| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Physics, University of California at San Diego, La Jolla, California; Max-Planck Institut für Bioanorganische Chemie, Mülheim an der Ruhr, Germany; and Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas and INTEC, Universidad Nacional del Litoral and CONICET, Santa Fe, Argentina
Correspondence: Address reprint requests to George Feher, E-mail: gfeher{at}ucsd.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSEXPERIMENTAL RESULTS AND...CONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
in the well-characterized reaction center from the photosynthetic bacterium Rhodobacter sphaeroides R-26. We used electron paramagnetic resonance and electron nuclear double resonance techniques at 35 GHz at a temperature of 80 K. The goal of this study was to identify and assign sets of 1H-ENDOR lines to protons hydrogen bonded to each of the two oxygens. This was accomplished by preferentially exchanging the hydrogen bond on one of the oxygens with deuterium while concomitantly monitoring the changes in the amplitudes of the 1H-ENDOR lines. The preferential deuteration of one of the oxygens was made possible by the different 1H 
2H exchange times of the protons bonded to the two oxygens. The assignment of the 1H-ENDOR lines sets the stage for the determination of the geometries of the H-bonds by a detailed field selection ENDOR study to be presented in a future article. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSEXPERIMENTAL RESULTS AND...CONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
,2). Their biological function is related to their electrochemical properties, which are influenced by their local protein environment. Quinones can be reduced in two successive reversible one-electron steps. The first step leads to an intermediate, a semiquinone (quinone anion radical); the second step, followed by protonation, leads to the fully reduced quinol (3,4). These properties make quinones ideally suited as electron and proton carriers in the bioenergetic processes of respiration and photosynthesis (5).
Two ubiquinones, QA and QB, are present in the reaction centers (RCs) of photosynthetic purple bacteria. QA accepts one-electron and is not protonated, whereas QB accepts, sequentially, two electrons. The second electron transfer is coupled with protonation leading to the formation of ubiquinol, which is weakly bound and diffuses out of the binding site to participate in the formation of the proton gradient required for ATP synthesis (5). QA and QB form hydrogen bonds to the RC protein. The bonds contribute to the binding and to the chemical properties and function of the quinones. It is, therefore, important to characterize the hydrogen bonds in detail.
Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies are ideally suited techniques to study the transient paramagnetic species involved in the primary events of photosynthesis, e.g., the ubiquinone anion radicals, and 
These studies provide information about the electronic and spatial structure of the transient radicals. In particular, in the photosynthetic RC of Rhodobacter sphaeroides has been investigated extensively using EPR and ENDOR spectroscopies (6–12). These investigations showed that the ENDOR lines of are due to three classes of protons: 1), protons associated with the protein in the vicinity of the binding site (matrix lines);2), nonexchangeable protons on the quinone, e.g., methyl, methoxy, or methylene protons; and 3), exchangeable protons, the most important of which are the protons forming hydrogen bonds with the two carbonyl oxygens of the quinone. The ENDOR lines corresponding to the exchangeable protons were identified by comparing ENDOR spectra of RCs in H2O and D2O buffer (6).
The initial ENDOR results suggested that is bound to the RC protein by two nonequivalent hydrogen bonds to the carbonyl oxygens of the quinone (7). Confirming evidence of two H-bonds was obtained by electron spin echo envelope modulation (ESEEM) (13–15). Additional, independent evidence of two nonequivalent hydrogen bonds was obtained by Paddock et al. (16), who showed that there are two different proton-deuterium exchange times associated with two distinct hydrogen bonded protons.
