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Two-dimensional NMR studies of electron transfer in cytochrome c3
JOURNAL
OF MAGNETIC
RESONANCE
Two-Dimensional
59, 177-l 80 (1984)
NMR Studies of Electron Transfer in Cytochrome c3*
HELENA SANTOS,DAVID L. TURNER, ANDANT~NIO
V-XAVIER
CentrO de Quimica Estrutural, Complexo I. IST, Av. Rovisco Pais, 1000 Lisbon, Portugal AND
JEANLE GALL Department of Biochemistry, University of Georgia, Athens, Georgia 30602 Received March 27, 1984
The biological role of electron-transfer proteins justifies the amount of work devoted to the understanding of their mechanism of action (I). Sulfate-reducing bacteria provide suitable systems for the investigation of the electron-transfer mechanisms occurring in vivo (2); in particular, cytochrome c3 (MW 13,000) one of the components of the electron transfer chain of the Desulfovibrio bacteria is an attractive candidate for these studies since a considerable amount of information on this protein is now available (J-11). This information has been obtained using a wide range of techniques that includes X-ray (3, 4), NMR (58), EPR (9, lo), and electrochemical methods (I I). Also, both intramolecular and intermolecular electron transfer mechanisms may be probed since each molecule contains four redox centers. Proton NMR proved to be a suitable technique to provide specific information about each redox center at each of the five oxidation steps obtained from the fully reduced state by successive losses of one electron, according to the following scheme (7, 8): -If?
-le
-It?
-1e
Step 0 P Step I P Step II Z Step III P Step IV The steps are numbered 0, I, II, III, and IV according to the number of oxidized hemes in any one molecule. For Desulfovibrio gigas cytochrome c3 the intramolecular electron exchange rate is fast on the NMR time scale (> lo5 s-r), but the intermolecular electron exchange is slow at 273 K and protein concentrations of the order of 3 mA4 (8). Under these conditions, the fast intramolecular electron exchange implies that each heme methyl group originates only one resonance in the NMR spectrum for each oxidation step, its chemical shift depending on the ratio between the molar fractions of the states in * Presented at the FNRS NMR Symposium, Brussels, December 1983. 177
0022-2364184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
178
COMMUNICATIONS
which the heme considered is oxidized and the states in which the same heme is reduced. A full characterization of the heme methyl group resonances in the different oxidation steps is important for the understanding of the intramolecular and intermolecular electron transfer mechanisms and this has been partly achieved (7, B), using several series of selective saturation transfer experiments (12) at different solution redox potentials. However, this is a time consuming task since 16 heme methyl groups are present, each one giving rise to five resonances in a complete oxidation/reduction cycle. Also, selective irradiation is required and this is difficult to achieve in the crowded regions of the spectrum. Therefore, the analogous two-dimensional experiment (13) should be more efficient for the study of this complex system. The present article reports for the first time the application of 2D exchange NMR to obtain the crossassignments of the resonances in a multisite electron transfer system. The solution of Desulfovibrio gigus cytochrome q was prepared as reported elsewhere (7, 8). The protein concentration was 4.5 mM and the pH was adjusted to 8.1 with NaOD. A few crystals of sodium dithionite were used as reducing agent and the intermediate oxidation stages were obtained by introducing a few ~1 of air into the NMR tube with a Hamilton syringe through serum caps. The NMR spectra were obtained using a Bruker CXP-300 spectrometer equipped with an Aspect 2000 computer. The two-dimensional NMR spectra were recorded with the pulse sequence: ~90”-t~-90”-~,-90”-~~,I, (13), where 7, is the mixing time and tl and t2 are, respectively, the evolution period and the observation period. The