J. Phys. Chem. 1996, 100, 10433-10442
Hydrogen-Bonding Effect on Ubisemiquinones in Vitro
13C
10433
and Proton Hyperfine Couplings of [4-13C]-Labeled
Tatyana N. Kropacheva,† Willem B. S. van Liemt,‡ Jan Raap,‡ Johan Lugtenburg,‡ and Arnold J. Hoff*,§ Department of Chemistry, Udmurt State UniVersity, IzheVsk, 426037 Russia, Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502, 2300 RA Leiden, The Netherlands, and Department of Biophysics, Leiden UniVersity, P.O. Box 9504, 2300 RA Leiden, The Netherlands ReceiVed: January 2, 1996; In Final Form: April 5, 1996X
EPR spectra of ubiquinone-0 (UQ0) and UQ10 anion radicals selectively labeled with 13C at the 4-CdO position are reported. The environmental effect on the 4-13C splitting constant was studied in mixed solvents in which the molar fraction of the protic component was changed over a wide range. The quinones were electrochemically reduced, with cyclic voltammetry and concomitant optical spectroscopy being performed as controls. The value and the sign of the 4-13C hyperfine splitting constant strongly depend on the H-bonding properties (proticity) of the solvent, due to the formation of mono- and disolvates through hydrogen bonding of the carbonyl oxygens in protic solvents. The formation of disolvates is less favorable by a factor of 80 than formation of the monosolvated anion. The results are discussed in light of recent EPR, FTIR, and NMR experiments on the primary 13C-labeled acceptor quinone QA in bacterial photosynthetic reaction centers. We propose that in the neutral state both carbonyls of QA are not or only weakly hydrogen bonded to the protein and that formation of anionic QA•- results in a much stronger H-bond for the 4-carbonyl only. The large shift of the 4-CdO IR signal reported for UQ in ViVo is in our view mostly due to a change in bond order induced by binding of UQ to the protein, perhaps through a change in sp2 hybridization at the 4-C position.
Introduction The electron transfer chain of bacterial photosynthetic reaction centers (RCs) from Rhodobacter (Rb.) sphaeroides contains ubiquinone-10 (UQ10) molecules as the primary (QA) and secondary (QB) electron acceptors:1 hν
(BChl)2BPhAQAQB 98 (BChl)2*BPhAQAQB f (BChl)2•+BPhA•-QAQB f (BChl)2•+BPhAQA•-QB f (BChl)2•+BPhAQAQB•- f f Here, (BChl)2 is a bacteriochlorophyll dimer, which upon photoexcitation donates in 2.8 ps an electron to BPhA, a bacteriopheophytin molecule, which in turn donates in ∼200 ps an electron to QA. From QA•- the electron is transferred to QB in ∼100 µs. QA is a strict one-electron acceptor. QB, however, can accept two electrons: after two flashes and double protonation the hydroquinone leaves the reaction center, and takes part in further metabolic processes. The different properties of QA and QB (redox potentials, one- or two-electron gate, protonation) are induced by their interaction with the protein matrix. Hence, it is of interest to compare the spectroscopic properties of QA and QB with those of UQ10 in Vitro. From such comparison one may hope to learn about the specific interactions endowing QA and QB their special function in photosynthetic electron transport. The ubi(semi)quinones in Vitro and in ViVo have been investigated with several types of spectroscopy including UV/ visible,2-12 X- and Q-band EPR,13-15 ENDOR,16-19 IR20-31 and †
Udmurt State University. Leiden Institute of Chemistry, Leiden University. § Department of Biophysics, Leiden University. X Abstract published in AdVance ACS Abstracts, May 15, 1996. ‡
S0022-3654(96)00059-7 CCC: $12.00
NMR.32 These studies suggested that both QA•- and QB•- have the spectral characteristics of anion radicals with one or two hydrogen-bonded carbonyl oxygens. The absorbance spectrum of UQ10 in methanol is characterized by an intense band at 274 nm (extinction coefficient ) 15 mM-1 cm-1) and that of the ubisemiquinone anion radical in alkaline methanol by bands at 320 nm ( ≈ 10.5 mM-1 cm-1) and 445 nm ( ) 6.4 mM-1 cm-1), while the neutral protonated form in acidic methanol absorbs at 275 nm ( ≈ 7.6 mM-1 cm-1) and 420 nm ( ) 3 mM-1 cm-1).4-6,33 The absorbance spectra for UQ0 are close to those of UQ10.6 The difference spectra of both QA•--QA and QB•--QB, measured for reaction centers of purple bacteria,9-12 are very similar to the UQ10•-UQ10 spectrum, showing a band near 450 nm with a shoulder at 430 nm. EPR spectroscopy of benzo- and ubisemiquinones gave evidence that effects of polar solvents on the hyperfine interaction (hfi) are mostly due to hydrogen bonding to the carbonyl groups.19,34-41 Solution proton ENDOR studies of the UQ10 anion in dimethoxyethane (DME) and in basic ethanol showed only lines due to nonexchangeable protons (methyl, methylene, methoxy).17 In contrast, for QA•- and QB•- two different (anisotropic) hfi’s were found that were attributed to exchangeable protons, presumably hydrogen-bonded to the two CdO groups. Surprisingly, the inequivalency of the two hf couplings seemed to be somewhat stronger for QB•- than for QA•-.17,18 FTIR studies showed that for UQ10 in Vitro (a film dried from a solution in n-pentane), the CdO and CdC stretches are strongly mixed and that the 1- and 4-positions (and the 2- and 3-positions) are practically equivalent.28,30 In contrast, for QA in RCs of Rb. sphaeroides the bands predominantly arising from the CdO stretches are nonequivalent, the 4-CdO band being shifted some 60 cm-1 to lower frequency compared to the 1-CdO band.28,30 For QB, the 1- and 4-CdO positions were © 1996 American Chemical Society
10434 J. Phys. Chem., Vol. 100, No. 24, 1996 again practically equivalent.42,43 If the 60 cm-1 shift of the 4-CdO band of QA is due to H-bonding, then this H-bond is exceptionally strong, having an energy of 6-8 kcal/mol.44 On the basis of the X-ray crystallography, several candidates for a 4-CdO hydrogen bond have been advanced. For example, for Rb. sphaeroides the 4-CdO is close to the side chain of ThrM222 or to the Nδ(1) of HisM219.45-47 Recently, ESEEM spectroscopy has shown that QA•- is bound to a nitrogen, whose quadrupole coupling parameters provide strong evidence that it is the Nδ(1) of HisM219.48 Solid-state NMR experiments on 1- and 4-13CdO containing QA showed a pronounced difference in the temperature dependence of the 13C signal of the two carbonyls, suggesting that their difference in bond order as revealed by FTIR is at least partly dynamic in character.32 Note that in addition to H-bonding configurational distortions of QA connected with binding to the protein may also play a role in generating the observed asymmetry