Abstract
We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli. Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His′ δ1 protons near 100 ppm, and β and α protons collectively between 20 and 50 ppm, in Fe(III)SOD. In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically. A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the β protons of Asp 156, superseding earlier assignments (Ming et al. (1994) Inorg. Chem., 33, 83–87). We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of δ2 and ε1 protons. These are the closest protein protons to Fe(II) and therefore they have the broadest (∼4000 Hz) and most difficult to detect resonances. Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state. Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand of Rhodopseudomonas palustris cytochrome c′ (Bertini et al. (1988) Inorg. Chem., 37, 4814–4821) suggesting that Fe(II)SOD's equatorial His2Asp− ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+.
Similar content being viewed by others
References
Balch, A.L., Chan, Y.-W., La Mar, G.N., Latos-Grazynski, L. and Renner, M.W. (1985) Inorg. Chem., 24, 1437–1443.
Banci, L., Bertini, I., Luchinat, C. and Viezzoli, M.S. (1990) Inorg. Chem., 29, 1438–1440.
Bertini, I., Dikiy, A., Luchinat, C., Macinai, R. and Viezzoli, M.S. (1998) Inorg. Chem., 37, 4814–4821.
Bertini, I., Gori, G., Luchinat, C. and Villa, A.J. (1993a) Biochemistry, 32, 776–783.
Bertini, I., Jonsson, B.-H., Luchinat, C., Pierattelli, R. and Vila, A.J. (1994) J. Magn. Reson., B104, 230–239.
Bertini, I., Luchinat, C., Parigi, G. and Walker, F.A. (1999) J. Biol. Inorg. Chem., 4, 515–519.
Bertini, I., Luchinat, C. and Tarchi, D. (1993b) Chem. Phys. Lett., 203, 445–449.
Bertini, I., Turano, P. and Vila, A.J. (1993d) Chem. Rev., 93, 2833–2932.
Bertini, L. and Luchinat, C. (1986) NMR of Paramagnetic Molecules in Biological Systems, Benjamin Cummings, New York, NY.
Bougault, C.M., Dou, Y., Masao, I.-S., Langry, K.C., Smith, K.M. and La Mar, G.N. (1998) J. Am. Chem. Soc., 120, 2113–2123.
Browne, D.T., Kenyon, G.L., Packer, E.L., Sternlicht, H. and Wilson, D.M.J. (1973) J. Am. Chem. Soc., 95, 1316–1323.
Bull, C. and Fee, J.A. (1985) J. Am. Chem. Soc., 107, 3295–3304.
Carlioz, A., Ludwig, M.L., Stallings, W.C., Fee, J.A., Steinman, H.M. and Touati, D. (1988) J. Biol. Chem., 263, 1555–1562.
Cheng, H. and Markley, J.L. (1995) Annu. Rev. Biophys. Biomol. Struct., 24, 209–237.
Fee, J.A., McClune, G.J., Lees, A.C., Zidovetzki, R. and Pecht, I. (1981) Isr. J. Chem., 21, 54–58.
Feig, A.L. and Lippard, S.J. (1994) Chem. Rev., 94, 759–805.
Goff, H. and La Mar, G.N. (1977) J. Am. Chem. Soc., 99, 6599–6605.
Gueron, M. (1975) J. Magn. Reson., 19, 58–66.
Hegg, E.L. and Que, L.J. (1997) Eur. J. Biochem., 250, 625–629.
Hu, J.-S. and Redfield, A.G. (1993) Biochemistry, 32, 6763–6772.
Inubishi, T. and Becker, E.T. (1983) J. Magn. Reson., 51, 128–133.
Lah, M.S., Dixon, M.M., Pattridge, K.A., Stallings, W.C., Fee, J.A. and Ludwig, M.L. (1995) Biochemistry, 34, 1646–1660.
La Mar, G.N., Budd, D.L. and Goff, H. (1977) Biochem. Biophys. Res. Commun., 77, 104–110.
La Mar, G.N., Horrocks, W.D., Jr. and Holm, R.H. (1973) NMR of Paramagnetic Molecules, Academic Press, New York, NY.