measurement was repeated for a set of 128 equidistant t, values, with 5 12 points accumulated in t2. To improve the signal to noise ratio, 384 transients were accumulated for each value of tl . The mixing time was 50 ms and full phase cycling was used to eliminate experimental artifacts (14). Prior to Fourier transformation, the time-domain data matrix was multiplied in both dimensions with a sine-bell function. This affects the two dimensions differently because of the different acquisition times. The temperature was kept at 17 f 0.5”C, and 2,2dimethyl-2-silapentane-5-sulfonate was used as an internal standard. For Desuljixibrio gigus cytochrome c3, the Tr’s of the heme methyls are in the range of 25 to 80 ms in the fully oxidized form of the protein at 290 K, and the exchange lifetimes determined using saturation transfer techniques (12) are of the order of 100 ms. The section of the 2D NMR spectrum shown in Fig. 1 was obtained at an intermediate oxidation stage and shows several pairs of peaks linked by exchange cross-peaks which have been confirmed by selective experiments. The resonances are due to the heme methyl groups in species belonging to either Step II or III and the spectrum allows cross-assignment of the resonances connected by electron exchange between these steps (sites A, B). The cross-assignments in the region below 17 ppm are reported now for the first time. Measurements in a later oxidation stage (Steps III and IV) also confirm selective results. Some heme methyl resonances are assigned on the spectrum as Mf according to the nomenclature previously used (8). A@ is the resonance due to heme methyl group i in an oxidation Step k. The advantage of 2D exchange is that selectivity is not required and a single experiment may provide the information of many saturation-transfer experiments, which corresponds to a considerable sensitivity advantage in complex systems. A
179
COMMUNICATIONS
30
25
20
15 ppm
I%. 1. A contour plot of a section of the 2D exchange spectrum CXP-300 spectrometer from a 0.5 ml sample of 4.5 mM solution of 290 K. The signal was averaged over six 64-step phase cycles with increments in the pulse spacing, giving spectral widths of 18.5 kHz T,, was fixed at 50 ms. In this intermediate oxidation stage, each resonance frequency of the protons of a particular heme methyl group (Step II or Step III).
obtained
overnight
using
a Bruker q at 5 12 points acquired for each of 128 in each dimension. The mixing time, pair of diagonal peaks identifies the when two or three hemes are oxidized
Desuljbvibrio gigus cytochrome
disadvantage is that the mixing time 7, must serve a range of exchange rates and T1’s and the necessary compromise imposes some limitations on sensitivity. Also, a complex scheme of phase cycling is required to eliminate artifacts from the 2D exchange or 2D NOE experiments (14). If the signal is to be time averaged over several complete phase cycles, then it may be advantageous to use a variety of mixing times. These experiments are generally more difficult than coherent autocorrelation experiments (IS). The experiment shows particular advantages over selective saturation transfer for the assignment of peaks in crowded regions of the spectrum (cross sections are then particularly useful); also it is normally possible to observe exchange in both directions by 2D but this is not usual with saturation transfer. In fact, the exchange cross-peaks corresponding to magnetization transfer between two sites A, B, are of equal intensity for the forward and reverse reactions, though the relative peak heights may be modified by using different lineshape transformations in the two dimensions. Curiously, although some peaks on the diagonal (for instance My) have intensities smaller than the lowest contour shown, the exchange peaks are still visible (see Fig. 1). When 7, is optimized,
180
COMMUNICATIONS