of the change in bond order of its two carbonyl ends (discussed below). It is tempting to conclude from the above data that specific (hydrogen) bonds between the carbonyl groups, especially the 4-CdO, of the quinone/semiquinone and the protein environment are involved in conferring on the quinones their different functions in the primary processes of photosynthesis. It is therefore important to further assess the details of the interaction between QA and QA•- and the protein matrix. A particularly suitable spectroscopic technique for studying these interactions for QA•- is EPR spectroscopy of 13C-labeled quinones, because 13C hyperfine splitting constants are known to be sensitive to the environment.16,34-40 As a first example, earlier work from our laboratory showed conclusively that the z-component of the anisotropic 13C hyperfine splitting of the 4-13CdO group in reaction centers of Rb. sphaeroides was about 50% larger than for the 1-13CdO group.49 In contrast, the two carbonyls of UQ10•- in Vitro49 and of QB•- in ViVo50 show similar values for the z-component of the hyperfine interaction. For interpreting these and future results, it is essential to compare binding of the acceptor quinones in ViVo with ubi(semi)quinone solvation in various model systems. Up to now, however, only limited data are available for the naturally occurring ubiquinones in Vitro.19 This has prompted us to start a program aimed at a thorough characterization of ubiquinone radicals in various solvents, using site-selective 13C labeling and EPR and ENDOR spectroscopy.19,51 In the present work EPR spectra of ubiquinone-0 (UQ0) and UQ10 anion radicals selectively labeled with 13C at the 4-CdO position are reported. The environmental effect on the 4-13C splitting constant was studied in mixed solvents in which the molar fraction of the protic component, responsible for hydrogen bonding, was changed over a wide range. We included UQ0 to get further insight in the influence of the isoprenoid side chain of UQ10 on its spectroscopy properties. (It is well-known that the first isoprenoid unit has by far the most influence.29,52-54) The quinones were electrochemically reduced, because this method provides a wider solvent choice and better control of the reduction process than chemical reduction. In addition, the electrochemical method allows one to avoid side products from the reducing agents, which sometimes interfere with the measured spectroscopic signal. All electrochemical preparations were checked with optical spectroscopy for side-product contamination. Materials and Methods Unlabeled UQ0, UQ10, and tetrabutylammonium hexafluorophosphate (supporting electrolyte) were obtained from Sigma and were used without further purification. For IUPAC numbering, see Scheme 1. The synthesis of [4-13C]-labeled UQ0
Kropacheva et al. SCHEME 1
and UQ10 was performed as described.55 Dimethylformamide (DMF, J.T. Baker) was dried over 4 Å molecular sieves and then distilled under low pressure in an inert gas atmosphere. Dimethoxyethane (DME, Merck) was dried with metallic Na and distilled as DMF; isopropyl alcohol (IPA, Merck) was dried over 4 Å molecular sieves and distilled as above. All solvents were stored over molecular sieves until use. For some optical measurements tetrahydrofuran (THF, Merck) with extremely low water content (∼0.001%) was used, which was freshly distilled over LiAlH4 in a dry inert gas atmosphere and immediately used. Cyclic voltammetry was performed in DMF with an AUTOLAB-potentiostat (ECO CHEMIE) with platinum working and auxiliary electrodes against an aqueous saturated calomel reference electrode. The solutions (1 mM of ubiquinone and 50 mM of supporting electrolyte) were deaerated by bubbling pure dry argon. UV/visible spectra were recorded in DME and in THF in a thin-layer spectroelectrochemical cell with the potential applied to a transparent working electrode made from platinum mesh. EPR experiments were carried out in DME and in mixtures of DME and IPA using a special cell designed for simultaneous electrochemical/EPR measurements in vacuum. The solution of quinone (5 mM) and supporting electrolyte (50 mM) in appropriate solvent was degassed on a vacuum line in one of the cell compartments. Then the cell was sealed off, and the solution was transferred to another compartment where the electrolysis was performed. The radicals were generated on a platinum electrode at the potential chosen in accordance with preliminary cyclic voltammetry results. The reduction process was also visually controlled, and the colored radical-anion solution was tipped into the EPR tube (1 mm i.d.) for the EPR measurements. All experiments were done at room temperature with a JEOL (JES-RE2X) spectrometer. Experimental conditions: microwave frequency 9.5 GHz; microwave power 1 mW; modulation amplitude 0.02-0.2 G; modulation frequency 100 kHz; time constant 0.03 s; scan time 30-120 s. The spectra were simulated using software of the JEOL instrument. Hyperfine splittings were first determined directly from the spectrum as indicated in Figure 3 and then refined by accurately simulating the peak positions and base-line crossings of all hyperfine lines, giving an error margin for the hyperfine interactions of 0.02 G. No attempt was made to simulate the alternating line-broadening effect and the difference in linewidth for the components of the 13C splitting, which are easily discerned by comparing the relative amplitudes of the experimental hyperfine lines with those of the simulated lines. Results and Discussion Cyclic Voltammetry. The cyclic voltammograms of UQ0 and UQ10 in DMF show two well-separated reduction and two oxidation waves (Figure 1). From the peak position (repeatability (0.01 V) the half-reduction potentials for the formation of semiquinone (UQ•-) and the dianion (UQ2-) were calculated (Table 1). The potentials obtained for the UQ/UQ•- couple are in good agreement with the published data,20,25,56,57 which for comparison are given vs the solvent-independent potential of
Hyperfine Couplings of [4-13C]-Labeled Ubisemiquinones
J. Phys. Chem., Vol. 100, No. 24, 1996 10435
TABLE 1: Half-Reduction Potentials (V, 20 °C) for Q/Q•- and Q•-/Q2- in DMF (E°(SCE, 20 °C) ) 0.247 V vs NHE; E°(fe+/fe, 20 °C) ) 0.51 V vs SCE published values20,25,58 quinone
Q/Q•-
vs SCE
Q•-/Q2-
vs SCE
Q/Q•-
vs
fe+/fe
Q•-/Q2-
vs
fe+/fe
UQ0
-0.55
-1.40
-1.06
-1.91
UQ10
-0.62
-1.13
-1.13
-1.64
Figure 1. Cyclic voltammograms in DMF of UQ10 at scan rate 2 mV/s (a) and of UQ10 and UQ0 at scan rate 20 mV/s (b). Concentration of UQs is 1 mM.
