3942
Inorg. Chem. 1995, 34, 3942-3952
Sequence-Specific lH,13C, and 1 5 N NMR Assignments for Oxidized Ferredoxin from Clostridium pasteuriunum Sergio D. B. Scrofani,*" Robert T. C. Brownlee: Maruse Sadek,**' and Anthony G. Weddt School of Chemistry, University of Melbourne, Parkville, Victoria 3052, Australia, and School of Chemistry, La Trobe University, Bundoora 3083, Australia Received February 3, I995@
Sequence-specific I3C and I5N NMR assignments for the oxidized form of the 2[Fe4S4I2+ ferredoxin from Clostridium pasteurianum are presented. Partial or complete sequence-specific 'H NMR assignments for 52 of the 55 amino acid residues are also derived, extending those previously identified in this paramagnetic protein (Gaillard J.; Moulis, J.-M.; Kiimmerle, R.; Meyer, J. Magn. Reson. Chem. 1993,31, S27). Protons which remain unassigned are in regions close to the paramagnetic clusters. The 'H resonances for all but nine non-cysteinyl amide groups have been assigned specifically. Those which have not been assigned are all likely NH. * O Sdonors to either a cluster sulfide or cysteinyl S Y . Eight of the nine are symmetry-related (V9II38, G12lG41, A13/N42 and A22lA5 1). Eight paramagnetically shifted non-proline I5N resonances (140-170 ppm) exhibit a pairwise arrangement in two-dimensional I5N acquired INEPTexperiments. The NH functions of symmetry-relatedresidues Cys 47 and Cys 18 have been assigned specifically. Assignments for the NH functions for symmetry-related residues Cys 14 and Cys 43 are made on the basis of their close proximity to the paramagnetic centers.
Introduction At first sight, paramagnetic metalloproteins do not appear to be suitable candidates for structural determination by NMR' methods, since the effects of paramagnetically-induced relaxation for nuclei close to paramagnetic centers cannot be predicted easily.2 Although recent 'H NMR studies on ironsulfur proteins have been very useful in defining domains wellremoved from these center^,^-^ information on regions close to the metal sites has been difficult to obtain. This is due to the increased proton relaxation rates induced by the paramagnetic centers. For similar reasons, NOE information in these regions is greatly attenuatede2 Studies of oxidized plant ferredoxins failed to detect resonances of protons sited less than 7.8 8, from an iron atom of the [Fe$32]2+.5,6,8In contrast, many of the backbone amide proton resonances located less than 6.5
' University of Melbourne. La Trobe University. @Abstractpublished in Advance ACS Abstracts, June 15, 1995. (1) Abbreviations: CauFd, ferredoxin from Clostridium acidi-urici; Cp, Clostridium pasteurianum; CpFd, ferredoxin from C. pasteurianum; CSI, chemical shift index; DQ-COSY, double-quantum correlation spectroscopy; DQF-COSY, double-quantum filtered correlation spectroscopy; DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate; FeS, iron-sulfur; HiPIP, high potential iron protein; HMQC, heteronuclear multiple-quantum coherence; HSQC, heteronuclear singlequantum coherence; INEPT, insensitive nuclei enhanced by polarization transfer; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser spectroscopy; PaFd, ferredoxin from Peptococcus aerogenes; ROESY, rotating frame Overhauser spectroscopy; TOCSY, total correlation spectroscopy; TPPI, time proportional phase incrementation. (2) La Mar, G. N.; de Ropp, J. S. In Biological Magnetic Resonance, Volume 12: NMR of Paramagnetic Molecules; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1993. (3) Oh, B.-H.; Markley, J. L. Biochemistry 1990, 29, 3993. (4) Oh, B.-H.; Markley, J. L. Biochemistry 1990, 29, 4004. ( 5 ) Oh, B.-H.; Markley, J. L. Biochemistry 1990, 29, 4012. (6) Ye, X. M.; Pochapsky, T. C.: Pochapsky, S. S. Biochemistry 1992, 31, 1961. (7) Gaillard, J.; Albrand, J.-P. Moulis, J.-M.; Wemmer, D. E. Biochemistry 1992, 31. 5632. (8) Chae, Y. K.; Abildgaard, F.: Moobem. E. S.: Marklev. J. L. Biochemistry 1994, 313, 3278. (9) Teng, Q.; Zhour, 2. H.; Smith, E. T.; Busse, S. C.; Howard, J. B.; Adams, M. W. W.; La Mar, G. N. Biochemistry 1994, 33, 6316.