the exchange peak intensity will exceed that of the diagonal peak at the frequency of resonance A, if the exchange rate for that species, kA > kB > RB, where RB is the relaxation rate at the second site (16); however, it cannot exceed the intensity of the signal from the less abundant species obtained in an ordinary spectrum. Curvature of the base plane caused by the large intensity in the center of the spectrum necessitated strong line narrowing in order to obtain a useful contour plot since the somewhat primitive software allows only absolute value spectra. However, a much greater signal to noise ratio can be obtained from the same data by using matched filters and examining cross sections to determine line frequencies. In conclusion, we find that the 2D experiment can easily be set up for an overnight run and appears to be a more efficient method for identifying the resonance frequencies of exchanging species than a fully automated series of saturation-transfer experiments. With the improved 2D facilities of modem spectrometers, such experiments should be extremely useful in studies of electron-transfer proteins. REFERENCES 1. S. WHERLAND AND H. B. GRAY, in “Biological Aspects of Inorganic Chemistry” (A. W. Addison, W. R. Gullen, and B. R. James, Eds.) Wiley, New York, 1977. 2. H. D. PECK, JR., AND J. LEGALL, Philos. Trans. Roy. Sot. London Ser. B 298, 443 (1982). 3. R. HASER, M. PIERROT, M. FREY, F. PAYAN, J. P. ASTIER, M. BRUSCHI, AND J. LEGALL, Nature (London) 282, 806 (1979). 4. Y. HIGUSHI, S. BANDO, M. KUSUNOKI, Y. MATSUURA, N. YASUOKA, M. KAKUDO, T. YAMANAKA, T. YAGI, AND H. INOKUCHI, J. Biol. Chem. 89, 1659 (1981). 5. C. C. MCDONALD, W. D. PHILLIPS, AND J. LEGALL, Biochemistry 13, 1952 (1974). 6. C. M. D~BSON, N. J. HOYLE, D. F. GERALDES, M. BRUSCHI, J. LEGALL, P. E. WRIGHT, AND R. J. P. WILLIAMS, Nature (London) 249, 425 (1974). 7. J. J. G. MOURA, H. SANTOS, I. MOURA, J. LEGALL, G. R. MOORE, R. J. P. WILLIAMS, AND A. V. XAVIER, Eur. J. Biochem. 127, 15 1 (1982). 8. H. SANTOS, J. J. G. MOURA, I. MOURA, A. V. XAVIER, AND J. LEGALL, Eur. J. Biochem., in press. 9. D. V. DER~ARTANIAN, A. V. XAVIER, J. LEGALL, Biochimie (Paris) 60, 315 (1978). 10. A. V. XAVIER, J. J. G. MOURA, J. LEGALL, AND D. V. DER~ARTANIAN, Biochimie (Paris) 61, 689 ( 1979). II. K. NIKI, T. YAGI, AND H. INOKUCHI, in “Advances in Chemistry Series,” No. 201, “Electrochemical and Spectrochemical Studies of Biological Redox Components” (K. M. Kadish, Ed.) (1982). 12. S. FOR&N AND R. A. HOFFMAN, J. Chem. Phys. 39,2892 (1963). 13. J. JEENER, B. H. MEIER, P. BACHMANN, AND R. R. ERNST, J. Chem. Phys. 71,4546 (1979). 14. H. SANTOS, D. L. TURNER, AND A. V. XAVIER, J. Magn. Reson. 55,463 (1983). 15. W. AUE, E. BARTHOLDI, AND R. R. ERNST, J. Chem. Phys. 64,2229 (1976). 16. D. L. TURNER, Mol. Phys., submitted for publication.
OF MAGNETIC
RESONANCE
Two-Dimensional
59, 177-l 80 (1984)
NMR Studies of Electron Transfer in Cytochrome c3*
HELENA SANTOS,DAVID L. TURNER, ANDANT~NIO
V-XAVIER
CentrO de Quimica Estrutural, Complexo I. IST, Av. Rovisco Pais, 1000 Lisbon, Portugal AND
JEANLE GALL Department of Biochemistry, University of Georgia, Athens, Georgia 30602 Received March 27, 1984
The biological role of electron-transfer proteins justifies the amount of work devoted to the understanding of their mechanism of action (I). Sulfate-reducing bacteria provide suitable systems for the investigation of the electron-transfer mechanisms occurring in vivo (2); in particular, cytochrome c3 (MW 13,000) one of the components of the electron transfer chain of the Desulfovibrio bacteria is an attractive candidate for these studies since a considerable amount of information on this protein is now available (J-11). This information has been obtained using a wide range of techniques that includes X-ray (3, 4), NMR (58), EPR (9, lo), and electrochemical methods (I I). Also, both intramolecular and intermolecular electron transfer mechanisms may be probed since each molecule contains four redox centers. Proton NMR proved to be a suitable technique to provide specific information about each redox center at each of the five oxidation steps obtained from the fully reduced state by successive losses of one electron, according to the following scheme (7, 8): -If?
-le
-It?
