A Simple Proposal That Can Explain the Inactivity of Metal-Substituted Superoxide Dismutases

We propose that the apparent catalytic inactivity of Mnand Fe-substituted superoxide dismutases (SODs) reflects E°s that are either lower (Fe-sub-(Mn)SOD) or higher (Mn-sub-(Fe)SOD) than those of native Feor Mn-SODs. In support, we show that the E° of Fe-sub-(Mn)SOD (Fe substituted into Mn-SOD protein) is -240 mV vs NHE, almost 0.5 V lower than our E° of 220 mV for Fe-SOD. The E° of Fe-sub-(Mn)SOD is lower than that of O2/O2 and therefore is sufficient to explain Fe-sub-(Mn)SOD’s inactivity. Indeed, Fe-sub-(Mn)SOD is shown to be unable to oxidize O2. Alternate causes of inactivity are ruled out by our demonstration that Fe-sub-(Mn)SOD retains the ability to reduce O2. Thus, the active site remains active with respect to substrate binding and proton and electron transfer. Finally, we show that Fe-sub-(Mn)SOD’s inactivity with respect to O2 oxidation cannot be solely due to competitive inhibition by OH-. Thus, our proposal provides a simple chemical basis for the observed catalytic inactivity of metal-exchanged Mnor Fe-SODs and suggests that these strongly homologous enzymes may provide important insights into mechanisms of redox midpoint potential tuning in proteins. Feand Mn-containing superoxide dismutases (SODs1) constitute a family of closely related enzymes that catalyze the two-step disproportionation of O2: where M signifies the active site Fe or Mn ion.2,3 The mechanism and kinetic parameters of Fe-SOD and Mn-SOD are similar except that the latter is subject to reversible inhibition by a side reaction.4 Feand Mn-SODs share high amino acid sequence and structural homology, and the active sites are virtually identical.5 The Fe of Fe-SODs is coordinated in a trigonal bipyramid by three His, an Asp-, and in most cases a solvent molecule.6-8 Mn-SODs employ the same ligands9-12 * Phone: (410) 516-4951. Fax: (410) 516-8420. E-mail: afm@ intrepid.chm.jhu.edu. (1) Abbreviations: DCIP, dichloroindophenol; DMSO, dimethyl sulfoxide; E°, reduction midpoint potential, E°′, reduction midpoint potential at pH 7; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; LMCT, ligand-to-metal charge transfer; NHE, normal hydrogen electrode; SOD, superoxide dismutase, only Feand Mn-SODs are discussed in this paper. (2) Bull, C.; Fee, J. A. J. Am. Chem. Soc. 1985, 107, 3295-3304. (3) Lavelle, F.; McAdam, M. E.; Fielden, E. M.; Roberts, P. B.; Puget, K.; Michelson, A. M. Biochem. J. 1977, 161, 3-11. (4) Bull, C.; Niederhoffer, E. C.; Yoshida, T.; Fee, J. A. J. Am. Chem. Soc. 1991, 113, 4069-4076. (5) Lah, M. S.; Dixon, M. M.; Pattridge, K. A.; Stallings, W. C.; Fee, J. A.; Ludwig, M. L. Biochemistry 1995, 34, 1646-1660. (6) Carlioz, A.; Ludwig, M. L.; Stallings, W. C.; Fee, J. A.; Steinman, H. M.; Touati, D. J. Biol. Chem. 1988, 263, 1555-1562. (7) Stoddard, B. L.; Howell, P. L.; Ringe, D.; Petsko, G. A. Biochemistry 1990, 29, 8885-8893. (8) Cooper, J. B.; McIntyre, K.; Badasso, M. O.; Wood, S. P.; Zhang, Y.; Garbe, T. R.; Young, D. J. Mol. Biol. 1995, 246, 531-544. O2 •+ M-SOD f O2 + M -SOD (1a) O2 •+ M-SOD + 2 H f H2O2 + M -SOD (1b) VOLUME 120, NUMBER 3 JANUARY 28, 1998 © Copyright 1998 by the American Chemical Society S0002-7863(97)02060-X CCC: $15.00 © 1998 American Chemical Society Published on Web 01/28/1998 in the same geometry with only very slight differences in ligand side chain rotations or distances to the metal ion.5 Thus the Feand Mn-SODs appear to be variants of the same enzyme. Nonetheless, with the exceptions of a few so-called cambialistic SODs,13-15 when Mnor Fe-SOD