Based on the RC structure (17) the two hydrogen bonds were assigned to the imidazole nitrogen of His (M219) (N
-H
O4) and the NH group of Ala (M260) (N-HO1) (see Fig. 1). However, there remained two problems: 1), in none of the previous experiments (6,7,16) could the ENDOR lines be assigned to protons associated with specific oxygens (i.e., O1 or O4, Fig. 1); and 2), there remained the puzzle why only three pairs of 1H-ENDOR lines were observed instead of the expected four pairs (in randomly oriented molecules each proton gives rise to two pairs of lines corresponding approximately to the parallel and perpendicular components of the axially symmetric hyperfine tensors). In this work we addressed both of these problems. We first determined the different 1H 2H exchange times of the two hydrogen bonds by incubating protonated (or deuterated) RCs for different times in D2O and monitoring the amplitudes of the 1H-ENDOR lines. By using the results of these experiments we prepared RCs whose hydrogen bonds were preferentially deuterated (or protonated) at either O1 or O4. This enabled us to determine which set of ENDOR lines belong to a given oxygen. This solved the problem of the assignment of the observed three pairs of lines. Using the known structure of the RC (17) we postulated an assignment of each set of lines to a particular oxygen. A detailed simulation of the ENDOR spectra and the determination of the geometry of the H-bonds will be presented in a future article.
|
and the magnetic moments of protons or deuterons that form hydrogen bonds to the quinone oxygens O1 and O4 (see Fig. 1). The observed ENDOR spectra were interpreted using a spin Hamiltonian, 
, containing the electron and nuclear Zeeman interactions with the applied magnetic field Bo, the hyperfine coupling (hfc) and the nuclear quadrupole coupling (nqc) terms (e.g., Weil et al. (18)):
 | (1) |
The first term in Eq. 1 represents the electronic Zeeman term that gives rise to the observed EPR spectrum. The other three terms represent nuclear interactions that give rise to the ENDOR spectra. These can be calculated to first order from Eq. 1 using an expansion in powers of the ratio hA/(gßeBo) (18,19). At the microwave frequency of 35 GHz used in this work, second order terms are small and can be neglected. Furthermore, at 35 GHz the ENDOR free frequency of protons (or deuterons) is much larger than its respective hfc interaction, i.e., gNßNBo/h >> A/2. Thus, the resonance frequency of the ENDOR transition M
M – 1 is given to first order for a given direction of the magnetic field by (18,19):
 | (2) |
) and M the magnetic quantum number corresponding to the lower energy levels of the nucleus, i.e., + for protons and +1, 0 for deuterons.
For protons M = + making the term containing the nuclear quadrupole interaction P zero. Thus, for a given magnetic field direction with respect to the molecular axes, one expects two ENDOR lines separated by the hfc A centered around the proton Larmor frequency gNßNBo/h. For deuterons M = +1, 0 and each line is additionally split by 3P. In a sample having randomly oriented molecules, the anisotropies of A and P smear out the spectrum and sharp lines are observed only at the extrema (e.g., approximately at A// and A
).
At 35 GHz (Q-band) the anisotropies of the electronic g-value gives rise to a spectrum shown in Fig. 2. At the positions labeled gx, gy, and gz, the majority of the molecules contributing to the spectrum have their molecular axes along the principal directions x, y, and z. However, it should be noted that at each position there is a significant admixture of molecules having different directions. Thus, only a partial magnetic field selection of orientations is obtained.
|
1.25 T and the ENDOR lines for protons and deuterons are observed in different frequency ranges, 53 MHz for 1H and 8 MHz for 2H. This enabled us to separate the signals due to protons in H-bonds from those in the quinone ring by using samples of deuterated RCs in H2O buffer. To observe deuterons in H-bonds we used samples of protonated RCs in D2O buffer. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSEXPERIMENTAL RESULTS AND...CONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
). The paramagnetic nonheme Fe2+ was chemically removed and replaced with diamagnetic Zn2+ to reduce the EPR line width of the semiquinone, following the procedure of Utschig et al. (20). The ratio of Zn/RC was determined by atomic absorption spectroscopy and Q-band EPR spectroscopy to be 
0.90.
Deuteration of reaction centers
Fully deuterated RCs were obtained by growing Rb. sphaeroides R-26 bacteria in a D2O-based modified Hutner's medium using perdeuterated succinic acid (98% D, Aldrich, Milwaukee, WI) as the sole carbon source as described previously by van der Est et al. (21).