the ferricenium/ferrocene (fe+/fe) redox couple. Under our experimental conditions in DMF, E°(fe+/fe) ) 0.51 V vs SCE. The potential for the second electron transfer step of UQ0 differs somewhat from that given in the literature;25,58 the corresponding value for UQ10 was not determined earlier. We studied the first electron transfer step for UQ10 at different scan rates ν, switching the direction of the scan at a potential of about 0.3 V past the cathodic peak. Under these conditions, the shapes of the cathodic (I) and anodic (IV) waves are almost identical, with the ratio of peak currents IV/I ) 0.93 ( 0.04. An adsorption prewave at ∼0.6 V, which is more pronounced at the slower scan rate, accompanies the first electron transfer step. For waves I and IV the peak positions and peak current function (Ip/V1/2) were independent of the scan rates (0.002-0.2 V/s). Such behavior corresponds to reversible charge transfer with the absence of coupled chemical reactions.59 Investigation of the second electron transfer step is more difficult because of the uncertainty in the base lines. When the scan rate is increased, the position of peak II is cathodically shifted and the anodic wave III becomes smaller and less definite. These results can be explained by a mechanism including reversible charge transfer followed by a reversible chemical reaction.59 In our case this reaction probably represents the protonation of UQ2-, which can take place in DMF in the presence of residual water. In general, however, for a potential scan in the whole range, when both steps of electron transfer take place, the ratios of peak currents I/IV and II/III are still very close to unity, indicating a high degree of reversibility of the UQ0 and UQ10 reduction/oxidation cycle in DMF. Regarding this point, our results are different from those for UQs in MeCN,20,25 where the strong decrease of anodic current was attributed to irreversible reactions of the UQ anions with the solvent. Comparison of the half-reduction potentials of semiquinone formation for
Q/Q-·
vs fe+/fe
-1.06 (DMF) -1.00 (MeCN) -1.13 (DMF) -1.50 (THF)
Q-·/Q2- vs fe+/fe -1.77 (DMF) -1.46 (MeCN)
UQ0 and UQ10 (Table 1) shows that the polyisoprenyl chain reduces the electron affinity of substituted quinones. Thus, the isoprenoid chain acts like other alkyl substituents, which shift the reduction potentials to more negative values due to their ability to donate electron density. A remarkable feature of the UQ0 cyclic voltammogram is the appearance of a small additional wave between the two major peaks (Figure 1). The same phenomenon was observed in refs 20 and 25 and was explained by the formation and subsequent reduction of protonated semiquinone (QH•). This feature is absent for UQ10, suggesting that the proton affinity of the semiquinone obtained from UQ0 is larger than that of UQ10. This observation will be confirmed below by our UV/visible spectral data. Optical Spectroscopy. Figure 2 shows the optical spectra of UQ0 in DME in a thin-layer spectroelectrochemical cell during stepwise change of the applied voltage. During the reduction the absorption band at 262 nm of the initial UQ0 (spectrum 1) disappears and new bands in the UV/visible region develop. Spectra 2 and 3 correspond to the potentials where the first electron transfer reaction takes place. The maxima are due to π-π* (323 nm) and n-π* transitions (414, 440 nm) of the semiquinone UQ0•- formed during the first reduction wave.2 When the potential is shifted past the first peak (Figure 1), the bands at 414 and 440 nm disappear, while the broad maximum at 660 nm, which gives the solution a green color, remains (spectrum 4, Figure 2). This band starts to decrease after the potential is changed to more negative values, so that at the end of the electrolysis a single maximum exists at 320 nm (π-π* transition of UQ02-). Exposure of the green solution to air also causes loss of the green color. The above data allows one to draw a tentative conclusion about the origin of the 660 nm maximum. It is clear from Figure 2 that although the appearance of the green product is closely connected to semiquinone formation, it cannot be attributed to UQ0•-. Protonation of UQ0 semiquinone gives rise to a neutral radical absorbing close to the semiquinone band. Moreover, the EPR spectrum of the green solution shows that the only radical product formed is the radical anion of UQ0, its EPR spectrum agreeing well with literature data.19 Therefore, we conclude that protonated semiquinone radicals are not stable under our conditions and rapidly undergo subsequent reactions, such as for example the formation of donor-acceptor complexes via the dismutation reaction: 2UQ0H• ) UQ0‚UQ0H2.60 The energy of the n-π* transition in such complexes is usually decreased,60 which results in a new absorbance maximum at longer wavelength. To check whether a donor-acceptor complex could be formed via protonation of UQ0•- by residual water in the DME stock solution, the same electrolysis experiment was performed in superdry THF. In this case no absorbance above 550 nm was observed (Figure 2, spectrum 5). The absorbance changes at 262 nm due to the UQ0 a UQ0•reduction/oxidation cycle were more than 90% reversible. The absence of the 660 nm band when superdry THF is used as solvent strongly suggests that this band is indeed due to a product formed via a protonated UQ0•- intermediary. When protonation of UQ0•- is not possible, no side reactions take place and no 660 nm band is formed (spectrum 5 in Figure 2).
10436 J. Phys. Chem., Vol. 100, No. 24, 1996
Kropacheva et al.
Figure 2. Absorbance spectra of UQ0 in DME during the reduction (1-4). See text for details. Absorbance spectrum of the UQ0 radical anion in THF (5). Inset: absorption spectra of UQ10 in DME; initial spectrum (1), radical anion (2).
