Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part I, pp. 3292-3295, December 1973

Dependence of the Proton Magnetic Resonance Spectra on the Oxidation State of Flavodoxin from Clostridium MP and from Peptostreptococcus elsdenii (natural spin label/paramagnetic line broadening/peak assignments/species dependence/conformation) THOMAS L. JAMES*, MARTHA L. LUDWIGt, AND MILDRED COHN* * Department of Biophysics and Physical Biochemistry, School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania 19174; and t Biophysics Research Division, Institute of Science and Technology, University of Michigan, Ann Arbor, Mich. 48105 Contributed by Mildred Cohn, July 25, 1973

ABSTRACT The broadening of protein nuclear mag- In this paper, the proton nuclear magnetic resonance netic resonances in the spectra of the semiquinone forms (NMR) spectra of flavodoxins from P. elsdenii and Cl. MP in of flavodoxins derived from Clostridium MP and Pepto- streptococcus elsdenii relative to the resonances in the each of the three oxidation states were compared. The phe- oxidized and reduced forms is highly selective. Spectra nomena observable in the present investigation differ from from both species of flavodoxin indicate that conforma- previous NMR investigations of iron-containing electron tional differences between the oxidized and fully reduced carriers (16, 17) in two essential ways: (1) three oxidation states are minor and, consequently, the broadening in the semiquinone form is ascribed to the paramagnetic effect states of flavodoxins are available for comparison, two dia- of the flavin free radical. The chemical shifts of the para- magnetic species, the oxidized and fully reduced forms, and magnetically broadened lines are used in conjunction with one paramagnetic species, the semiquinone form; (2) the x-ray crystallographic models to assign peaks to amino- dominant effect of the unpaired electron in the flavodoxin acid residues in the proximity of the flavin mononucleo- is manifested in the nuclear relaxation as moni- tide. Species-dependent differences in the spectra can semiquinone generally be attributed to differences in amino-acid com- tored by linewidths rather than contact shifts since its elec- position and sequence. The spectra from both species of tron spin relaxation time, unlike that of iron, is very long; a flavodoxin indicate that there is slow exchange between value of the order of 10-8 sec can be estimated from the elec- oxidized and semiquinone forms or reduced and semi- tron spin resonance linewidth (18). The flavin free radical quinone forms of the flavodoxins with a limit of kex < 50 on sec'I for the exchange rate. selectively broadens the resonances of protons amino acids located near the isoalloxazine ring, permitting, in principle, Flavodoxins are small flavoproteins (molecular weight mapping of amino acids in the vicinity of the active site. The 15,000-22,000) that replace ferredoxins as low-potential elec- semiquinone form of flavodoxin thus provides a natural site- tron carriers. Several organisms, including anaerobes (1-5), specific probe of the active-site environment. photosynthetic bacteria (6), blue-green (7, 8) and eukaryotic Recently, paramagnetic nitroxide radicals (spin labels) have algae (9), nitrogen-fixing aerobes (10, 11), and Escherichia coli been attached at specific sites on enzymes to perturb the (12), synthesize flavodoxins. The purified proteins all contain proton NMR spectra by broadening nuclear resonances on one flavin mononucleotide per molecule, with no other pros- those amino-acid residues near the unpaired electron of the thetic groups or bound metal ions. radical (19-21). For flavodoxins, we have a natural spin label Flavodoxins do