Proton-deuterium exchange
We used two sets of samples in which protons were exchanged with deuterons—one for the determination of the exchange times and the other for obtaining detailed ENDOR spectra, which required a higher signal/noise ratio, i.e., a higher RC concentration.
For the determination of the exchange times, fully protonated and fully deuterated RCs were purified and concentrated to an optical absorbance of 
The RC samples were diluted into D2O buffer for different lengths of time, at the end of which was generated photochemically (see next section) and frozen by immersing the sample in liquid nitrogen. We used a fivefold dilution for deuterated RCs and a fourfold dilution for protonated RCs. The final optical absorbance 
was 40 and 50, respectively.
To obtain ENDOR spectra in which one of the H-bonds was preferentially deuterated, we prepared the following high concentration (
) samples:
The incubation times (see section on ENDOR results) and dilutions were chosen to maximize the preferential deuteration of one of the H-bonds to QA. After dilution the samples were concentrated by centrifuging in a Millipore (5–50k) filter to an optical absorbance At the end of the incubation was generated chemically (see next section) and the sample frozen by immersing it in liquid nitrogen.
Generation of semiquinone radical anion
We used two methods to generate : 1), for low concentration samples (
) we illuminated the RCs with a single, saturating laser flash (Lumen-X DL2100C, 
= 590 nm) in the presence of excess cyt c2 to reduce the donor, D+, and stigmatellin to inhibit electron transfer to QB (photochemical method). After the laser flash the sample was plunged into liquid nitrogen. 2), For high concentration samples (
), we used Na2S2O4 to reduce QA before freezing (chemical method) (6). The quartz sample tubes were type 705PQ (Wilmad, Buena, NJ) (OD 3 mm, ID 2 mm).
Nomenclature
Since we used several combinations of protonated and deuterated RCs and buffer, we define the following notation: a), (
) for in fully protonated RCs and (
) for in fully deuterated RCs; b), (H2O) for fully protonated buffer and (D2O) for fully deuterated buffer; and c), (
) for RCs prepared in H2O and incubated later in D2O for a specific time (in minutes) and (
) for the reverse.
EPR instrumentation
EPR and ENDOR measurements were performed at 35 GHz and 80 K. The spectrometer is a home-built superheterodyne-type instrument with a Varian klystron, a cylindrical TE011 brass cavity, and an immersion dewar system for temperature control. The cavity and coupler is similar to one described by Jaworski et al. (22). A Li:LiF sample was used as a primary g-value standard (g = 2.00229) (23), and P-doped Si as a secondary standard (g = 1.99891 at 80 K) (24,25). The P-Si marker was permanently attached to the bottom wall of the cavity. ENDOR experiments were performed with the EPR spectra 50% saturated. ENDOR spectra were recorded at the magnetic field positions as indicated in the corresponding traces, using frequency modulation (FM) of ±140 kHz for protons and ±30 kHz for deuterons, at FM rates as indicated in the figure captions. The output of the RF amplifier (ENI 3100L) feeding the ENDOR coils was 50 W for protons and 25 W for deuterons. To improve the signal/noise ratio of the ENDOR signal, many traces were averaged for up to 3 h for protons and 42 h for deuterons.
|
EXPERIMENTAL RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSEXPERIMENTAL RESULTS AND...CONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
in protonated buffer (H2O), at T = 80 K, is shown in Fig. 2. The broadening of the spectrum is mainly due to protons in solution (exchangeable protons), including the H-bonds to the carbonyl oxygens. Exchanging H2O with D2O, decreases the broadening (data not shown).