0020- 166919511334-3942$09.00/0
hl from an iron atom in the single [Fe4S4I2' cluster of reduced HiPIPs are identified.7$'0This difference in distance threshold is apparently controlled by variations in the relaxation processes induced by the two different clusters. There are two [Fe4S4I2+ clusters in oxidized ferredoxin from Clostridiumpasteurianum (CpFd), complicating the situation further. The residual paramagnetism present at room temperature beff 2 p~ per cluster") is responsible for dramatic differences in chemical shifts and for increased line widths for resonances of nuclei close to paramagnetic centers compared to values observed in diamagnetic proteins. 2,1 The iron atoms in each of the two Fe-S clusters in CpFd are linked to the protein backbone through cysteinyl thiolate ligands (Figure l).I43l5 Together with orientation of peptide amide groups and access to solvent water, NH..*S hydrogen bonding involving the sulfur atoms of the Fe-S clusters and their cysteinyl ligands modulate both redox potentials and electron transfer pathways in such proteins.I5-' Our aim is to define the structure of CpFd using NMR methods to assist identification and study of sites involved in electron transfer with redox partners. We detail here a multinuclear NMR study of CpFd employing both native and I5N labeled protein. Studies of the vegetative ferredoxin from Anabaena 7120 have revealed resonances with shifts of up to 310 ppm in
-
(10) Bertini, I.; Felli, I. C.; Kastrau, D. H. W.; Luchinat, C.: Piccioli, M.
Eur. J. Biochem. 1994, 225, 703. (11) Gaillard, J.; Moulis, J.-M.; Meyer, J. Znorg. Chem. 1987, 26, 320. (12) Bertini, I.; Capozzi, F.; Luchinat, C.; Piccioli, M.; Vila. A. J. J. Am. Chem. SOC. 1994, 116, 651. (13) Scrofani, S. D. B.; Brereton, P. S.; Hamer, A. M.; Lavery, M. J.; McDowell, S. G.; Vincent, G. A.; Brownlee, R. T. C.: Hoogenraad, N. J.; Sadek, M.; Wedd, A. G. Biochemistry 1994, 33, 14486. (14) Adman. E. T.; Sieker, L. C.; Jensen, L. H. J. Biol Chem. 1973, 248, 3987. (15) Backes, G.; Mino, Y.; Loehr, T. M.; Meyer, T. E.; Cusanovich, M. A.; Sweeney, W. V.; Adman, E. T.: Sanders-Loehr. J. J. Am. Chem. SOC. 1991, 113. 2055. (16) Jensen, G. M.; Warshel, A.; Stephens, P. J. Biochemistry 1994, 33, 10911. (17) Ueyama, N.; Okamura, T.; Nakamura, A. J. Chem. Soc., Chem. Commun. 1992, 1019.
0 1995 American Chemical Society
Inorganic Chemistry, Vol. 34, No. 15, 1995 3943
NMR of Oxidized Ferredoxin from C. pasteurianum
Experimental Section
25
10 V
tv3
Figure 1. Simple two-dimensionalrepresentation of CpFd. NMR data predicts that the shaded residues participate in B-sheet structure. The crystal structure of PaFdlSpredicts that residues outlined in bold will be likely NH hydrogen bond donors to cluster sulfides, while circles with horizontal lines represent residues which are NH* *S donors to cysteinyl thiolates. Although not discussed ~pecifically,~~ Cys 11 and Cys 40 are likely NH* *S donors to cluster sulfides.