UV/visible spectra obtained for single-electron reduction of UQ10 in DME (Figure 2, inset) and in THF (not shown) show that here no green product is formed; that is, no coupled reactions occurred. This indicates that the proton affinity of carbonyl oxygen in UQ10 is less than for UQ0. This result agrees with the cyclic voltammetry results described above, where only UQ0 had an additional wave attributed to UQ0H reduction. The UQ10 radical anion was found to be very stable in dried, degassed solvents (DMF, DME, THF); no change in EPR spectra was noticed during several hours. The electrochemical and UV/visible/EPR spectroscopic results reveal two important differences in UQ0 and UQ10 behavior: (i) The difference between the potentials of the first and second electron reduction of UQ10 is smaller than that of UQ0. In other words, the first electron transfer is more difficult for UQ10 than for UQ0, while the second electron transfer occurs more readily in the case of UQ10. (ii) The proton affinity of the UQ10 semiquinone is less strong than that of the UQ0 radical anion. This property increases the stability of the radical anion obtained from UQ10.51 The substituent effect of the isoprenoid chain on the potential of the UQ/UQ•- couple indicates that the side chain causes an increase of electron density in the quinone ring at the carbonyl positions. The same conclusion was drawn in ref 58, where it was also pointed out that the chain length (UQ1 and UQ10) is not important. UQ10•- can be stabilized owing to the possible formation of an intramolecular hydrogen bond between the H atom of the methylene group and the negatively charged 1-C carbonyl oxygen atom, which is not possible for UQ0•-. Such an intramolecular H bond, which has also been proposed for vitamin K1,61 must reduce the proton affinity of UQ and facilitate the second electron transfer due to the decrease in the effective negative charge of UQ10•-. EPR Spectroscopy. High-resolution EPR spectra were recorded of the anion radical of UQ0 and [4-13C]UQ0 in DME and in several DME isopropyl alcohol mixtures. Representative spectra for DME/2%IPA are shown in Figure 3, together with their simulations. As reported earlier,16 the spectrum of UQ0•is particularly simple due to accidental (near)-degeneracy of the splittings of the methyl group and the ring proton. These splittings are relatively insensitive to the addition of alcohol62 (Table 2). The spectra of [4-13C]-labeled UQ0•- show an additional splitting due to the hyperfine interaction with the 13C (I ) 1/2). Two aspects strike the eye. The amplitude of the high-field line of the 13C splitting is considerably higher than that of the low-field component, and the splitting itself depends
Figure 3. (Left) EPR spectra of unlabeled UQ0•- (top) and of [4-13C]UQ0•- (bottom) in DME + 2% IPA. (Right) Simulation of the experimental spectra at left for unlabeled (top) and labeled (bottom) UQ0. The 4-13C hyperfine splitting (0.97 G), the splitting of the R-proton at C-6 (2.16 G), and the methyl splitting (2.32 G) were first evaluated from the experimental spectrum as indicated and then refined by simulating accurately the peak positions and base-line crossings of the spectrum. Estimated error in the hyperfine splittings is 0.02 G.
TABLE 2: Hyperfine Coupling Constants (G) of Ubiquinone Anion Radicals as a Function of Solvent Composition 4-13C Ubisemiquinone-0 molar ratio of isopropyl alcohol to DME
a(H)
a(CH3)
a(13C)
0 0.028 0.166 0.643 3.50
2.25 2.16 2.13 2.11 2.10
2.25 2.32 2.34 2.35 2.36
-1.17 -0.97 -0.84 -0.57 -0.18
4-13C Ubisemiquinone-10 molar ratio of isopropyl alcohol to DME
a(CH3)
a(CH2)
a(13C)
0 0.014 0.028 0.057 0.152 0.585 1.50 3.50 13.5
2.11 2.10 2.09 2.08 2.08 2.08 2.08 2.08 2.08
1.06 1.05 1.05 1.04 1.04 1.04 1.04 1.04 1.04
-0.93 -0.64 -0.42 -0.36 -0.21 0.00 +0.26 +0.48 +0.63
strongly on the alcohol content of the solvent. The amplitude difference of the 13C lines, first observed by De Boer and Mackor,63 is due to the coupling of the anisotropic 13C hyperfine interaction with an anisotropic g-tensor, which gives rise to a
Hyperfine Couplings of [4-13C]-Labeled Ubisemiquinones
J. Phys. Chem., Vol. 100, No. 24, 1996 10437
SCHEME 2: Resonance Structures of Ubisemiquinone (R ) H, Ubiquinone-O, Decaprenyl, Ubiquinone-10)
line-broadening term proportional to the spin quantum number > > 1 for aCFC < 0, where ∆HF and ∆LF mS:63-65 ∆HF/∆LF < are the line widths of the high- and low-field component of the 13C splitting, respectively, a the isotropic hyperfine interaction C of the 13C nucleus, and FC its (possibly negative) spin density. Because FC is with certainty positive for the C-4 carbon,51,66 the ratio ∆HF/∆LF immediately gives the sign of aC: for [4-13C]UQ0•- it is negative for all IPA concentrations studied, up to 90%. The sensitivity of carbonyl 13C splittings to the hydrogenbonding character of the solvent was first demonstrated by Stone and Maki34,37 and later by others.19,35,36 It is attributed to increased electronegativity of the carbonyl group induced by the positively charged hydrogen-bonding proton, which in turn gives rise to a redistribution of the spin density on the carbonyl C and O atoms (see below). Representative EPR spectra of anion radicals prepared from UQ10 and [4-13C]-labeled UQ10 in DME and in several DME isopropyl alcohol mixtures are shown in Figure 4, together with their simulations. The nine-line spectrum of UQ10•- in DME arises because of accidental degeneracy of the methyl and methylene proton splittings, which are practically insensitive to the alcohol content of the solvent.62 The 13C splitting of [4-13C]UQ10•-, however, is even more sensitive to hydrogenbond formation with alcohol molecules than the corresponding splitting for UQ0•-. It becomes zero at 30% alcohol and rises again for higher alcohol percentages. The sign of the hyperfine coupling constant, however, is then reversed and is positive, as seen immediately by comparing the ∆HF/∆LF ratio for the spectra displayed in Figure 4. The values of aC for [4-13C]UQ0•- and [4-13C]UQ10•- derived from the simulations are given in Table 2 and displayed in Figure 5 as a function of the IPA content of the solvent. Another marked difference between the EPR spectra of UQ10•- and the UQ0•- spectra is the alternating line-width effect, dramatically illustrated in the spectrum of unlabeled UQ10•- of Figure 4. This effect arises because of hindered rotation of the methylene group.64,67 The difference between two inequivalent positions of the two methylene protons is then insufficiently motionally averaged, which leads to severe broadening of hyperfine lines with mI ) mH1 + mH2 ) 0 (because when a proton moves from one site to the other, the positions of the (+1/2,-1/2) and (-1/2,+1/2) lines are interchanged, whereas the mI ) (1 lines remain in place). The simulations of Figures 3 and 4 do not take into account the alternating line-width effect and/or unequal 13C splitting amplitudes. We have included the simulations to illustrate the determination of the hyperfine splittings by accurate simulation of the line positions and for assessing the importance of the line-broadening effects.