ENDOR results
1H ENDOR spectrum corresponding to the H-bonds
Fig. 3 shows the Q-band 1H ENDOR spectrum at T = 80 K of in H2O (chemically reduced), arising from 1H-bonds and from protons in the solvent. The spectrum was recorded at the magnetic field corresponding to gy (see Fig. 2). The splittings of the 1H-ENDOR peaks associated with the H-bonds are A1 (4.82 MHz), A2 (6.43 MHz), and A3 (9.07 MHz). Similar splittings were previously observed at X-band by comparing ENDOR spectra of in H2O and D2O (6,12).
|
) components of an axially symmetric hfc tensor (7,26–28), i.e., each proton should give rise to two pairs of lines. For benzoquinone anion radicals in solution, the ENDOR experiments showed that the H-bond hfc tensor is approximately axially symmetric. This is typical for a proton in a normal H-bond. Deviations are expected only for very short H-bonds as described in (29). Since in reaction centers there are two nonequivalent H-bonds to (12–16), in principle four pairs of 1H ENDOR lines should be observed in the spectrum. However, the experiment shows only three pairs, despite a thorough search for a fourth set of lines. This problem is discussed in a subsequent section. There are two problems to be solved in the identification of the ENDOR lines associated with the hydrogen-bonded protons: 1), which pair of lines belong to the same proton; and 2), with which oxygen (O1 or O4) is a particular set of ENDOR lines associated.
To answer the first question we make use of the selective deuteration of the oxygens as discussed in the next section.
Determination of the 1H 2H exchange times of the two protons H-bonded to QA
To preferentially deuterate one of the H-bonds to QA, it is necessary to determine their respective 1H 2H exchange times. We used ENDOR to measure the 1H 2H exchange times of the two protons H-bonded to QA, in fully deuterated RCs and in fully protonated RCs (R-26). In the fully deuterated RCs in H2O we measured the amplitudes of the 1H-ENDOR lines L1 and L2 corresponding to the two protons (Fig. 3) at different times of incubation in D2O. The incubation time started with a fivefold dilution of the H2O sample in D2O. The semiquinone was produced photochemically (see Materials and Methods) at the end of the incubation period.
In view of the fivefold dilution of the sample, 20% of the bonds remain protonated and only 80% are subject to exchange with deuterium. Thus, the normalized ratio of amplitudes of L1 to L2 is given by:
 | (3) |
and 
are the respective initial intensities and 
1 and 2 are the respective 1H 2H exchange times for the two protons. For protonated RCs, the H2O samples were diluted fourfold in D2O.
Fig. 4 shows the observed time dependence of the normalized intensity ratio of the L1 and L2 lines for fully deuterated and fully protonated RCs. The 1H 2H exchange times were determined by fitting the experimental data with Eq. 3, with 1 and 2 as adjustable parameters. The resultant values of 1 and 2 are shown in the inserts of Fig. 4.
|
), who measured the exchange times in protonated RCs of the HC (M266) mutant, found an order of magnitude different times than in the wild-type, suggesting again a structural change. Thus, the exchange times are very sensitive to the details of the structure and may provide a useful probe in investigating structural changes.
Assignment of pairs of lines belonging to the same proton
To solve this problem we used the differential exchange of one proton with respect to the other as discussed in the previous section. The lines associated with a particular proton should show the same reduction in amplitude when the proton is exchanged with deuterons. Fig. 5 shows the low frequency part of the 1H-ENDOR spectrum of in H2O (top) and after 27 min of exchanges in D2O (bottom), both samples were produced chemically. Deuterated RCs were used to eliminate ENDOR signals from protons of the protein. Using the exchange times for the two protons of 10 min and 89 min, respectively (see Fig. 4 a), we predict that one proton should be 80% exchanged and the other 20%. The reduction in amplitudes of L1 and L2 correspond roughly to these values. Thus, the peaks L1 and L2 are associated with two different protons, as had been postulated previously (7,16).