not react directly with pyridine nucleotides probe of the active-site environment without the possible or other oxidizable substrates but rather act as substrates for complications arising from introduction of an extrinsic bulky other proteins. The oxidation-reduction potential for the one- group. Unlike the nitroxide free radical, however, the fiavin electron reduction of the semiquinone radical form is free radical suffers from electron delocalization (22), which in- about -0.4 V (13), in the range typical for ferredoxins. In terferes with absolute quantitation of distances. flavodoxins derived from Peptostreptococcus elsdenii and Amino-acid residues near the active site whose geometry is Clostridium MP, the potentials of the two one-electron steps conserved in both flavodoxins should be discernible by NMR. are separated by more than 0.2 V (5, 13) so that the oxi- Assignment of resonances to these residues may be made in dized, semiquinone, and fully reduced forms of the protein are conjunction with the x-ray data on Cl. MP flavodoxin. Com- each accessible for chemical and structural studies. Re- parison of the proton NMR spectra of the diamagnetic oxi- cent x-ray studies have shown that the three-dimensional dized and completely reduced forms also provides a sensitive structures of flavodoxins from Desulfovibrio vulgaris (14) and means of monitoring any conformational changes that may Cl. MP (15) are very similar in spite of the differences in com- occur on reduction. In addition, the NMR measurements position and the greater chain length (about 10 residues) of yield information concerning the rate of exchange between the D. vulgaris protein. In both structures the isoalloxazine the oxidized or fully reduced flavodoxins and the semiquinone ring is bound near the surface, but the flavin-protein interac- form. tions differ somewhat in the two species. EXPERIMENTAL The flavodoxins from C1.MP and P. elsdenii were prepared as Abbreviations: DSS, sodium 2,2-dimethyl-2-silapentanesulfo- described (3, 5). After lyophilization of solutions in phosphate nate; NMR, nuclear magnetic resonance. buffer (pH 7.5), the samples were dissolved in 99.97% deu- 3292 Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Proton NMR of Flavodoxins 3293

terium oxide, allowed to stand from 4 to 16 hr at room tem- perature to permit proton-deuterium exchange, lyophilized, and redissolved in deuterium oxide. This procedure was re- peated three times. The fully reduced form of flavodoxin was obtained by addition of an excess of the required 1 mol of sodium dithionite per mol of flavodoxin under a nitrogen atmosphere. The violet semiquinone form was quickly gen- Reduced ,, erated from the pale yellow reduced form by injecting small volumes of air through a septum seal on the NMR tube. Reoxidation of the semiquinone form is slow (13) and re- quired that the sample be spun for about 1 hr after exposure to air. The NMR spectra of the initial oxidized samples and of the reoxidized samples were identical. Semiquinone A Varian HA-220 spectrometer equipped with a pulse unit and computer was used to obtain continuous wave spectra Odi with time-averaging and Fourier transform spectra at 220 MHz and 190. Chemical shifts were measured relative to the methyl resonance of external sodium 2,2-dimethyl-2-silapen- Oxidized tanesulfonate (DSS). I RESULTS AND DISCUSSION The well-resolved proton NMR spectra of P. elsdenii flavo- 9 8 7 6 5 4 3 2 1 0 -1 doxin in various oxidation states are shown in Fig. 1, and the Chemical Shift (ppm from DDS) FIG. 2. Proton NMR spectra of 4.5 mM flavodoxin (Cl. MP) in potassium phosphate buffer (pD 7.5) in three different oxida- tion states. Each spectrum shown is an average of 10 scans.