Figure 4. (Left) EPR spectra of unlabeled UQ10•- in DME (top) and of [4-13C]UQ10•- in DME and in DME + IPA with molar ratio IPA/ DME as indicated. (Right) Simulations of the experimental spectra at left, with the hyperfine splittings of Table 2. Note the strong alternating line-width effect and the high-field/low-field amplitude ratio for the 4-13C splitting, which is >1 for the spectra at IPA/DME < 0.58 (a(13C) < 0) and 0.58 (a(13C) > 0).
As noted above, the solvent effect on the 13C splitting at the carbonyl position of different semiquinones has been described in the literature19,34-37 and explained by the change of radicalsolvent interactions, which determine the distribution of spin density in the radical. The addition of protic solvent to an apolar solvent may promote the formation of quinone-solvent complexes through H-bonding of protic solvent molecules to the carbonyl oxygens. Note that the formation of protonated semiquinone species can be neglected in the solvents studied here, because of the low acidity of isopropyl alcohol (pKa ) 16.9).25 Thus, even in a pure alcohol the fraction of QH is only about 0.2%. Assuming that the negative charge is mostly located at one of the oxygen atoms, one may suggest the resonance structures in Scheme 2 for the UQ radical anion. Although the formation of hydrogen bonds is possible via all four oxygen atoms of the carbonyl and methoxy groups of
10438 J. Phys. Chem., Vol. 100, No. 24, 1996
Kropacheva et al.
Figure 5. Observed 13C hyperfine coupling constants vs the molar ratio of isopropyl alcohol to DME (W). Curves were fitted with eq 4. The values of the parameters are given in Table 3. (b) [4-13C]UQ10•-; (O) [4-13C]UQ0•-. Inset: the initial parts of the same curves.
UQ10, in the anion radical the strongest bond by far is formed with the negatively charged oxygen of the carbonyl groups. The decrease of effective negative charge of the anion due to H-bonding must reduce the spin density near the opposite oxygen atom and increase it inside the quinone ring, including the H-bonding carbonyl carbon. Thus, the hydrogen bonding stabilizes the structures 2-4, and 6-8 compared to structures 1 and 5, which predominate in aprotic solvents. According to ref 68, the carbonyl 13C splitting constant depends on the spin densities of the carbonyl carbon, the two other carbons to which it is bonded, and the oxygen. Depending on the magnitude of their values and the carbonyl σ-π parameters, the splitting can have a different sign.68 In our case the increase of the [4-13C] splitting in UQ10•- from negative to positive values indicates the redistribution of electron density from the carbonyl oxygen to the carbonyl carbon upon addition of alcohol to DME, such that it becomes higher on the oxygen and lower on the carbon (see below). Solvation of Ubisemiquinone. The dependence of the 13C coupling constant on the concentration of isopropyl alcohol was used to obtain quantitative information on the association of alcohol with UQ0•- and UQ10•-. The following assumptions were made. (i) Depending on the solvent composition, a number of solvated complexes between the quinone radical (Q) and the alcohol molecule (S) can exist: Q, QS1, QS2, ... QSn. (ii) The complexes are formed due to the re-exchange of DME (D) by alcohol molecules via the following reactions: K1
QDi + S {\} QDi-1S1 + D K2
QDi-1S1 + S {\} QDi-2S2 + D
(1)
Kn
QDSn-1 + S {\} QSn + D (iii) Each complex is characterized by one carbonyl coupling constant ai. Because the specific interaction between Q and D is weak, the splitting constant is independent of the number of D molecules in the complex.69 Thus, for simplicity we can consider QSi instead of QDn-iSi. The observed coupling constant (a) is the average of the ai of all unsolvated and solvated species:
a ) x0a0 + x1a1 + ... + xnan
(2)
where xi is the mole fraction of the QSi complex,
xi ) [QSi]/([Q0] + [QS1] + [QS2] + ... + [QSn])
(3)
Using the above assumptions the experimental value a is given by70
a)
a0 + a1K1W + a2K1K2W2 + ... + anK1K2 ... KnWn 1 + K1W + K1K2W2 + ... K1K2 ... KnWn
(4)
where W is the molar ratio of alcohol to DME, W ) [S]/[D], and the Ki’s are the equilibrium constants of the species formed in the solution. To find the best model of complex formation (number of associated solvent molecules) and to determine the Ki’s, the parameters of eq 4 have been determined by numerical analysis of the data of Table 2 and Figure 5, using a homewritten least-squares fitting program. It was found that for the model with n ) 1 no agreement was possible within reasonable limits of deviation. The best fit of the experimental data corresponds to the model with n ) 2 (Table 3, Figure 5). Models with more solvated complexes (QS3, QS4) yielded significantly worse fits. Thus, the model containing only monoand disolvates of ubiquinone adequately describes the behavior of the system studied in the total range of alcohol content (090% by volume). The distribution diagram of the species calculated with the equilibrium constants obtained from the fit for UQ10•- is shown in Figure 6. The nonsolvated radical exists in appreciable amounts only at very low alcohol concentrations. The yield of QS1 is maximal at 0.5% of isopropyl alcohol by volume. It is reasonable to assume that complex QS1 corresponds to the quinone radical, with one alcohol molecule hydrogen-bonded to one of the negatively charged carbonyl oxygen. Several arguments allow us to conclude that the monosolvate of UQ0•is formed via the O-1 not the O-4 atom. First, we have observed that for ratios W ) [S]/[D] between 0 and 1, where QS1 exists, a(H) decreases and a(CH3) increases with increasing the solvent polarity (Table 2). The same phenomenon was observed for UQ10•- in another binary solvent (acetonitrile H2O).62 This corresponds with the spin density at C-6 decreasing and that at
Hyperfine Couplings of [4-13C]-Labeled Ubisemiquinones
J. Phys. Chem., Vol. 100, No. 24, 1996 10439
TABLE 3: Parameters of Eq 4 for the Model with n ) 2 quinone