-1e
Step 0 P Step I P Step II Z Step III P Step IV The steps are numbered 0, I, II, III, and IV according to the number of oxidized hemes in any one molecule. For Desulfovibrio gigas cytochrome c3 the intramolecular electron exchange rate is fast on the NMR time scale (> lo5 s-r), but the intermolecular electron exchange is slow at 273 K and protein concentrations of the order of 3 mA4 (8). Under these conditions, the fast intramolecular electron exchange implies that each heme methyl group originates only one resonance in the NMR spectrum for each oxidation step, its chemical shift depending on the ratio between the molar fractions of the states in * Presented at the FNRS NMR Symposium, Brussels, December 1983. 177
0022-2364184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
178
COMMUNICATIONS
which the heme considered is oxidized and the states in which the same heme is reduced. A full characterization of the heme methyl group resonances in the different oxidation steps is important for the understanding of the intramolecular and intermolecular electron transfer mechanisms and this has been partly achieved (7, B), using several series of selective saturation transfer experiments (12) at different solution redox potentials. However, this is a time consuming task since 16 heme methyl groups are present, each one giving rise to five resonances in a complete oxidation/reduction cycle. Also, selective irradiation is required and this is difficult to achieve in the crowded regions of the spectrum. Therefore, the analogous two-dimensional experiment (13) should be more efficient for the study of this complex system. The present article reports for the first time the application of 2D exchange NMR to obtain the crossassignments of the resonances in a multisite electron transfer system. The solution of Desulfovibrio gigus cytochrome q was prepared as reported elsewhere (7, 8). The protein concentration was 4.5 mM and the pH was adjusted to 8.1 with NaOD. A few crystals of sodium dithionite were used as reducing agent and the intermediate oxidation stages were obtained by introducing a few ~1 of air into the NMR tube with a Hamilton syringe through serum caps. The NMR spectra were obtained using a Bruker CXP-300 spectrometer equipped with an Aspect 2000 computer. The two-dimensional NMR spectra were recorded with the pulse sequence: ~90”-t~-90”-~,-90”-~~,I, (13), where 7, is the mixing time and tl and t2 are, respectively, the evolution period and the observation period. The measurement was repeated for a set of 128 equidistant t, values, with 5 12 points accumulated in t2. To improve the signal to noise ratio, 384 transients were accumulated for each value of tl . The mixing time was 50 ms and full phase cycling was used to eliminate experimental artifacts (14). Prior to Fourier transformation, the time-domain data matrix was multiplied in both dimensions with a sine-bell function. This affects the two dimensions differently because of the different acquisition times. The temperature was kept at 17 f 0.5”C, and 2,2dimethyl-2-silapentane-5-sulfonate was used as an internal standard. For Desuljixibrio gigus cytochrome c3, the Tr’s of the heme methyls are in the range of 25 to 80 ms in the fully oxidized form of the protein at 290 K, and the exchange lifetimes determined using saturation transfer techniques (12) are of the order of 100 ms. The section of the 2D NMR spectrum shown in Fig. 1 was obtained at an intermediate oxidation stage and shows several pairs of peaks linked by exchange cross-peaks which have been confirmed by selective experiments. The resonances are due to the heme methyl groups in species belonging to either Step II or III and the spectrum allows cross-assignment of the resonances connected by electron exchange between these steps (sites A, B). The cross-assignments in the region below 17 ppm are reported now for the first time. Measurements in a later oxidation stage (Steps III and IV) also confirm selective results. Some heme methyl resonances are assigned on the spectrum as Mf according to the nomenclature previously used (8). A@ is the resonance due to heme methyl group i in an oxidation Step k. The advantage of 2D exchange is that selectivity is not required and a single experiment may provide the information of many saturation-transfer experiments, which corresponds to a considerable sensitivity advantage in complex systems. A
179
COMMUNICATIONS
30
25
20
15 ppm
I%. 1. A contour plot of a section of the 2D exchange spectrum CXP-300 spectrometer from a 0.5 ml sample of 4.5 mM solution of 290 K. The signal was averaged over six 64-step phase cycles with increments in the pulse spacing, giving spectral widths of 18.5 kHz T,, was fixed at 50 ms. In this intermediate oxidation stage, each resonance frequency of the protons of a particular heme methyl group (Step II or Step III).