|
|
Additional evidence for the overlap is provided by the 2H-ENDOR spectra of protonated RCs in D2O shown in Fig. 6 (high concentration samples; see Materials and Methods). Each 2H-ENDOR line is split into a doublet by the nuclear quadrupole interaction, 3P (Eq. 2). The center of each doublet (solid arrows in Fig. 6) corresponds to the hfc (18). The top trace shows the low frequency 2H-ENDOR spectrum after incubating the RC for 50 min in D2O. In these experiments protonated RCs were used to eliminate signals from the protein. With the exchange times of 50 min and 1200 min (Fig. 4 b), we expect one oxygen (Oa) to be 50% deuterated, whereas the other (Ob) only 3% (see Eq. 3 and inset in Fig. 6, top). Thus, the spectrum is essentially due to deuterons on Oa. In the lower trace the deuterons were exchanged with protons giving rise to an 2H-ENDOR spectrum associated with deuterons on Ob (see inset in Fig. 6, bottom). Comparing the two spectra we see that the low frequency line (at –0.8 MHz) of P3 is shifted by 30 kHz from that of P4 showing that the two lines belong to different D-bonds. The shift between the two lines is smaller than the line width which explains why the two lines are not resolved when both oxygens are protonated (or deuterated). These results clearly show that the line at –0.8 MHz is associated with deuterons on both Oa and Ob.
|
) that Ala (M260), i.e., O1, is closer to the surface of the RC and is, therefore, expected to exchange its proton faster than the buried His (M219), i.e., O4. Furthermore, EPR (8,10,30) and FTIR (31,32) experiments on 13C substituted quinones showed that the hydrogen bond to O1 is weaker than to O4 (reviewed in Lubitz and Feher (12). We, therefore, postulate that the ENDOR line L1 and L3 (Fig. 5) are associated with O1 and L2 and L4 with O4. This assignment will be further validated in a subsequent publication. |
CONCLUSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSEXPERIMENTAL RESULTS AND...CONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
in reaction centers of Rb. sphaeroides. In a forthcoming study, these assignments will be used to determine the exact geometries of the hydrogen bonds. |
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSEXPERIMENTAL RESULTS AND...CONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
This work was supported by National Science Foundation MCB 99/82186 and National Institutes of Health GM13191 and by Max Planck Society, and Fonds der Chemischen Industrie (to W.L.).
Submitted on November 16, 2005; accepted for publication January 18, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSEXPERIMENTAL RESULTS AND...CONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
2. Trumpower, B. L. 1982. Function of Quinones in Energy Conserving Systems. Academic Press, New York
3. Chambers, J. Q. 1988. Electrochemistry of quinones. In The Chemistry of Quinonoid Compounds. S. Patai and Z. Rappoport, editors. John Wiley & Sons, Chichester; New York. 719–757.
4. Morrison, L. E., J. E. Schelhorn, T. M. Cotton, C. L. Bering, and P. A. Loach. 1982. Electrochemical and spectral properties of ubiquinone and synthetic analogs: Relevance to bacterial photosynthesis. In Function of quinones in energy conserving systems. B. L. Trumpower, editor. Academic Press, New York. 35–58.
5. Cramer, W. A., and D. B. Knaff. 1990. The quinone connection. In EnergyTransduction in Biological Membranes. C. R. Cantor, editor. Springer-Verlag, New York. 193–238.
6. Lubitz, W., E. C. Abresch, R. J. Debus, R. A. Isaacson, M. Y. Okamura, and G. Feher. 1985. Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides. Biochim. Biophys. Acta. 808:464–469.[Medline]
7. Feher, G., R. A. Isaacson, M. Y. Okamura, and W. Lubitz. 1985. ENDOR of semiquinones in RCs from Rhodopseudomonas sphaeroides. In Antennas and Reaction Centers of Photosynthetic Bacteria-Structure, Interactions and Dynamics. M. E. Michel-Beyerle, editor. Springer-Verlag, Berlin. 174–189.