a0 (G)
a1 (G)
a2 (G)
K1
K2
[4-13C]UQ10•[4-13C]UQ0•-
-0.93 -1.17
-0.24 -0.96
+0.73 +0.24
64.1 205.5
0.7 0.7
C-5 increasing with W (for carbon numbering see Scheme 1), which with the resonance structures of Scheme 2 indicates that the negative charge is located on O-1. A further argument for preferential solvation of UQ0•- at O-1 is the observation that only one O-1 protonated UQ0H• radical can be detected.51 This conclusion is corroborated by calculations of the spin density at the C-5 and C-6 positions of UQ0•-, which yielded changes in spin density going from UQ0•- to UQ0H• similar to those observed only when the proton was bound to O-1.19 For UQ10•the effective negative charge on both carbonyl oxygens is almost the same, because the electron-donating effects of methyl and polyprenyl groups are practically equal. This higher symmetry means that for the QS1 complex of UQ10•- both O-1 and O-4 can be hydrogen-bonded, with a rapid equilibrium between them. Moreover, the isoprenoid chain makes the O-1 atom of UQ10 less accessible for external hydrogen donors than that of UQ0 due to the formation of an intramolecular hydrogen bond between the methylene proton and the oxygen of the 1-CdO group. Also the steric hindrance caused by the long side chain could make H-bonding with the O-1 atom more difficult for UQ10 than for UQ0. All this explains the lower stability constant K1 for QS1 in the case of UQ10•- compared to UQ0•- (Table 3). Our data show that, for both quinones studied, binding of the second alcohol molecule (by QS1) is much less favorable than that of the first alcohol molecule (K1/K2 ) 90 for UQ10•- and K1/K2 ) 300 for UQ0•-), and takes place via the remaining carbonyl oxygen of the ubisemiquinone. Spin Density Calculations. It is possible to calculate the spin density on the carbonyl carbon from its isotropic hf splitting aCO, using the treatment of Karplus and Fraenkel.68 The hf coupling is a function of the polarization of the 1s orbital and the sp2 hybrid bonds due to spin density in the pz orbitals of the carbonyl carbon and of the neighboring ring carbons and oxygen atom. With the experimental proportionality constants (Q-factors) given in refs 35 and 68 one obtains for the hf coupling π aCO ) 33.8FCO - 27.8FCπ - 27.1FOπ G
(5)
where FπC is the spin density on the two neighboring ring carbons (which, since they are small anyway, are taken equal). π , and the The spin densities on the carbonyl carbon, FCO π carbonyl oxygen, FO, when changed by hydrogen bonding, are to a good approximation linearly related:65,71 π ) -1.115FOπ + 0.3406 FCO
(6)
Substituting (6) in (5), we obtain π - 27.8FCπ - 8.28 G aCO ) 58.1FCO
(7)
For UQ10•- eq 7 can be further simplified using the experimental value of the hf coupling of the methyl group, which is practically independent of hydrogen bonding to the carbonyls: aCH3(UQ10•-) ) 2.08-2.11 G,62 which with the Q-factor for a rotating methyl group (0.5 × 300 MHz, i.e. 53.6 G72) gives FπC ) 0.04 and π - 9.39 G aCO ) 58.1FCO
(8)
The experimental values of aCO(UQ10•-) given in Table 3 are -0.93 and +0.73 G for DME (nonprotic) and IPA, respectively,
Figure 6. Distribution diagram of UQ10•-/isopropyl alcohol complexes as a function of the molar ratio of isopropyl alcohol to dimethoxyethane. π π which with eq 8 gives FCO (DME) ) 0.146 and FCO (IPA) ) •0.172. For UQ0 we may take for aCH3 2.25 G (DME) and 2.36 G (80% IPA), and for aCO -1.17 G (DME) and +0.24 G π π (IPA), yielding FCO (DME) ) 0.142 and FCO (IPA) ) 0.168. (The third decimal place is given only for comparative purposes.) Thus, for both ubisemiquinones the hydrogen-bondπ induced change in FCO is small, but sufficient to change the sign of the hf coupling, leading to the observed striking difference in alternating line-width effect of the solution EPR spectra. Comparison with in Vivo Experiments. The above EPR experiments were all carried out on ubisemiquinones in solution and consequently yielded the isotropic hf couplings. For the ubiquinones in the QA,B binding sites of the RC protein in liquid or solid solution, EPR experiments yield the powder line shape, as the rotational correlation time of the protein is much too slow to average out the anisotropic parts of the g- and hf-tensors. Individual hf lines are smeared out, and hf coupling constants can only be estimated by spectral simulation, involving the principal elements of all tensors and their mutual orientations. Fortunately, the resulting large parameter space can be greatly reduced by recording EPR spectra at high microwave frequency, such that the field range spanned by the g-tensor is so large that at the low- and high-field edges practically only RCs are observed that have the x- or z-axis of their g-tensor oriented along the magnetic field. In favorable cases one may then directly measure the hf-splittings along these directions, either directly from the EPR spectrum or with ENDOR. As a first step along this line of approach, we recently recorded the Q-band (35 GHz) EPR spectrum of QA•- in Zn2+-substituted RCs of Rb. sphaeroides R26.49 (The presence of the paramagnetic nonheme iron severely broadens the EPR line of QA•-; it is therefore necessary to remove the iron and to reconstitute the RCs with a diamagnetic divalent ion as Zn2+.) The spectra of 1- and 4-13C-labeled QA•- in ref 49 clearly show a splitting of the peak at gzz, due to the z-component of the 13C hf-tensor (the z-axis of which is to good approximation parallel to the z-axis of the quinone g-tensor). The two splittings are quite different (8.0 ( 0.3 and 12.7 ( 0.3 G, respectively). This is in marked contrast with the two splittings for UQ10•- in frozen solution,