'H-decoupled 15N spectra, with line widths greater than 400 H z . ~ However, due to its lower magnetogyric ratio, such paramagnetic effects are not as dramatic for 15Nas they are for 'H or 13Cnuclei in similar environments.18 In the present work, the advantages of acquiring NMR data through 15Nnuclei has been exploited, allowing all NH functions in CpFd to be identified. A preliminary report has been comm~nicated.'~ Specifically designed mutant forms of CpFd have been prepared to assist in identification of regions closer to the paramagnetic centers.13 In addition, a structural model of CpFd13 has been derived from the crystal structure of the 2[Fe4S4I2+ferredoxin from Peptococcus aerogenes (PaFd; 70% hom010gy)~5to assist interpretation of the structural information obtained from NMR results. Sequence-specific 'H NMR assignments of CpFd are presented here which confirm and extend assignments presented by Gaillard and co-workers.20A comprehensive list of 3 J ~ vicinal ~ a coupling constants, amide proton exchange rates, and observed sequential and long-range NOE connectivities are described. These results allow development of a more detailed view of the secondary structure of CpFd. (18) Bloembergen, N.; Morgan, L. 0. J. Chem. Phys. 1961, 34, 842. (19) Sadek, M.; Scrofani, S. D. B.; Brownlee, R. T. C.;Wedd, A. G. J. Chem. SOC., Chem. Commun. 1995, 1, 105. (20) Gaillard, J.; Moulis, J.-M.; Kummerle, R.; Meyer, Mugn. Reson. Chem. 1993, 31, S27.
Bacterial Growth and Isolation. Growth of native C. pasteuriunum (Cp) was initiated by inoculating 20 mL cultures containing dehydrated minced beef (5 g), Schaedler's anaerobic broth (25.6 g L-I, Oxoid Co.) and glucose (5 g L-I), with a single colony from a freshly streaked plate. The seed colony was grown until late log phase at 37 "C and subsequently used to inoculate a 16 L culture2*purged with high purity (1.5 g L-I) was the argon gas. For I5N labeled cultures, (15NH4)2S04 sole I5N source and the CaCO3 content was doubled (2 g L-'). All cultures were harvested at an ODm of approximately 3, yielding about 110 g of wet cells. The isolation of wild-type CpFd has been described previously?1 Preparation of recombinant and Gly to Ala mutant forms of CpFd are as described previou~ly.~~ Preparation of the Asp 39 to Asn (D39N) mutant will be described elsewhere?2 Deuteration of protein samples was performed by first exchangingbuffer in an Amicon ultrafiltration cell equippedwith a YM3 membrane, and then an Amicon Centricon 3 microconcentrator. All samples were degassed and sealed under an atmosphere of dinitrogen gas. N M R Experimental Data. NMR spectra were recorded on oxidized CpFd dissolved in 100% 2H20or 90% IH20/10% 2H20(3-7 mM; 50 mM phosphate buffer, pH 6.2-7.8). Unless otherwise specified, all spectra were recorded at 298 K. All 'H NMR experiments were recorded on a Bruker AM 400NJ3 spectrometerequippedwith an ASPECT 3000 computer at 400.13 MHz. Tailored acquisition parameters were used to remove base line distortion^.^^ For 1D spectra, a sweep width of 11905 Hz was used to prevent spectral folding. The FID was collected with 8192 real data points resulting in an acquisition time of 0.69 s. All homonuclear 2D spectra were acquired in the phase-sensitive mode, with sequential acquisition in the ~ 0 dimension, 2 and quadrature detection in the 01 dimension achieved by TPPI." 'H DQF-COSY, TQF-COSY,25 ROESY,26 TOCSY," and NOESv8 spectra were generally recorded with spectral widths of 4950 Hz in both dimensions. This increased digital resolution in the diamagnetic spectral region at the expense of folding in the paramagnetic region. 'H DQ-COSY experimentswere carried out at three different DQ preparation periods (t= 30,60, and 90 ms) to maximize excitation for geminal (-14 Hz), vicinal (-7 Hz) and smaller couplings (-3 Hz). All 2D experiments were recorded with 384-768 01 increments of 2048 real data points. Low power irradiation of residual water was normally required. H-D exchange studies were carried out by exchanging IH20 with 2H20. Usually, 3-4 h was required to ensure complete solvent exchange, replacing all fast exchanging pr0tons.2~As a result, only nonlabile and slowly exchanging protons are observed in all spectra recorded and are referenced to the initially recorded spectrum (taken as t = 0 min). The H-D exchanged CpFd sample was characterized by a series of 1D and 2D spectra recorded over a period of 48 h. For NOESY (50-250 ms) spectra, a 180" composite pulse placed in the middle of the mixing time was employed to prevent further relaxation recovery of the residual water peak.30 Zero quantum coherences were not suppressed. TOCSY (40-80 ms) spectra were acquired in reverse mode using the MLEV- 17 mixing scheme," flanked by 500 ps trim pulses. ROESY spectra were recorded at 50 and 100 ms for both 'H20 and 2H20 samples and were obtained in Bruker Rabinowitz, J. C . Methods Enzymol. 1972,24,431. Brereton, P. S.;Sadek, M.; Scrofani, S. D. B.; Brownlee, R. T. C.; Wedd, A. G. 1995, Manuscript in preparation. Sadek, M.; Brownlee, R. T. C.;Scrofani, S. D. B.; Wedd, A. G. J. Magn. Reson., Ser. B 1993, 101, 309. Marion, D.; Wuthrich, K. Biochem Biophys. Res. Commun. 1983,113, 967. Rance, M.; Wrensen, 0. W.; Bodenhausen, G.; Wagner, G.; Emst, R. R.; Wuthrich, K. Biochem. Biophys. Res. Commun. 1983, I 17,458. Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 63, 207. Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355. Macura, S.; Emst, R. R. Mol. Phys. 1980, 41, 95. Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley-Interscience Publications: New York, 1986. Brown, S. C.;Weber, P. L.; Mueller, L. J. Magn. Reson. 1988, 77, 166.