Lauffer, R.B., Antanaitis, B.C., Aisen, P. and Que, L., Jr. (1983) J. Biol. Chem., 258, 14212–14218.
Miller, A.-F. (2001) Fe-superoxide dismutase. In Handbook of Metalloproteins, Wieghardt, K., Huber, R., Poulos, T.L. and Messerschmidt, A., Eds. Wiley and Sons, New York, NY.
Miller, A.-F. and Sorkin, D.L. (1997) Commun. Mol. Cellular Biophys., 9, 1–48.
Ming, L.-J., Lynch, J.B., Holz, R.C. and Que, L., Jr. (1994) Inorg. Chem., 33, 83–87.
Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C.A. and Chen, V.J. (1991) Biochemistry, 30, 11653–11659.
Muchmore, D.C., McIntosh, L.P., Russell, C.B., Anderson, D.E. and Dahlquist, F.W. (1989) Methods Enzymol., 177, 44–73.
Pappu, K. and Serpersu, E.H. (1994) J. Magn. Reson., B105, 157–166.
Parker, J. and Friesen, J.D. (1980) Mol. Gen. Genet., 177, 439–445.
Qin, J., Delaglio, F., La Mar, G.N. and Bax, A. (1993) J. Magn. Reson., B102, 332–336.
Que, L., Jr. and Ho, R.Y.N. (1996) Chem. Rev., 96, 2607–2624.
Redfield, A.G. and Gupta, R.K. (1971) Cold Spring Harbor Symp. Quant. Biol., 36, 541–550.
Renault, J.P. and Morgenstren-Badarau, I. (1999) Inorg. Chem., 38, 614–615.
Roach, P.L., Clifton, I.J., Fulop, V., Harlos, K., Barton, G.J., Hajdu, J., Andersson, I., Schofield, C.J. and Baldwin, J.E. (1995) Nature, 375, 700–704.
Sadek, M., Brownlee, R.T.C., Scrofani, S.D.B. and Wedd, A.G. (1993) J. Magn. Reson., B101, 309–314.
Satterlee, J.D. (1986) Ann. Rep. NMR Spectrosc., 17, 79–178.
Sorkin, D.L. and Miller, A.-F. (1997) Biochemistry, 36, 4916–4924.
Sorkin, D.L., Duong, D.K. and Miller, A.-F. (1997) Biochemistry, 36, 8202–8208.
Stallings, W.C., Metzger, A.L., Pattridge, K.A., Fee, J.A. and Ludwig, M.L. (1991) Free Rad. Res. Commun., 12–13, 259–268.
Thanabal, V. and La Mar, G.N. (1989) Biochemistry, 28, 7038–7044.
Tierney, D.L., Fee, J.A., Ludwig, M.L. and Penner-Hahn, J.E. (1995) Biochemistry, 34, 1661–1668.
Vance, C.K., Kang, Y.M. and Miller, A.-F. (1997) J. Biomol. NMR, 9, 201–206.
Vathyam, S., Byrd, R.A. and Miller, A.-F. (1999) J. Biomol. NMR, 14, 293–294.
Vathyam, S., Byrd, R.A. and Miller, A.-F. (2000) Magn. Reson. Chem., 38, 536–542.
Vaughan, J.D., Mughrabi, Z. and Wu, E.C. (1970) J. Org. Chem., 35, 1141–1145.
Weiss, M.A., Redfield, A.G. and Griffey, R.H. (1986) Proc. Natl. Acad. Sci. USA, 83, 1325–1329.
Whittaker, J.W. and Solomon, E.I. (1988) J. Am. Chem. Soc., 110, 5329–5339.
Wu, F.-J. and Kurtz, D.M., Jr. (1989) J. Am. Chem. Soc., 111, 6563–6572.
Wüthrich, K. (1969) Proc. Natl. Acad. Sci. USA, 63, 1071–1078.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sorkin, D.L., Miller, AF. Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli. J Biomol NMR 17, 311–322 (2000). https://doi.org/10.1023/A:1008344210662
Issue Date:
DOI: https://doi.org/10.1023/A:1008344210662