10440 J. Phys. Chem., Vol. 100, No. 24, 1996 which are virtually identical (11.3 ( 0.4 and 11.0 ( 0.4 G, respectively49). For evaluating the carbon spin density of the 13CdO group from the above hyperfine couplings we first note that they represent anisotropic hf couplings and that therefore the formalism for evaluating the carbon spin density given above does not directly apply. Instead, one may to a first approximation attribute the carbonyl 13C-azz component to the sum of the isotropic coupling and the axial component of a uniaxial dipolar coupling between the unpaired electron spin in the pz orbital of the 13C nucleus and its nuclear spin, neglecting the coupling with the pz spin density on the neighboring ring carbons, which is low (Vide supra). Thus, azz ) aiso + a|, where a| is the axial component of the purely dipolar hyperfine coupling tensor. Once the value for the isotropic coupling is known, F(pz) follows directly using the calculated value for the axial coupling for F(pz) ) 1 (a| ) 76.6 G73). The isotropic hyperfine coupling can be evaluated from the sum of the principal components of the hf-tensor, following the reasoning by Isaacson et al.50 that the large azz component is certainly positive,19,66 and since the trace of the dipolar part of the hf-tensor must be zero, the axx and ayy components must be both negative. For [1-13C]UQ10 as QA this gives aiso ) (8.0 - 5.5 - 6.5)/3 ) - 1.3 G,49 so π that FCO ) 9.3/76.6 ) 0.121; for [4-13C]UQ10 we obtain similarly a lower limit of aiso ) +2.6 G and a corresponding π upper limit for FCO ) 10.1/76.6 ) 0.132. The spin density for 1-13CdO of UQ10 as QA is somewhat higher than that found by Isaacson et al.50 for RCs reconstituted with the non-native [1-13C]UQ3 (0.113), the difference seeming to be outside the margin of error and possibly due to the difference in side chain. The upper limit for the spin density on the 4-13C is likewise a little lower than the value quoted in ref 50 (0.138), but here the difference is within the margin of error. Both spin densities are significantly smaller than the spin densities obtained above for UQ10•- in DME or in IPA. Whether this is real, and pointing to a somewhat different spin density distribution for UQ10•- in ViVo compared to liquid solution, possibly because of a protein-induced distortion, or an artifact caused by inaccuracies in the numerical values of the constants in eq 7 is at present difficult to decide. The calculated values of aiso can be compared with the measured values for UQ10•- in Vitro, -0.93 G for DME and +0.63 G for DME 80% IPA.62 For 1-13C the calculated aiso values are negative, -1.3 G (-0.5 G), pointing to a non- (or weakly) hydrogen-bonded carbonyl; those for 4-13C are positive, g2.6 G (+1.9 G), pointing to a strong (or medium-strong) hydrogen bond (values in parentheses from ref 50). We note that the relative strength of the C-1 and C-4 hydrogen bonds is opposite to that found above for UQ0•- in protic solvents. Apparently, for UQ10•- in ViVo the local properties of the protein matrix, such as the presence of a hydrogen donor at a suitable distance, determine hydrogen bonding to the two carbonyls. The above conclusion that the semiquinone QA•- is hydrogenbonded to the protein finds strong support in ENDOR experiments,17 which show signals due to exchangeable protons, and in recent Fourier-transform electron spin echo envelope modulation (FT-ESEEM) spectroscopy of QA•- in Zn2+-substituted reaction centers.48 In the latter work, three strong lines were found that corresponded to the quadrupole coupling of 14N, with the signature coupling and asymmetry parameters (e2qQ and η, respectively) of the imidazole Nδ(1) of a histidine. With help from the high-resolution crystal structure of reaction centers of Rb. sphaeroides R26,47 this histidine was identified as HisM219 binding to the 4-carbonyl of QA•-.
Kropacheva et al. Is Neutral QA Hydrogen-Bonded to the Protein? The hydrogen-bonding pattern of neutral QA has been assessed with Fourier-transform infrared spectroscopy (FTIR)28,30 and magic angle spinning solid-state (MAS) 13C-NMR,32 utilizing reaction centers reconstituted with 1- and 4-13C-labeled UQ1028,32 or UQ3.30 FTIR showed a strong asymmetry between the band positions of the carbonyl stretches of the 1- and 4-CdO groups, at 1660 and 1601 cm-1, respectively. The former is close to the position of the two (symmetric) carbonyl stretches for UQ in Vitro; the large shift of the latter was attributed to a strong hydrogen bond. With MAS-NMR, however, the resonance positions of the two labeled carbonyls were virtually identical (183.8 ( 0.2 and 183.1 ( 0.2 ppm for the 1- and 4-13C, respectively), which is a strong indication that there are no significant differences in the electrostatic environment of the two 13C nuclei. Because a strong hydrogen bond to a carbonyl group makes the participating oxygen nucleus considerably more electronegative than the nonbonded oxygen, the similarity of the chemical shift of the two carbonyl 13C-nuclei strongly argues against such a hydrogen bond to the 4-carbonyl. The MASNMR results also provided the asymmetry parameter η of the chemical shielding tensor of the two labeled carbonyls. The two values of η were 0.6 ( 0.2 and 0.3 ( 0.2 for 1- and 4-13C, respectively; that is, the statistical probability that they are different is 86% (normal error distributions). Such a difference could be interpreted as resulting from a difference in sp2 hybridization,32 perhaps caused by the C-5 methoxy group being forced out of the quinone plane by the protein matrix. Because FTIR spectroscopy measures only differences in bond order, which could equally well result from a change in sp2 hybridization as from the presence of a strong hydrogen bond, the results of the two spectroscopies taken together suggest in our opinion a binding mode in which the neutral QA is distorted by, but not hydrogen-bonded to, the protein. If one accepts the above arguments, then the question arises why the experiments on the semiquinone QA•- all point to a strong hydrogen bond to the 4-carbonyl. In our opinion the answer is given by the quantum chemistry calculations of Samoilova et al.51 and the experimental evidence collected by Meot-Ner,74 that hydrogen bonds to an anionic species are considerably stronger than those to