protein, (Mn)SOD or (Fe)SOD, is prepared with the other’s metal ion in the active site, the resulting Fe-sub-(Mn)SOD (Fe substituted into Mn-SOD protein) or Mn-sub-(Fe)SOD is unable to catalyze disproportionation of O2 in the standard assay.16-19 Relatively little information is available to explain why many metal-exchanged SODs appear to be catalytically inactive. Possible reasons include distortion of the active site by the nonnative metal ion, inability to bind substrate, and inability to supply the required protons. The non-native metal ion has been reported to bind in the active site similarly to the native metal ion based on EPR and NMR studies.20,21 Oxidized Fe3+-sub(Mn)SODs also retain the ability to coordinate substrate analogues20-22 and thus presumably the substrate itself. However, Yamakura et al. have observed that the pK of 8.5-9 of the oxidized active site,23 which is ascribed to coordination of OHto Fe3+ in Fe3+-SOD,24 is depressed to near 7 in Fe3+sub-(Mn)SOD from S. marcescens20 and lower in Fe3+-sub(Mn)SOD from E. coli.25 We note that since OHacts as a competitive inhibitor, the higher affinity for OHcould render Fe3+-sub-(Mn)SOD more susceptible to inhibition by OHthan native Fe3+-SOD and thus account for its lower activity which increases at decreasing pHs. Alternately, or in addition, metalsubstituted SODs might be unable to oxidize or reduce substrate. We propose that Fe-sub-(Mn)SODs and Mn-sub-(Fe)SODs appear inactive, at least in part, because the E° of the substituent metal ion is either too low or too high, respectively, to mediate both half-reactions effectively.26 Specifically, because the E°s of the 3+/2+ couple of high-spin Mn compounds are typically significantly higher than the E°s of analogous Fe complexes,27 we note that Mn-specific SOD proteins must depress the E° of Mn3+/Mn2+ considerably more than Fe-specific proteins depress that of Fe3+/Fe2+, to achieve the optimal E°′ of ≈0.36 V (vs NHE) and the E°s of 0.2-0.4 V observed in most Feand Mn-SODs.28 Therefore, we conjecture that, when Fe is bound in the (Mn)SOD protein, its E° is depressed to a value well below 0.2-0.4 V and, similarly, the E° of Mn bound in (Fe)SOD is insufficiently depressed, to a value well above 0.2-0.4 V. This simple, chemically rational hypothesis is both informative and testable and is supported by the experiments described below. Materials and Methods Mn-SOD and Fe-SOD were purified from E. coli 29,30 and had activities of 4500-6000 and 6000-7000 units/mg of protein, respectively. The activities of native and metalexchanged SODs were measured at pH 7.8 in the “standard” indirect assay of McCord and Fridovich.16 Fe-sub-(Mn)SOD was prepared from Mn-SOD with a yield close to 100%, as was Mn-sub-(Fe)SOD, building on published examples.17,18 Briefly, Fe-sub-(Mn)SOD was prepared by partially unfolding Mn-SOD protein in 3.5 M guanidinium HCl and 10 mM EDTA at pH 3.1, dialyzing against EDTA in the presence of 2.5 M guanidinium HCl at pH 8.0, reconstituting with Fe2+ at pH 8.0 under N2, and removing extraneous Fe by dialysis against 1 mM EDTA and 1 mM ascorbate under N2. Mn-sub-(Fe)SOD was prepared by removing Fe at pH 11 and 37 °C and reconstituting with Mn2+ by dialysis at pH 8.5. Oxidation of Fe2+-SOD and Fe2+-sub-(Mn)SOD by O2 was conducted at 25 °C in a medium of 100 mM phosphate, 100 mM KBr, and 0.5 mM glucose supplemented with 500 units of catalase (Sigma No. C-3155) and 50 units of glucose oxidase (Sigma No. G-6891) to eliminate any effects of H2O2 and O2 formed upon spontaneous disproportionation of O2. A pH of 7.8 was used, except where stated otherwise. The