3944 Inorganic Chemistry, Vol. 34, No. 15, I995
Scrofani et al.
I3c (PPm)
c
- 40.0
1 1
- 44.0
4
I
5.0
I
I
I
4.5
4.0
3.5
I
'H (ppm) Figure 2. Partial 400-MHz {'H-I3C}-HMQC
spectrum of CpFd showing the (CH)I region of the four Gly spin systems.
reverse mode with mixing achieved using small flip angle pulses of 20" and an effective spin-locking field of 3 ~ H z . ~ ~ ('H-I3C}-HMQC spectra were recorded at natural abundance as described previo~sly.'~All I5N experiments were recorded on I5N labeled CpFd. Two-dimensional { 15N- 'H} INEPT experiments were acquired as described previo~s1y.l~A {1H-15N}-HMQC-NOESY spectrum was recorded on a Bruker AMX.500 spectrometer at the Biomolecular Research Institute, Melboume, Australia, in the phasesensitive mode with simultaneous acquisition in c.02.'~ Spectral widths of 6024 and 5069 Hz were used in the 'H and I5N dimensions, respectively and an evolution period of 3 ms was utilized. A total of 4096 complex data points were collected with 512 r l increments and 96 transients per increment. The water signal was suppressed by low power irradiation for 100 ms during the relaxation delay and the mixing time. One dimensional I5N spectra were recorded with gated decoupling to minimize NOE effects. Data were processed on a SUN SPARCstation-2 using FELIX software (1.1) from Hare Research Inc. For spectral analysis, zero filling to 2048 real points in W I was used. Windows were optimized iteratively for each 2D spectrum, however 60-80" shifted square sine bells were generally used in both dimensions, zero filled to form 2K x 2K real matrices. Proton chemical shifts were referenced to the IHzO signal at 4.78 ppm (298 K), and I3C chemical shifts were
referenced indirectly to DSS in IHzO. I5N resonances were referenced ~ a coupling indirectly to I5NH4C1 (24.93 ppm, 2.9 M). 3 J ~ vicinal constants were measured by the method of Kim and P r e ~ t e g a r d . ~ ~
Results Methods for Detection and Assignment of Resonances. The large variation in relaxation rates and hence line widths in this protein requires a much broader range of experiments than is normal for diamagnetic proteins. As well as the normal suite of 2D NMR experiments (DQF-COSY, TOCSY and NOESY) used to study low molecular weight proteins, a series of heteronuclear (HMQC and INEPT)and less commonly used 'H COSY-type (DQ-COSY and TQF-COSY) experiments have been utilized. In addition, TOCSY23 and NOESY34 experiments, embodying shorter evolution times have been used to identify many of the faster relaxing nuclei present in CpFd. Dispersion of broad and sharp resonances in crowded regions has resulted from the use of both directly and indirectly detected heteronuclear experiments. The {IH- 13C}-HMQCexperiment has been very effective in detecting a large number of paramagnetically affected resonances at natural 13Cabundance. For example, {'H-I3C}-HMQC experiments have been used to identify almost all cysteinyl (CH)a and (CHY functions in clostridial ferredoxin^.'^,'^ This technique also readily identifies the four Gly spin systems present in CpFd (Figure 2) and shows that two of the four systems are broadened significantly, most likely due to paramagnetic effects (vide infra). I5N detected NMR experiments have been utilized to identify all I5N resonances in CpFd.lg The I5N detected 2D INEPT experiment has been found to be useful for identification of 15N and, consequently, 'H resonances of NH functions both remote from and close to the paramagnetic centers (Figure 3). In contrast, 'H detected heteronuclear 2D NMR experiments (HMQC and HSQC) acquired under a variety of conditions have failed to detect a number of key NH groups.I9
P19,48 1
I
I
1
170
160
P52
1
I
1
I
I
150
I40
130
120
110
I
@pm) Figure 3. 400-MHz {'5N-1H}-2D INEPT spectrum of CpFd. The 'H decoupled 1D spectrum is also shown. The asterisk denotes an electronic spike.