neutral groups. The calculated enthalpy of complex formation of two methanol molecules with UQ10•- is about 19 kcal/mol,51 whereas the energy found for H-bonds of the acetate carboxyl to various anions is 24-31 kcal/mol.74 These values compare with energies of formation of hydrogen bonds to neutral species of about 3-5 kcal/mol.75 Thus, we think that generating the semiquinone QA•- promotes the formation of a strong hydrogen bond with HisM219, pulling as it were the M-polypeptide chain a little closer to the 4-carbonyl. It is noteworthy that in the best resolved crystal structure of reaction centers of Rb. sphaeroides (resolution 2.75 Å)47 the distances between the 4-carbonyl oxygen and the hydrogen donor (oxygen and nitrogen, respectively) of the two candidates for H-bonding (ThrM222, 3.6 Å, and HisM219, 3.2 Å) are both somewhat larger than normally found for such bonds, viz., 2.5-3.1 and 2.6-3.1 Å, respectively.75 Conclusions 1. Cyclic voltammetry and optical spectroscopy show that the proton affinity of the semiquinone obtained from UQ0 is larger than that of UQ10. A stable product formed from protonated UQ0•-, absorbing at 660 nm and absent for superdry solvents, is not formed from UQ10•-. 2. EPR results obtained with anions of UQ0 and UQ10 labeled with 13C in the [4-C]-carbonyl position show that the value and
Hyperfine Couplings of [4-13C]-Labeled Ubisemiquinones the sign of the 13C hyperfine splitting constant strongly depend on the H-bonding properties (proticity) of the solvent. 3. The changes in 13C splitting are caused by complex formation of the anion radical and protic solvent molecules. Numerical analysis of our data shows that mono- and disolvates of ubisemiquinones are formed, presumably through hydrogen bonding of the carbonyl oxygens. 4. The equilibrium constant for the formation of monosolvated radical is about three times higher for UQ0•- than for UQ10•-. The formation of disolvates is less favorable compared to binding the first protic solvent molecule by a factor of about 300 and 90 for UQ0•- and UQ10•-, respectively. 5. The strong dependence of the carbonyl 13C splitting constant on the environmental interactions makes the labeled quinone a sensitive probe of the quinone-binding pockets in photosynthetic reaction centers. From EPR experiments on reaction centers reconstituted with 1- and 4-13C-labeled UQ10 as QA we conclude that QA•- is bound to the protein via a strong hydrogen bond to the 4-CdO group, while its 1-carbonyl is not, or only weakly hydrogen-bonded. The opposite tendency for H-bonding to the C-1 and C-4 carbonyl in ViVo compared to that in protic solvents indicates that the protein matrix, and not the properties of UQ10•- itself, determines H-bonding in ViVo. 6. Comparing our present and published EPR results on anionic QA•- and the results of FTIR and NMR experiments on 13C-labeled neutral QA, we propose that in the neutral state both carbonyls of QA are not or only weakly hydrogen-bonded to the protein. Formation of anionic QA•- then results in a much stronger H-bond for the 4-carbonyl only. Acknowledgment. We are indebted to Dr. F. Hartl of the Chemistry Department of the University of Amsterdam for allowing us to use his opto-electrochemical facility. This research was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organization for Scientific Research (NWO). T.N.K. acknowledges a travel grant from NWO in the framework of the collaborative grant 07-30-036. References and Notes (1) Feher, G.; Okamura, M. Y. In The Photosynthetic Bacteria; Clayton, R. K., Sistrom, W. I., Eds.; Plenum Press: New York, 1978; p 349. (2) Bridge, N. K.; Porter, G. Proc. R. Acad. Ser. A 1958, 244, 259. (3) Diebler, H.; Eigen, M.; Mathies, P. Z. Elektrochem. 1961, 65, 634. (4) Land, E. J.; Swalow, A. J. J. Biol. Chem. 1970, 245, 1890. (5) Land, E. J.; Simic, M.; Swalow, A. J. Biochim. Biophys. Acta 1971, 226, 239. (6) Bensasson, R.; Land, E. J. Biochim. Biophys. Acta 1973, 315, 175. (7) Mayer, J.; Krasiukanis, R. Radiat. Phys. Chem. 1991, 37, 273. (8) Mayer, J.; Krasiukanis, R. J. Chem. Soc., Faraday Trans. 1991, 87, 2943. (9) Slooten, L. Biochim. Biophys. Acta 1972, 275, 208-218. (10) Clayton, R. K.; Straley, S. C. Biophys. J. 1972, 12, 1221. (11) Verme´glio, A. Biochim. Biophys. Acta 1977, 459, 516. (12) Wraight, C. A. Biochim. Biophys. Acta 1977, 459, 525. (13) Handbook of EPR Spectra from Quinones and Quinols; Pedersen, J. A., Ed.; CRC Press: Boca Raton, FL, 1985. (14) Feher, G.; Okamura, M. Y.; McElroy, J. D. Biochim. Biophys. Acta 1972, 267, 222. (15) Gast, P.; de Groot, A.; Hoff, A. J. Biochim. Biophys. Acta 1983, 723, 52. (16) Das, M. R.; Connor, H. D.; Leniart, D. S.; Freed, J. H. J. Am. Chem. Soc. 1970, 92, 2258. (17) Feher, G.; Isaacson, R. A.; Okamura, M. Y.; Lubitz, W. In Antennas and Reaction Centers of Photosynthetic Bacteria; Michel-Beyerle, M. E., Ed.; Springer-Verlag: Berlin, 1985; p 174. (18) Lubitz, W.; Abresch, E. C.; Debus, R. J.; Isaacson, R. A.; Okamura, M. Y.; Feher, G. Biochim. Biophys. Acta 1985, 808, 464. (19) Samoilova, R. I.; van Liemt, W. B. S.; Steggerda, W. F.; Lugtenburg, J.; Hoff, A. J. J. Chem. Soc., Perkin Trans. 2 1994, 609.
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10442 J. Phys. Chem., Vol. 100, No. 24, 1996 (64) Freed, J. H.; Fraenkel, G. K. J. Chem. Phys. 1963, 39, 326; J. Chem. Phys. 1964, 40, 1815. (65) Das, M. R.; Fraenkel, G. K. J. Chem. Phys. 1965, 42, 1350. (66) Straus, H. L.; Fraenkel, G. K. J. Chem. Phys. 1961, 35, 1738. (67) Carrington, A.; McLachlan, A. D. Introduction to Magnetic Resonance; Harper & Row: New York, 1969; p 213. (68) Karplus, M. A.; Fraenkel, G. K. J. Chem. Phys. 1961, 35, 1312. (69) The weak influence on the spin density distribution of solvents that nonspecifically interact with UQ anion radicals is demonstrated by the solvent independence of UQ•- proton and 13C splittings for solvents such as DME, DMF, and acetonitrile (data not shown and Table 1 of ref 19).
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