spectra of Cl. MP flavodoxin in three oxidation states are presented in Fig. 2. Many of the differences between the spectra of the oxidized forms of the two flavodoxin species F Reduced 1i II (Figs. 1A and 2A) can be attributed to differences in amino- acid composition. For example, the alanine peak at 1.3-1.4 in with I I ppm is much larger in the P. elsdenii spectrum, accord the presence of 18 Ala (23) rather than the 6 Ala (24) found I'1 in Cl. MP flavodoxin. The aromatic region appears less well- E. -,-50% Semiquinone + - 50% Reduced resolved in the Cl.MP spectrum, partly as a result of one addi- Ai tional Tyr and one additional Phe in the molecule. Differences between the spectra shown here and that of oxidized Cl. pasteurianum flavodoxin (25) also reflect alterations in amino- I acid composition (1, 26). Spectra of the oxidized and reduced forms of P. elsdenii flavodoxin are nearly identical (Fig. 1A and F). The peak at -0.81 ppm in the spectrum of the oxidized form is shifted by C Sermquinone 0.07 ppm downfield in the spectrum of the reduced form. There is some possibility that the other perturbed peaks are shifted by 0.01-0.02 ppm, but that cannot be stated with certainty. The small shift of the -0.81 ppm peak is probably caused by a small change in the electron density of the flavin Ii ring in going from the oxidized to the reduced form, such that the ring current effect on the chemical shift is slightly at- A. Oxidized 1J4 tenuated in the reduced flavin relative to the oxidized flavin. It is also conceivable that a slight change in the orientation of the flavin ring may occur between the oxidized and reduced 10 9 8 7 6 5 4 3 2 0 -I species, in addition to the electron density alteration. Chemical Shift (ppm from DSS) The spectra of the oxidized and fully reduced forms of Cl. FIG. 1. Proton NMR spectra of 4.8 mM flavodoxin (P. MP flavodoxin are virtually indistinguishable (Fig. 2A and elsdenii) in potassium phosphate buffer (pD 7.5) under various C). Thus, the spectral data imply that there can be no major conditions of oxidation or reduction. In each case the Fourier conformational differences between the oxidized and reduced transform NMR spectrum is shown for the sum of 3000 transients contrast to with an- with a 0.2-sec sampling time. The vertical scale for each spectrum, forms of flavodoxin, in the observations however, is not the same. The continuous wave NMR spectrum, other electron carrier, cytochrome c (27-29). Comparison of obtained for some samples, was the same as the Fourier transform the diffraction patterns from crystals of Cl. MP flavodoxin in spectrum. the semiquinone and fully reduced states leads to the con- Downloaded by guest on September 23, 2021 3294 Biochemistry: James et al. Proc. Nat. Acad. Sci. USA 70 (1973)

Reduced peared consistent with the results of chemical modification and maintained some homology with the flavin-binding regions A, of D. vulgaris flavodoxin. However, at 1.9-A resolution the electron density clearly corresponds to tryptophan. The tryptophan-90 proton peaks would be upfield from the usual Oxidized aromatic peaks for a random-coil protein due to the effect of ring currents resulting from the stacking interaction between the flavin and the aromatic amino acid. A very approximate 75 70 6.5 6.0 estimate, using the ring-current tables of Haigh and Mallion (31) and assuming the flavin ring system could be approxi- Chemical Shift (ppm from DSS) mated by three benzene rings, would give tryptophan peaks FIG. 3. Comparison of the aromatic region of the proton NMR at 6.12-6.19 ppm and 6.70-6.80 ppm, in rough agreement with spectra of 4.5 mM flavodoxin (Cl. MP) in potassium phosphate the observed positions. buffer (pD 7.5) in the three oxidation states. Each continuous Peaks at -0.81 and -0.27 ppm in the upfield region of the wave spectrum shown is an average of 10 scans. P. elsdenii spectrum disappear in the presence of the para- magnetic flavin radical, and have been tentatively assigned to clusion