NMR of Oxidized Ferredoxin from C. pasteurianum In the present application, the DQ-COSY35,36 experiment has been found to be a powerful tool for the identification of spin systems. One of the significant advantages of this technique over other COSY-type experiments is the unambiguous assignment of HN-Ha cross peaks. Water presaturation effects do not interfere with detection of coupling between those Ha resonances located beneath or near the water resonance and their corresponding backbone amide protons.37 In addition, remote and combination peaks allow unambiguous identification of spin systems, including those of magnetically equivalent s p e ~ i e s . ~Seven ~ , ~ ~of, the ~ ~eight Ala spin systems have been identified in DQ-COSY experiments by noting the presence of a remote peak indicative of the magnetic equivalence of (CH@ protons. The TQF-COSY experiment in 2H20 is useful in helping to identify AMX-type residues, and residues with long side chains. For example, in CpFd, three of the four GldGlu spin systems exhibit unusually large separation between HBsT resonances, attributable to paramagnetic shifting and/or aromatic ring current effects. Spin systems were identified using the strategy developed by Chazin and Wright.@ Backbone amide and terminal side chain proton resonances are used as initial points of assignment. The former feature a larger chemical shift dispersion compared to Ha resonances, are relatively isolated from other resonances (6- 10 ppm), and are sensitive to pH and temperature changes. This fact can then be exploited to resolve overlapped spin systems. When no backbone amide proton resonance is observed, spin systems can normally be assigned to residue types through their characteristic resonance p a t t e m ~ . ~ ~ CpFd contains 55 amino acids (Figure l), eight of which are cysteinyl ligands to the two Fe-S clusters. A total of 28 residues possess unique spin topology, (four Gly, eight Ala, one Thr, six Val, five Ile, one Lys, and three Pro), while there are 15 non-cysteinyl AMX-type spin systems (five Ser, five Asp, three Asn, one Tyr, one Phe). In addition, four nonunique longer-side chain residues are also present (two Glu, two Gln). Spin system assignments are presented in Table 1. Sequencespecific assignment of the cysteinyl Ha and HB resonances has been described e l ~ e w h e r e . ' ~ .Systems '~ which could not be identified unambiguously due to a lack of connectivity are listed in Table 2. Assessment of the Amide Fingerprint Region. 'H DQCOSY and TOCSY spectra acquired in 'H20 under a variety of conditions show the presence of about 80% of the expected H N to Ha intraresidue connectivities. In contrast, onedimensional I5N 'H-decoupled spectra of I5N labeled CpFd detect all 61 possible IsN resonances: 54 backbone, one Lys N*, one N-terminal, and five carboxamide resonances. Twodimensional I5N detected INEPTexperiments allow the corresponding 'H amide chemical shifts associated with each IsN resonance to be identified (Figure 3). All but one of the anticipated IsN-'H cross peaks have been detected.I9 Sequence-Specific Resonance Assignments. The following section describes the sequential and long-range assignments made in CpFd using homonuclear and heteronuclear NOESY experiments. ROESY experiments were used to support these assignments and also to identify labile protons exchanging with solvent water. The unique Lys and Thr residues at positions 3 and 36, respectively (Figure 1) provide convenient sequence markers since they are on opposite sides of the m01ecule.I~The amide fingerprint region of a 100 ms NOESY spect" is shown in Figure 4, and sequential assignments are identified in Figure 5 . Long-range NOE connectivities are presented in Figure 6. Tyr 2 to Ser 7 Segment. A series of daN(i, i 1) connec-