8. van den Brink, J. S., A. P. Spoyalov, P. Gast, W. B. S. van Liemt, J. Raap, J. Lugtenburg, and A. J. Hoff. 1994. Asymmetric binding of the primary acceptor quinone in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides R26, probed with Q-Band (35 GHz) EPR spectroscopy. FEBS Lett. 353:273–276.[CrossRef][Medline]
9. Isaacson, R. A., F. Lendzian, E. C. Abresch, W. Lubitz, and G. Feher. 1995. Electronic structure of in reaction centers from Rhodobacter sphaeroides. 1. Electron paramagnetic resonance in single crystals. Biophys. J. 69:311–322.
10. Isaacson, R., E. C. Abresch, F. Lendzian, C. Boullais, M. Paddock, C. Mioskowski, W. Lubitz, and G. Feher. 1996. Asymmetry of the binding sites of and 
in reaction centers of Rb. sphaeroides probed by Q-band EPR with 13C-labeled quinones. In The Reaction Center of Photosynthetic Bacteria: Structure and Dynamics. M. E. Michel-Beyerle, editor. Springer-Verlag, Berlin. 353–367.
11. Rohrer, M., F. MacMillan, T. F. Prisner, A. T. Gardiner, K. Mobius, and W. Lubitz. 1998. Pulsed ENDOR at 95 GHz on the primary acceptor ubisemiquinone in photosynthetic bacterial reaction centers and related model systems. J. Phys. Chem. B. 102:4648–4657.
12. Lubitz, W., and G. Feher. 1999. The primary and secondary accepters in bacterial photosynthesis III. Characterization of the quinone radicals and by EPR and ENDOR. Appl. Magn. Reson. 17:1–48.
13. Bosch, M. K., P. Gast, A. J. Hoff, A. P. Spoyalov, and Y. D. Tsvetkov. 1995. The primary acceptor quinone QA in reaction centers of Rhodobacter-sphaeroides R26 is hydrogen-bonded to the N(1)-H of His M219. An electron-spin echo study of 
Chem. Phys. Lett. 239:306–312.[CrossRef]
14. Lendzian, F., J. Rautter, H. Kass, A. Gardiner, and W. Lubitz. 1996. ENDOR and pulsed EPR studies of photosynthetic reaction centers: protein-cofactor interactions. Ber. Bunsenges. Phys. Chem. 100:2036–2040.
15. Spoyalov, A. P., R. J. Hulsebosch, S. Shochat, P. Gast, and A. J. Hoff. 1996. Evidence that Ala M260 is hydrogen-bonded to the reduced primary acceptor quinone in reaction centers of Rb. sphaeroides. Chem. Phys. Lett. 263:715–720.[CrossRef]
16. Paddock, M. L., E. C. Abresch, R. A. Isaacson, W. Lubitz, M. Y. Okamura, and G. Feher. 1999. Identification of hydrogen bonds to in RCs of Rb. sphaeroides by ENDOR spectroscopy. Biophys. J. 76:A141–A141.
17. Stowell, M. H. B., T. M. McPhillips, D. C. Rees, S. M. Soltis, E. Abresch, and G. Feher. 1997. Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer. Science. 276:812–816.
18. Weil, J. A., J. R. Bolton, and J. E. Wertz. 1994. Electron Paramagnetic Resonance. Elementary Theory and Practical Applications. Wiley, New York.