that the semiquinone and reduced structures must be residues in the vicinity of the prosthetic group (see below). nearly identical (24). Furthermore, electron density maps of Unassigned peaks at 1.06, 1.15, and 2.10 ppm, which stand the flavin region show the conformations of the oxidized and out from the protein envelope in the spectra of the oxidized radical forms to be very similar (15). Taken together, the and reduced forms, disappear in the semiquinone spectrum, NMR and x-ray results establish that all three oxidation as do peaks at 6.32 and 7.10 ppm in the aromatic region. states of flavodoxin have closely similar conformationsT. The general similarity of the three-dimensional structures The spectra of the semiquinone forms (Figs. 1C and 2B) of flavodoxins from organisms as unrelated as D. vulgaris and are less well resolved than those of the diamagnetic forms and Cl. MP makes it reasonable to assume that the more closely show the selective disappearance of certain resonances and related P. elsdenii and Cl. MP flavodoxins will have nearly the broadening of several other peaks. Since the conforma- identical structures. The N-terminal 52 residues of the Cl. tional change with state of oxidation is minimal, this behavior MP protein show strong sequence homology with the P. can be ascribed to the paramagnetism of the flavin free radical elsdenii and Cl. pasteurianum proteins in those regions where which provides an efficient relaxation mechanism for the pro- the residues are near the flavin mononucleotide or are "in- ton spins. The magnitude of the effect of the unpaired elec- side" the molecule (23, 26, and Yasunobu, K. T., personal tron on the linewidth of the proton varies inversely with r6, communication). Fig. 4 is a drawing of the flavin-binding re- where r is the distance between the unpaired electron and the gion of the current model of the Cl. MP structure with residues proton (30). On the assumption that the Solomon equation identified according to the P. elsdenii sequence. It thus repre- (30) is valid for free radicals and that the pertinent correlation sents the predicted structure of P. elsdenii flavodoxin. Using time is 1 X 10-8 sec, then every proton less than 10 A from this model, we can tentatively assign certain resonances which the unpaired electron on the free radical should have a disappear from the spectrum of the semiquinone form. In the resonance linewidth > 100 Hz; such broad resonances are at the limit of detection at the signal-to-noise levels of these ex- Trp 7 L periments. The general lack of resolution is due to broadening in resonances of other protons farther from the isoalloxazine group. Some distinctive peaks, such as the one at 0.31 ppm Ser upfield from sodium 2,2-dimethyl-2-silapentanesulfonate in Pro the Cl. MP flavodoxin spectrum and the one at 0.43 ppm upfield in the P. elsdenii spectrum, are not perturbed by the Leu Tyr presence of the flavin radical. Except for the broadening of Aa some resonances due to the unpaired electron, there are no Met Glu Gl Tp obvious differences between the spectrum of the semiquinone and that of the oxidized form. The aromatic region of the NMR spectra of ClU MP flavo- 14-0N- Glulu ~Trp9 ~~~~er doxin in the three oxidation states is expanded in Fig. 3. The Gly 58 peaks occurring at 5.95 and 6.39 ppm with the oxidized or re- Ser duced forms are broadened beyond detection in the semi- FIG. 4. A drawing of the flavin-binding region, based on the quinone form. The structure of the Cl. MP flavodoxin derived three-dimensional model of Cl. MP flavodoxin, but incorporating from x-ray crystallographic studies (11) suggests that these the residues found in P. elsdenii flavodoxin. The residue numbers two resonances are due to the protons of an aromatic residue (at alpha carbons) are for the P. elsdenii chain and differ from those for Cl. MP flavodoxin. The ribosyl phosphate chain has that is approximately parallel to the isoalloxazine ring system and (Fig. 4). This been identified as