+
Inorganic Chemistry, Vol. 34, No. 15, 1995 3945 tivities are observed for the segment Tyr 2 to Ser 7. In addition, a number of NOE connectivities exist between Lys 3 side chain protons. A weak d"(i, i 1) cross peak is observed between Asp 6 and Ser 7, and Ala 5 HB resonance exhibits dpN connectivity with Ser 7 and a Gly spin system. Comparison with the CpFd structural model suggests that the most likely candidate for the latter is Gly 50. Ala 15 to Glu 17 Segment. Spin systems Ala 15 and Ser 16 exhibit d&i, i 1) and d m connectivities. This assignment is extended further to Glu 17 through the observation of a d" cross peak with Ser 16. Ile 23 to Thr 36 Segment. Strong daN(i, i 1) and dpN(i, i 1) connectivities are observed for the unique Ser 24-Gln 25-Gly 26 segment. A weaker dyN(i, i -t 1) connectivity is also observed between Gln 25 and Gly 26. A strong d&i, i 1) cross peak involving Sei 24 H N allows the identification of 1) Ile 23. Both a-protons of Gly 26 show d&i, i connectivity with Asp 27 HN. Although not apparent on spectra recorded at room temperature, NOESY spectra recorded at 287 K display d d ( i , i 1) connectivity between Asp 27 and Ser 28. Weak daN(i, i 4- 1) interactions are observed between Ser 28 and Ile 29, as well as strong d" (at 120 ms mixing time) and dpN connectivities. Strong d"(i, i 1) and daN(i, i 1) connectivities are observed for the unique tripeptide segment, Ala 34-Asp 35Thr 36, where Asp 35 also exhibits dNg(i, i - 1) and dgN(i, i 1) interactions in this segment. Specific assignment is extended further with the observation of dau(i, i 1) connectivities for the Ile 32-Asp 33-Ala 34 segment. Also observed is a weak NOE interaction between Asp 33 HB and Ala 34 HN. Ile 32 Ha exhibits dNa(i, i - 1) connectivity with the HN resonance of one of two Val spin systems at 9.17 ppm. Spectra recorded at lower temperatures allow the Val systems to be distinguished and confirm daN(i, i 1) connectivity with Val 31Ha. dNa(i, i - 1) connectivity is also identified between Val 31 HN and Phe 30 Ha. Strong d" connectivity is observed between Val 31 and Ser 24 and Gly 26 HNexhibits NOE connectivities with both Phe 30 Ha and a Val 31 HY resonance. Ala 44 to Val 46 Segment. Medium daN, dpN, and day connectivities allow the unique Asn 45-Val 46 segment to be assigned specifically. Strong dpN connectivity is observed between Asn 45 and an Ala spin system. The PaFd crystal structureI5 suggests that the adjacent residue Ala 44 is the only Ala residue close to Asn 45. The assignment of Ala 44 is confirmed through NOE cross peaks with Tyr 2 H' resonance in native and mutant forms of CpFd (vide infra). NOE cross peaks between the high field Val 46 HyJ' resonances and both HP resonances of Glu 17 are also observed, as is a dNN connectivity between Asn 45 and Glu 17. Pro 52 to Glu 55 Segment. A daN connectivity within a Pro-Val segment is apparent. Although all three Pro residues in CpFd are part of such a sequence, an assignment Pro 52Val 53 is possible as long-range NOE interactions between Val
+
+
+
+
+
+
+
+
+
+
+
+
Kessler, H.; Griesinger, C.; Kerssebaum, R.; Wagner, K.; Emst, R. R. J. Am. Chem. Soc. 1987, 109, 607. States, D. J.; Haberkom, R. A.; Ruben, D. J. J. Magn. Reson. 1982, 48, 286. Kim, Y.; Prestegard, J. H. J. Magn. Reson. 1989, 84, 9. Busse, S. C.; La Mar, G. N.; Howard, J. B. J. Biol. Chem. 1991, 266,
23714. Bax, A.; Freeman, R.; Kempsell, S. P. J. Am. Chem. SOC. 1980, 102, 485 1. Braunschweiler, L.; Bodenhausen, G.; Emst, R. R. Mol. Phys. 1983, 48, 535. Otting, G.; Wuthrich, K. J. Magn. Reson. 1986, 66, 359. Bax, A.; Freeman, R.; Moms, G. J. Magn. Reson. 1981, 42, 164. Mareci, T . H.; Freeman, R. J. Mugn. Reson. 1983, 51, 531. Chazin, W. J.; Wright, P. E. Biopolymers, 1987, 26, 973.
