interactions and mode of action have remained unclear. In the study presented here, (113)Cd magic-angle spinning solid-state NMR spectroscopy is used to probe the Ca(2+)-binding site in the water-oxidizing complex of (113)Cd(2+)-substituted PS2. A single NMR signal 142 ppm downfield from Cd(ClO(4))(2).2H(2)O was recorded from Cd(2+) present at the Ca(2+)-binding site. The anisotropy of the signal is small, as indicated by the absence of spinning side bands. The signal intensity is at its maximum at a temperature of -60 degrees C. The line width of the proton signal in a WISE (wide-line separation) two-dimensional (1)H-(113)Cd NMR experiment demonstrates that the signal arises from Cd(2+) in a solid and magnetically undisturbed environment. The chemical shift, the small anisotropy, and the narrow line of the (113)Cd NMR signal provide convincing evidence for a 6-fold coordination, which is achieved partially by oxygen and partially by nitrogen or chlorine atoms in otherwise a symmetric octahedral environment. The absence of a (113)Cd signal below -70 degrees C suggests that the Ca(2+)-binding site is close enough to the tetramanganese cluster to be affected by its electron spin state. To our knowledge, this is the first report for the application of solid-state NMR in the study of the membrane-bound PS2 protein complex.

Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 13(1)-deoxo-13(1)-hydroxy-pheophytin a and comparison of their 6 K absorption spectra.

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 13(1)-deoxo-13(1)-hydroxy-pheophytin a (13(1)-OH-Pheo). The absorption bands of 13(1)-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC(1x)) or twice (RC(2x)). HPLC analysis and Pheo Q(X) absorption at 543 nm show that in RC(1x) about 50% of Pheo is replaced and in RC(2x) about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo(-) is unmodified in RC(1x) and decreased only in RC(2x), suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the Q(Y) region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC(1x) or RC(2x), showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength.

Triplet properties and interactions of the primary electron donor and antenna chromophores in membranes of Heliobacterium chlorum, studied with ADMR spectroscopy.

The triplet states of antenna and reaction center bacteriochlorophyll (BChl) g in membranes of Heliobacterium chlorum were studied by optically detected magnetic resonance in zero magnetic field, using absorbance detection. A variety of triplet states was detected, which were all localized on single BChl g chromophores as concluded from a comparison with the triplet state of monomeric BChl g in organic solvents. With the aid of the microwave-induced absorbance difference spectra, we assign a triplet state with zero-field splitting parameters |D| = 727.5 and |E| = 254. 5 MHz to that of the primary donor. The low |E| value indicates that the BChls of the primary donor are monoligated. The intensities of the zero-field transitions were strongly dependent on the redox state of the secondary electron acceptors. A triplet state with |D| = 690-705 MHz and |E| =230 MHz, present under all redox conditions, is associated with antenna BChl g absorbing at 814 nm. Its triplet yield was independent of the redox conditions; we conclude therefore that the antenna chromophores absorbing at 814 nm are not connected with the reaction center at cryogenic temperatures (1.2 K). In addition, relatively strong signals were detected belonging to triplet states with |D| and |E| of 663-680 and 220-227 MHz, respectively, whose amplitudes were dependent on the redox conditions. Triplet states with these zero-field splitting parameters are located on antenna chromophores absorbing between 798-814 nm; their zero-field transitions and absorbance difference spectra indicate a considerable heterogeneity. The concentration of triplet states of antenna chromophores absorbing around 800 nm decreased markedly upon prolonged excitation at 1.2 K. This phenomenon is attributed to quenching of excitations on antenna pigments by stable charge separation in the closely connected reaction center, possibly involving a low-quantum yield menaquinone electron acceptor.

Effect of bicarbonate on the S2 multiline EPR signal of the oxygen-evolving complex in photosystem II membrane fragments.

Removal of bicarbonate from spinach photosystem II BBY particles by means of washing in a CO2-free medium results in the loss of their capability to accumulate the S2 multiline EPR signal upon continuous illumination at 190 K. Addition of 1 mM NaHCO3 before illumination leads to a 50-60% restoration of the multiline signal. Similarly, in BBY particles depleted of Mn by treatment with 1 M Tris-HCl (pH 8.0) and 0.5 M MgCl2, re-addition of MnCl2 in the presence of 1 mM NaHCO3 results in a partial restoration (approximately 30%) of the S2 multiline EPR signal of the Mn cluster, while in the absence of NaHCO3 no restoration is observed. The results provide further evidence that bicarbonate is essential for maintaining the Mn-containing oxygen-evolving complex of PS II in a functionally active form.