tyro- been omitted for clarity. Except for Trp-7, Met-57, Trp-91, residue had tentatively only the ,B-carbon of each residue is shown. The circle describes a sine in a 3.25-A electron density map. That assignment ap- radius of about 10 A from the N-5 position of the isoalloxazine ring. The Trp-91 corresponds to position 90 in the Cl. MP species, t The significant changes in x-ray intensities, observed upon which has a tryptophan ring stacked with the isoalloxazine ring oxidation of crystals of the semiquinone form of Cl. MP flavo- system. Residues 92 and 93 should be Gly and Ser, respectively doxin, remain to be explained by further crystallographic analysis. (23). Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Proton NMR of Flavodoxins 3295

aromatic region, by an argument analogous to that used with National Science Foundation, and Grant RR 00542 from the the Cl. MP species, we ascribe the peaks at 6.32 and 7.10 ppm National Institutes of Health for the Middle Atlantic NMR Re- search Facility. This work was done during the tenure of a to protons of Trp-91 which is stacked with the isoalloxazine Special Fellowship from the National Institute of General Medi- ring system. The other tryptophan resonances are probably cal Sciences (T.L.J.), of a Career Investigatorship from the buried under the larger peaks at 6.05 and 6.5-6.8 ppm. American Heart Association (M.C.) and of a Career Development Peaks occurring in the region upfield of sodium 2,2-dimethyl- Award (GM 06611) from the United States Public Health Service 2-silapentane sulfonate (see Fig. 1) are located at -0.27, (M.L.L.). and -0.81 ppm and have intensity ratios of 3:11:3, -0.43, 1. Knight, E., Jr., D'Eustachio, A. J. & Hardy, R. W. F. respectively, as determined from intensity measurements with (1966) Biochim. Biophys. Acta 113, 626-628. a continuous wave spectrum. The two peaks at -0.27 and 2. LeGall, J. & Hatchikian, E. C. (1967) C. R. Acad. Sci. Ser D. -0.81 ppm are not present in the spectrum of the semiqui- 264,2580-2583. none. From the model (Fig. 4) it is plausible to suggest that 3. Mayhew, S. G. & Massey, V. (1969) J. Biol. Chem. 244, one resonance, Met-57 794-802. is the Ala-56 methyl and the other the 4. Dubourdieu, M. & LeGall, J. (1970) Biochem. Biophys. Res. methyl group, since no other methyls seem close enough to be Commun. 38, 965-972. shifted upfield by flavin ring currents. However, neither of 5. Mayhew, S. G. (1971) Biochim. Biophys. Acta 235, 276-288. these two peaks (-0.27 and -0.81 ppm) is observed in the 6. Cusanovich, M. A. & Edmonson, D. E. (1971) Biochem. Cl. MP spectrum, even though the electron density at 1.9-A Biophys. Res. Commun. 45, 327-335. 7. Smillie, R. M. (1965) Biochem. Biophys. Res. Commun. 20, resolution corresponds to Ala and Met at the positions shown 621-629. in Fig. 4. Calculations based on approximate measurements 8. Crespi, H. L., Norris, J. R. & Katz, J. J. (1972) Nature New of the orientation of these methyl groups in the Cl. MP struc- Biol. 236, 178-180. ture indicates that the methyl protons should not be shifted 9. Zumft, W. G. & Spiller, H. (1971) Biochem. Biophys. Res. upfield enough to be resolved from the aliphatic envelope. Commun. 45, 112-118. 10. Van Lin, B. & Bothe, H. (1972) Arch Mikrobiol. 82, 155-172. These findings suggest the possibility that the relative orienta- 11. Shethna, Y. I., Wilson, P. W. & Beinert, H. (1966) Biochim. tions of the flavin and methyls 56 and 57 are sufficiently dif- Biophys. Acta 113, 225-234. ferent in the two flavodoxins to be distinguished by the 12. Vetter, H., Jr. & Knappe, J. (1971) Z. Physiol. Chem. 352, spectra. Alternative assignments would include other groups 433-446. 13. Mayhew, S. G., Foust, G. P. & Massey, V. (1969) J. Biol. within about 10 A of the flavin ring whose resonances are Chem. 244,803-810 shifted upfield by adjacent aromatic residues. Although the 14. Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., Legall, J. & model corresponding to Fig. 4 suggests some candidates, it Dubourdieu, M. (1972) Proc. Nat. Acad. Sci. USA 69, seems premature to discuss these before the identities and 3185-3188. orientations of all the residues in Cl. ALP flavodoxin are 15. Andersen, R. D., Apgar, P. A., Burnett, R. M., Darling, G. 1)., LeQuesne, M. E., Mayhew, S. G. & Ludwig, M. L. established. (1972) Proc. Nat. Acad. Sci. USA 69, 3189-3191. It is apparent from this investigation that the NMIR spectra 16. Kowalsky, A. (1965) Biochemistry 4, 2382-2388. of proteins containing "natural" organic radicals can yield 17. Poe, M., Phillips, W. D., McDonald, C. C. & Lovenberg, W. useful structural information. Flavodoxin is a favorable pro- (1970) Proc. Nat. Acad. Sci. USA 65, 797-804. 18. Palmer, G., Muller, F. & Massey, V. (1971) in Flavins and tein for study because of its low molecular weight. It is also Flavoproteins, ed. Kamin, H. (University Park Press, fortunate that the flavin prosthetic group is located at one Baltimore, Md.), pp. 123-140. end of the molecule (15). If the free radical were "buried" in 19. Sternlicht, H. & Wheeler, E. (1967) in Magnetic Resonance in the center of the protein, a very large number of peaks would Biological Systems, eds. Ehrenberg, A., Malmstrom, G. C., & broaden considerably with little selectivity. Vanngard, T. (Pergamon Press, New York), pp. 325-334. 20. Roberts, G. C. K., Hannah, J. & Jardetzky, 0. (1969) The perturbed resonances lose intensity rather than broaden Science 165, 504-505. as the semiquinone form is approached from either the oxi- 21. Wien, R. W., Morrisett, J. D. & McConnell, H. M. (1972) dized or reduced state. This is indicative of slow exchange Biochemistry 11, 3707-3716. between oxidized and semiquinone forms or between reduced 22. Muller, F., Hemmerich, P., Ehrenberg, A., Palmer, G. & Massey, V. (1970) Eur. J. Biochem. 14, 185-196. and semiquinone forms. From the linewidth, a limit of kex < 50 23. Tanaka, M., Haniu, M., Yasunobu, K. T., Mayhew, S. G. & sec'1 can be placed on the exchange rate of the electron. Massey, V. (1973) J. Biol. Chem. 248, 4354-4366. Electron exchange rates of 104 sec1 were found for cyto- 24. Ludwig, M. L., Andersen, R. D., Apgar, P. A., Burnett, R. chrome c (16) and ferredoxin (32). More detailed information M., LeQuesne, M. E. & Mayhew, S. G. (1971) Cold Spring could be derived from those systems where rapid exchange Harbor Symp. Quant. Biol. 36, 369-380. 25. McDonald, C. C. & Phillips, W. D. (1971) in Fine Structure between diamagnetic and paramagnetic species obtains. In of Proteins and Nucleic Acids, eds. Fasman, G. D. & Tima- such cases, it may be possible to poise the oxidation-reduction sheff, S. N. (Mercel Dekker, New York), pp. 1-48. potential such that equilibrium mixtures with any ratio of 26. Fox, J. L. & Brown, J. R. (1971) Fed. Proc. 30, 1242. the free radical to the fully oxidized or fully reduced form 27. Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, 0. B., Samson, L., Cooper, A. & Margoliash, E. i(1971) J. Biol. could be achieved. By studying the line broadening as a func- Chem. 246, 1511-1533. tion of that ratio, one could quantitatively map the protons 28. Takano, T., Swanson, R., Kallai, 0. B. & Dickerson, R. E. at increasing distances from the free radical by increasing the (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 397-404. fraction of free radical. 29. Wuthrich, K., Aviram, I. & Schejter, A. (1971) Biochim. Biophys. Acta 253, 98-103. We thank Dr. Stephen G. Mayhew who isolated the flavo- 30. Solomon, I. (1955) Phys. Rev. 99, 559-565. doxins used in this study and Dr. J. S. Leigh for helpful discus- 31. Haigh, C. W. & Mallion, R. B. (1972) Org. Alagn. Resonance sion. This investigation was supported in part by Grants GM 4, 203-228. 12446 and GM 16429 from the National Institutes of Health, 32. Poe, M., Phillips, W. D., McDoIoald, C. C. & Orme-Johnson, United States Public Health Service, Grant GB 18487 from the W. H. (1971) Biochem. Biophys. Res. Commun. 42, 705-713. Downloaded by guest on September 23, 2021