3946 Inorganic Chemistry, Vol. 34, No. 15, 1995
Scrofani et al.
Table 1. Chemical Shifts of Amino Acid Residues for Oxidized Ferredoxin from Clostridium pasreurinnum"
residue Ala 1 Tyr 2 Lys 3 Ile4 Ala 5 Asp6 Ser7 Cys 8 Val 9 Ser 10 cys 11 Gly 12 Ala 13 Cys 14 Ala 15 Ser 16 Glu 17 Cys 18 Pro19 Val 20 Asn 21 Ala 22 Ile 23 Ser 24 Gln 25 Gly 26 Asp 27 Ser28 Ile 29 Phe30 Val31 Ile 32 Asp33 Ala34 Asp35 Thr 36 cys 37 Ile 38 Asp 39 Cys 40 Gly 41 Asn 42 Cys 43 Ala 44 Asn45 Val 46 Cys47 Pro48 Val49 Gly50 Ala 51 Pro52 Val 53 Gln 54 Glu 55
NH
Ha 4.32 5.46 4.78 4.26 4.50 4.38 4.29 3.50 5.30 4.40 10.0 4.29, 3.48
Ca 51.3 55.6 53.0 60.7 49.7
7.40 3.91 4.45 4.40 5.50 5.17 4.89 4.70
92.9 53.1 56.9 52.6
7.89 7.69
166.8/165.2b 135.0 126.9 120.2 142.9 153.0/151.9b 119.8 121.7
7.54 9.07 8.54 7.83 8.54 8.62 7.69 7.07 9.17 8.83 9.11 8.88 8.21 7.33
120.8 123.8 123.2 118.4 137.4 114.5 118.0 133.0 117.8 127.8 129.7 134.2 115.9 114.0
5.08 4.66 2.93 4.45, 3.64 4.37 4.39 4.60 4.95 4.50 4.69 4.75 3.92 4.52 4.26 3.3 5.13
58.9 54.2 54.4 41.7
N
7.62 9.35 8.93 8.37 8.32 7.25
39.0/40.8b 137.9 120.1 126.9 134.5 117.4 112.7
8.50
122.6
6.47/6.12b 7.24 7.78 7.01 6.16
...
6.47/6.12b 6.49 7.36 6.82 6.34
...
7.98 7.15
... 9.18 8.30 8.32
55.2 58.7 98.1 43.1
63.0 52.1
56.9 58.4 56.8 57.3/58.9b 58.9 51.1 52.7 53.9 60.1 59.1
HP 1.72 2.53, 2.79 1.88 2.47 1.74 2.87, 2.76 3.86 12.0,g.O 2.94 3.88, 3.37 15.8, 9.4 14.9,4.9 1.58 4.03, 3.88 2.08, 1.32 12.4, 6.2 2.90, 2.50 2.90 3.03, 2.72 1.63 3.73 1.67, 1.20 2.71, 2.59 3.91, 3.85 Hy 1.42, 1.00; H6 0.93 2.70 1.95 2.47 2.99, 2.65 1.56 2.77, 2.66 3.90 11.2, 8.0 2.56 2.99, 2.86 17.3, 9.3
9.70 4.46, 3.57
83.3 44.3
166.8/165.2b 137.3 116.2 112.0 145.4 153.01151.9b 128.4 116.2
6.30 3.86 4.46 4.25 4.5 5.10 4.99 4.11, 3.79
89.5 52.3 52.7 58.6
139.3 124.3 126.6 132.5
5.39 4.50 3.64 4.10
61.4 2.59, 2.24 57.3/58.9b 2.10 57.7 1.73, 1.17 54.0 2.01, 1.92
62.4 57.7 44.6
2.84, 2.69 16.2, 5.0 1.69 2.80, 2.75 1.96 13.6, 6.8 2.69, 2.36 2.86
H (other)
N (other)
H6 7.64; HE6.46 Hy 1.40;HE2.98, 2.92; HN< 8.62 Hyl 2.18; H6 1.73
N< 39.0/40.8*
Hy 1.34, 1.20
Hy 1.82 Hy 3.04, 2.94 Hy 1.07 HN6 7.59, 7.13
N6 112.4
Hyl 1.42, 1.00;Hy2 0.93 Hy 2.16; HNE7.41, 6.68
NE 112.4
H6 7.34; HE7.08, H