Light-induced structural changes in photosynthetic reaction centres studied by ESEEM of spin-correlated D+QA- radical pairs.

Zn-substituted Rhodobacter sphaeroides R26 reaction centres (RCs) frozen in the dark and under illumination exhibit quite different recombination kinetics of the D+QA- radical pairs [Kleinfeld et al., Biochemistry, 23 (1984) 5780]. We have applied electron spin echo envelope modulation (ESEEM) of the spin-correlated D+QA- radical pairs to assess a possible light-induced change in the distance between the D and QA cofactors. The recombination kinetics and the field-swept spin-polarized EPR signal for the two preparations have been monitored by time-resolved EPR spectroscopy. For the samples frozen under illumination, a slight increase in the distance, 0.4+/-0.2 A, has been detected.

Characterization of pheophytin ground states in Rhodobacter sphaeroides R26 photosynthetic reaction centers from multispin pheophytin enrichment and 2-D 13C MAS NMR dipolar correlation spectroscopy.

The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from 13C chemical shift data. A new experimental approach involving multispin 13C labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly 13C biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state 13C magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state 13C-13C correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many 13C resonances of the [U-13C]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of 13C shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.

Formation of a long-lived P+BA- state in plant pheophytin-exchanged reaction centers of Rhodobacter sphaeroides R26 at low temperature.

Femtosecond transient absorption spectroscopy in the range of 500-1040 nm was used to study electron transfer at 5 K in reaction centers of Rhodobacter sphaeroides R26 in which the bacteriopheophytins (BPhe) were replaced by plant pheophytin a (Phe). Primary charge separation took place with a time constant of 1.6 ps, similar to that found in native RCs. Spectral changes around 1020 nm indicated the formation of reduced bacteriochlorophyll (BChl) with the same time constant, and its subsequent decay in 620 ps. This observation identifies the accessory BChl as the primary electron acceptor. No evidence was found for electron transfer to Phe, indicating that electron transfer from BA- occurs directly to the quinone (QA) through superexchange. The results are explained by a model in which the free energy level of P+Phe- lies above that of P+BA-, which itself is below P*. Assuming that the pigment exchange does not affect the energy levels of P* and P+BA-, our results strongly support a two-step model for primary electron transfer in the native bacterial RC, with no, or very little, admixture of superexchange.

Bicarbonate may Be required for ligation of manganese in the oxygen-evolving complex of photosystem II.

It was previously shown in the photosystem II membrane preparation DT-20 that photoxidation of the oxygen-evolving manganese cluster was blocked by 0.1 mM formate, unless 0.2 mM bicarbonate was present as well [Wincencjusz, H., Allakhverdiev, S. I., Klimov, V. V., and Van Gorkom, H. J. (1996) Biochim. Biophys. Acta 1273, 1-3]. Here it is shown by measurements of EPR signal II that oxidation of the secondary electron donor, YZ, is not inhibited. However, the reduction of is greatly slowed and occurs largely by back reaction with reduced acceptors. Bicarbonate is shown to prevent the loss of fast electron donation to . The release of about one or two free Mn2+ per photosystem II during formate treatment, and the fact that these effects are mimicked by Mn-depletion, suggests that formate may act by replacing a bicarbonate which is essential for Mn binding. Irreversible light-induced rebinding in an EPR-silent form of Mn2+ that was added to Mn-depleted DT-20 was indeed found to depend on the presence of bicarbonate, as did the reconstitution in such material of both the fast electron donation to and the UV absorbance changes characteristic of a functional oxygen-evolving complex. It is concluded that bicarbonate may be an essential ligand of the functional Mn cluster.

The association of different detergents with the photosynthetic reaction center protein of Rhodobacter sphaeroides R26 and the effects on its photochemistry.