obtained
overnight
using
a Bruker q at 5 12 points acquired for each of 128 in each dimension. The mixing time, pair of diagonal peaks identifies the when two or three hemes are oxidized
Desuljbvibrio gigus cytochrome
disadvantage is that the mixing time 7, must serve a range of exchange rates and T1’s and the necessary compromise imposes some limitations on sensitivity. Also, a complex scheme of phase cycling is required to eliminate artifacts from the 2D exchange or 2D NOE experiments (14). If the signal is to be time averaged over several complete phase cycles, then it may be advantageous to use a variety of mixing times. These experiments are generally more difficult than coherent autocorrelation experiments (IS). The experiment shows particular advantages over selective saturation transfer for the assignment of peaks in crowded regions of the spectrum (cross sections are then particularly useful); also it is normally possible to observe exchange in both directions by 2D but this is not usual with saturation transfer. In fact, the exchange cross-peaks corresponding to magnetization transfer between two sites A, B, are of equal intensity for the forward and reverse reactions, though the relative peak heights may be modified by using different lineshape transformations in the two dimensions. Curiously, although some peaks on the diagonal (for instance My) have intensities smaller than the lowest contour shown, the exchange peaks are still visible (see Fig. 1). When 7, is optimized,
180
COMMUNICATIONS
the exchange peak intensity will exceed that of the diagonal peak at the frequency of resonance A, if the exchange rate for that species, kA > kB > RB, where RB is the relaxation rate at the second site (16); however, it cannot exceed the intensity of the signal from the less abundant species obtained in an ordinary spectrum. Curvature of the base plane caused by the large intensity in the center of the spectrum necessitated strong line narrowing in order to obtain a useful contour plot since the somewhat primitive software allows only absolute value spectra. However, a much greater signal to noise ratio can be obtained from the same data by using matched filters and examining cross sections to determine line frequencies. In conclusion, we find that the 2D experiment can easily be set up for an overnight run and appears to be a more efficient method for identifying the resonance frequencies of exchanging species than a fully automated series of saturation-transfer experiments. With the improved 2D facilities of modem spectrometers, such experiments should be extremely useful in studies of electron-transfer proteins. REFERENCES 1. S. WHERLAND AND H. B. GRAY, in “Biological Aspects of Inorganic Chemistry” (A. W. Addison, W. R. Gullen, and B. R. James, Eds.) Wiley, New York, 1977. 2. H. D. PECK, JR., AND J. LEGALL, Philos. Trans. Roy. Sot. London Ser. B 298, 443 (1982). 3. R. HASER, M. PIERROT, M. FREY, F. PAYAN, J. P. ASTIER, M. BRUSCHI, AND J. LEGALL, Nature (London) 282, 806 (1979). 4. Y. HIGUSHI, S. BANDO, M. KUSUNOKI, Y. MATSUURA, N. YASUOKA, M. KAKUDO, T. YAMANAKA, T. YAGI, AND H. INOKUCHI, J. Biol. Chem. 89, 1659 (1981). 5. C. C. MCDONALD, W. D. PHILLIPS, AND J. LEGALL, Biochemistry 13, 1952 (1974). 6. C. M. D~BSON, N. J. HOYLE, D. F. GERALDES, M. BRUSCHI, J. LEGALL, P. E. WRIGHT, AND R. J. P. WILLIAMS, Nature (London) 249, 425 (1974). 7. J. J. G. MOURA, H. SANTOS, I. MOURA, J. LEGALL, G. R. MOORE, R. J. P. WILLIAMS, AND A. V. XAVIER, Eur. J. Biochem. 127, 15 1 (1982). 8. H. SANTOS, J. J. G. MOURA, I. MOURA, A. V. XAVIER, AND J. LEGALL, Eur. J. Biochem., in press. 9. D. V. DER~ARTANIAN, A. V. XAVIER, J. LEGALL, Biochimie (Paris) 60, 315 (1978). 10. A. V. XAVIER, J. J. G. MOURA, J. LEGALL, AND D. V. DER~ARTANIAN, Biochimie (Paris) 61, 689 ( 1979). II. K. NIKI, T. YAGI, AND H. INOKUCHI, in “Advances in Chemistry Series,” No. 201, “Electrochemical and Spectrochemical Studies of Biological Redox Components” (K. M. Kadish, Ed.) (1982). 12. S. FOR&N AND R. A. HOFFMAN, J. Chem. Phys. 39,2892 (1963). 13. J. JEENER, B. H. MEIER, P. BACHMANN, AND R. R. ERNST, J. Chem. Phys. 71,4546 (1979). 14. H. SANTOS, D. L. TURNER, AND A. V. XAVIER, J. Magn. Reson. 55,463 (1983). 15. W. AUE, E. BARTHOLDI, AND R. R. ERNST, J. Chem. Phys. 64,2229 (1976). 16. D. L. TURNER, Mol. Phys., submitted for publication.