19. Weil, J. A. 1975. Comments on second-order spin Hamiltonian energies. J. Magn. Reson. 18:113–116.
20. Utschig, L. M., S. R. Greenfield, J. Tang, P. D. Laible, and M. C. Thurnauer. 1997. Influence of iron-removal procedures on sequential electron transfer in photosynthetic bacterial reaction centers studied by transient EPR spectroscopy. Biochemistry. 36:8548–8558.[CrossRef][Medline]
21. van der Est, A., R. Bittl, E. C. Abresch, W. Lubitz, and D. Stehlik. 1993. Transient EPR spectroscopy of perdeuterated Zn-substituted reaction centers of Rhodobacter sphaeroides R-26. Chem. Phys. Lett. 212:561–568.[CrossRef]
22. Jaworski, M., A. Sienkiewicz, and C. P. Scholes. 1997. Double-stacked dielectric resonator for sensitive EPR measurements. J. Magn. Reson. 124:87–96.[CrossRef][Medline]
23. Stesmans, A., and G. Vangorp. 1989. Novel method for accurate g-measurements in electron-spin resonance. Rev. Sci. Instrum. 60:2949–2952.[CrossRef]
24. Feher, G., and E. Gere. 1959. Electron spin resonance experiments on donors in silicon. II. Electron spin relaxation effects. Phys. Rev. 114:1245–1256.[CrossRef]
25. Stesmans, A., and G. Devos. 1986. Electron-spin-resonance observation of temperature dependent g shifts in submetallic P doped Si at low temperatures. Phys. Rev. B. 34:6499–6502.[CrossRef]
26. O'Malley, P. J., and G. T. Babcock. 1986. Powder ENDOR spectra of para-benzoquinone anion radical principal hyperfine tensor components for ring protons and for hydrogen-bonded protons. J. Am. Chem. Soc. 108:3995–4001.[CrossRef]
27. MacMillan, F., F. Lendzian, and W. Lubitz. 1995. EPR and ENDOR characterization of semiquinone anion radicals related to photosynthesis. Magn. Reson. Chem. 33:S81–S93.[CrossRef]
28. Flores, M., R. A. Isaacson, R. Calvo, G. Feher, and W. Lubitz. 2003. Probing hydrogen bonding to quinone anion radicals by 1H and 2H ENDOR spectroscopy at 35 GHz. Chem. Phys. 294:401–413.[CrossRef]
29. Sinnecker, S., E. Reijerse, F. Neese, and W. Lubitz. 2004. Hydrogen bond geometries from electron paramagnetic resonance and electron-nuclear double resonance parameters: density functional study of quinone radical anion-solvent interactions. J. Am. Chem. Soc. 126:3280–3290.[CrossRef][Medline]
30. Hoff, A. J., T. N. Kropacheva, R. I. Samoilova, N. P. Gritzan, J. Raap, J. S. van den Brink, P. Gast, and J. Lugtenburg. 1996. Site-directed isotope labelling as a tool in spectroscopy of photosynthetic preparations. Investigations on quinone binding in bacterial reaction centers. In The Reaction Center of Photosynthetic Bacteria: Structure and Dynamics. M. E. Michel-Beyerle, editor. Springer-Verlag, Berlin. 405–420.
31. Breton, J., C. Boullais, J. R. Burie, E. Nabedryk, and C. Mioskowski. 1994. Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: assignment of the interactions of each carbonyl of QA in Rhodobacter sphaeroides using site-specific 13C-labeled ubiquinone. Biochemistry. 33:14378–14386.[CrossRef][Medline]
32. Brudler, R., H. J. M. de Groot, W. B. S. van Liemt, W. F. Steggerda, R. Esmeijer, P. Gast, A. J. Hoff, J. Lugtenburg, and K. Gerwert. 1994. 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. EMBO J. 13:5523–5530.[Medline]
This article has been cited by other articles:
 |
|
 |
|
  M. Flores, R. Isaacson, E. Abresch, R. Calvo, W. Lubitz, and G. Feher Protein-Cofactor Interactions in Bacterial Reaction Centers from Rhodobacter sphaeroides R-26: II. Geometry of the Hydrogen Bonds to the Primary Quinone Formula by 1H and 2H ENDOR Spectroscopy Biophys. J., January 15, 2007; 92(2): 671 - 682. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) for the low frequency 1H-ENDOR lines (see 
(a) and 50 (b).
(lower spectrum), at 35 GHz, taken at a magnetic field of 1248.3 mT (see arrow in 
in 
(upper spectrum) and in 
(lower spectrum), at 35 GHz, taken along the magnetic field position corresponding to gy (see