Detergent-free reaction centers from Rhodobacter sphaeroides R26 were used to study the solubilization of reaction centers in various detergents and their effects on reaction center photochemistry. 500 +/- 100 n-octyl-beta-D-glucopyranoside or 51 +/- 5 Triton X-100 molecules were associated with one reaction center. For N.N-alkylamine N-oxide detergents with chain lengths in the range from 8-13 carbon atoms, the number of detergent molecules associated with the reaction centers increased with decreasing chain length. The amount of detergent molecules associated with the reaction centers decreased almost tenfold if the pH was increased from pH 6 to pH 10. The addition of 5% 1,2,3-heptanetriol to various detergents lowered the detergent/reaction center ratio by a factor of two compared to that for the pure detergent. The detergent concentration at which solubilization of the reaction center occurs was close to the critical micelle concentration for all detergents studied. The absorption band at 865 nm of the primary donor in the reaction center shifts to 846 nm when detergent was removed from the reaction center; upon resolubilization with various detergents, this band shifts back to 865 nm. In 80-90% of the detergent-free reaction centers, the secondary electron transfer from QA to QB was inhibited: this electron transfer was restored after re-addition of detergent.

Influence of KF, DCMU and removal of Ca+ on the high-spin EPR signal of the cytochrome b-559 heme Fe(III) ligated by OH- in chloroplasts.

An EPR signal at g = 6.8 attributed to the cytochrome (Cyt) b-559 heme Fe(III) ligated by OH- (Fiege, R., Schrieber, U., Lubitz, W., Renger, G. and Shuvalov, V.A. (1995) FEBS Lett. 377, 325-329) was studied. This signal is observed in intact chloroplasts when oxidized by 10 mM 2,3-dicyano,5,6-dichloro-p-benzoquinone (DDQ), but not when 5 mM p-benzoquinone is added. Addition of KF (100 mM) or removal of Ca 21 for blocking the water-oxidizing complex considerably decreases the heme Fe(III)-OH- EPR signal. In contrast, DCMU does not decrease this signal and does not influence its photochemical changes at 140 K. Thus, the EPR spectrum of Cyt b-559 Fe(III) ligated by OH- is not affected by changes at the acceptor side of Photosystem 11, and its photochemical decrease is probably not due to reduction via the acceptor side. Comparison of the effect of KF on the model heme Fe(III) in myoglobin (Mb) at pH 10.5 shows that F- replaces OH- as a ligand at the sixth coordination position of the heme Fe(III) in both Mb and chloroplasts Cyt b-559. This replacement is accompanied by changes of the symmetry of the molecular field causing a disappearance of the EPR signals at g = 6.8 and 5.0. Our results provide further evidence for a possible participation of the Cyt b-559 heme Fe ligated by OH- in photosynthetic water oxidation.

FTIR spectroscopy shows weak symmetric hydrogen bonding of the QB carbonyl groups in Rhodobacter sphaeroides R26 reaction centres.

The absorption frequencies of the C = O and C = C (neutral state) and of the C...O (semiquinone state) stretching vibrations of QB have been assigned by FTIR spectroscopy, using native and site-specifically 1-, 2-, 3- and 4-13C-labelled ubiquinone-10 (UQ10) reconstituted at the QB binding site of Rhodobacter sphaeroides R26 reaction centres. Besides the main C = O band at 1641 cm-1, two smaller bands are observed at 1664 and 1651 cm-1. The smaller bands at 1664 and 1651 cm-1 agree in frequencies with the 1- and 4-C = O vibrations of unbound UQ10, showing that a minor fraction is loosely and symmetrically bound to the protein. The larger band at 1641 cm-1 indicates symmetric H-bonding of the 1- and 4-C = O groups for the larger fraction of UQ10 but much weaker interaction as for the 4-C = O group of QA. The FTIR experiments show that different C = O protein interactions contribute to the factors determining the different functions of UQ10 at the QA and the QB binding sites.

13C magic angle spinning NMR characterization of the functionally asymmetric QA binding in Rhodobacter sphaeroides R26 photosynthetic reaction centers using site-specific 13C-labeled ubiquinone-10.

Photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides R26 were reconstituted at the QA site with ubiquinone-10, selectively 13C-enriched on positions 1, 2, 3, 4, and 3-Me (IUPAC numbering). RCs dispersed in LDAO detergent were studied with 13C CP/MAS NMR spectroscopy at temperatures between 180 and 240 K, while RCs precipitated by removal of the detergent were investigated at ambient temperature and at temperatures down to 180 K. Electrostatic charge differences in QA induced by polarization from the protein are less than 0.02 electronic equivalent for any of the labeled positions. This includes the 4-carbonyl, which is therefore not significantly polarized by an electrostatic binding interaction with the protein. The QA site is slightly heterogeneous on the scale of the NMR as the observed line widths of the labels are between 150 and 300 Hz and inhomogeneous broadening is observed for the signals of positions 1, 2, and 3 upon cooling. This contrasts with earlier MAS observations for labels in the vicinity of the special pair. The chemical shifts are 184, 144, and 137 ppm for the labels at positions 1, 2, 3, and 12 ppm for the 3-methyl 13C. For the 4-carbonyl only at sample temperatures below approximately 255 K a CP/MAS response can be observed at 183 ppm. The principal components of the chemical shift tensors for the ring labels in QA were estimated using difference spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Asymmetric binding of the 1- and 4-C=O groups of QA in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10.

Using 1-, 2-, 3- and 4-13C site-specifically labelled ubiquinone-10, reconstituted at the QA site of Rhodobacter sphaeroides R26 reaction centres, the infra-red bands dominated by the 1- and 4-C = O vibration of QA are assigned in the QA(-)-QA difference spectra. The mode dominated by the 4-C = O vibration is drastically downshifted in the reaction centres as compared with its absorption frequency in free ubiquinone-10. In contrast, the mode dominated by the 1-C = O vibration absorbs at similar frequencies in the free and the bound forms. The frequency shift of the 4-C = O vibration is due to a large decrease in bond order and indicates a strong interaction with the protein microenvironment in the ground state. In the charge-separated state the mode dominated by the semiquinone 4-C = O vibration is characteristic of strong hydrogen bonding to the microenvironment, whereas the mode dominated by the 1-C = O vibration indicates a weaker interaction. The asymmetric binding of the 1- and 4-C = O groups to the protein might contribute to the factors governing different redox reactions of ubiquinone-10 at the QA site as compared with its reactions at the QB site.

QA binding in reaction centers of the photosynthetic purple bacterium Rhodobacter sphaeroides R26 investigated with electron spin polarization spectroscopy.

The relation between quinone (QA) binding and electron transport in reaction centers (RCs) of photosynthetic purple bacteria is investigated, using electron spin polarization (ESP) X-band (9 GHz) EPR as a tool to probe for structural changes resulting from charge separation and stabilization and from replacing the native QA molecule with other quinones. We present a study of possible changes in QA-binding that might be responsible for the remarkably prolonged lifetime of the charge-separated state at cryogenic temperatures for RCs of Rhodobacter sphaeroides R26 cooled under illumination [Kleinfeld, D., et al. (1984) Biochemistry 23, 5780-5786]. It is shown that this effect is not caused by a major reorientation of the chromophores. Furthermore, we studied the effects of structurally different quinones functioning as primary electron acceptor in different purple bacteria. With simulations of ESP X-band spectra of the spin-polarized secondary radical pair P.+QA.+- in menaquinone-reconstituted, Zn(2+)-substituted RCs of Rb. sphaeroides R26, we show that quinone reconstitution is highly selective for site and orientation. Furthermore, we find that a very small exchange interaction between P.+ and QA.+- (magnitude of JPQ approximately 1 microT) is needed to account accurately for the observed relative line intensities at X-band, without affecting the accuracy of the simulations of reported ESP K-band spectra [Füchsle, G., et al. (1993) Biochim. Biophys. Acta 1142, 23-35; Van der Est, A., et al. (1993) Chem. Phys. Lett. 212, 561-568]. This pronounced influence of small values for JPQ on the X-band ESP line shape results from cancellation effects of absorptive and emissive contributions to the spectrum, such that small shifts can be observed. The exchange interaction has opposite sign for the native, ubiquinone-containing RC [viz. JP.UQ = (-0.8 +/- 0.2) microT] and the menaquinone-substituted RC [JP.MK = (+0.3 +/- 0.2) microT]. The implications of these observations for electron-transport theory are discussed.