complete reaction medium including approximately 0.1 mM SOD dimers was kept anaerobic and SOD was reduced by titration with methylviologen or dithionite prior to oxidation by O2. One to four times stoichiometric31 aliquots of O2 were injected as a stock solution in dry DMSO. Initial additions were approximately one stoichiometric equivalent, but the addition volumes were increased as the system approached the steady state. Injection of DMSO alone produced no effect. Reduction of Fe3+-SOD and Fe3+-sub-(Mn)SOD by O2 was conducted similarly after degassing but not reducing the reaction mixture. Oxidation of Fe2+-SOD or Fe2+-sub-(Mn)SOD by O2 was achieved by injection of 60 mL of O2 at atmospheric pressure directly into the medium, which was prepared without glucose oxidase or glucose for these experiments, and reduced as above. Reduction by H2O2 was performed by injecting 1.5 stoichiometric equivalents of H2O2 in aqueous solution into the medium, initiating a drop in absorbance in Fe3+-SOD when catalase was absent. Potentiometric titrations were performed at 25 °C in an optical cell analogous to the one described by Stankovich.32 A combination Ag|AgCl and Pt electrode was inserted in one port, a syringe containing titrant was mounted in a second, and the third port was connected to a vacuum line and maintained a low flow of N2 gas treated to remove residual O2. The reaction mixture comprised 100 mM phosphate buffer at pH 7.4 (FeSOD) or pH 7.8 (Fe-sub-(Mn)SOD), 100 mM KBr, 0.1-0.2 (9) Ludwig, M. L.; Metzger, A. L.; Pattridge, K. A.; Stallings, W. C. J. Mol. Biol. 1991, 219, 335-358. (10) Parker, M. W.; Blake, C. C. F. J. Mol. Biol. 1988, 199, 649-661. (11) Borgstahl, G. E. O.; Parge, H. E.; Hickey, M. J.; Beyer, J., W. F.; Hallewell, R. A.; Tainer, J. A. Cell 1992, 71, 107-118. (12) Lim, J.-H.; Yu, Y. G.; Han, Y. S.; Cho, S.; Ahn, B.-Y.; Kim, S.H.; Cho, Y. J. Mol. Biol. 1997, 270, 259-274. (13) Martin, M. E.; Byers, B. R.; Olson, M. O. J.; Salin, M. L.; Aruneaux, J. E. L.; Tolbert, C. J. Biol. Chem. 1986, 261, 9361-9367. (14) Pennington, C. D.; Gregory, E. M. J. Bacteriol. 1986, 166, 528532. (15) Meier, B.; Barra, D.; Bossa, F.; Calabrese, L.; Rotilio, G. J. Biol. Chem. 1982, 257, 13977-13980. (16) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049-6055. (17) Yamakura, F. J. Biochem. 1978, 83, 849-857. (18) Ose, D. E.; Fridovich, I. Arch. Biochem. Biophys. 1979, 194, 360364. (19) Brock, C. J.; Harris, J. I. Biochem. Soc. Trans. 1977, 5, 15371539. (20) Yamakura, F.; Kobayashi, K.; Ue, H.; Konno, M. Eur. J. Biochem. 1995, 227, 700-706. (21) Vance, C. K.; Miller, A.-F. In preparation. (22) Whittaker, M. M.; Whittaker, J. W. Biochemistry 1997, 36, 89238931. (23) Fee, J. A.; McClune, G. J.; Lees, A. C.; Zidovetzki, R.; Pecht, I. Isr. J. Chem. 1981,21, 54-58. (24) Tierney, D. L.; Fee, J. A.; Ludwig, M. L.; Penner-Hahn, J. E. Biochemistry 1995, 34, 1661-1668. (25) Yamakura, F.; Matsumoto, T.; Kobayashi, K. in Frontiers of reactiVe oxygen species in biology and medicine; Asada, K., Yoshikawa, T., Eds.; Elsevier Science: Amsterdam, 1994; pp 115-118. (26) Miller, A.-F.; Sorkin, D. L. Comments Mol. Cell. Biophys. 1997, 9, 1-48. (27) Stein, J.; Fackler, J. P.; McClune, G. J.; Fee, J. A.; Chan, L. T. Inorg. Chem. 1979, 18, 3511-3519. (28) Barrette, J., W. C.; Sawyer, D. T.; Fee, J. A.; Asada, K. Biochemistry 1983, 22, 624-627. (29) Whittaker, J. W.; Whittaker, M. M. J. Am. Chem. Soc. 1991, 113, 5528-5540. (30) Sorkin, D. L.; Miller, A.-F. Biochemistry 1997, 36, 4916-4924. (31) Stoichiometries are relative to Fe sites, not SOD dimers. (32) Stankovich, M. T. Anal. Biochem. 1980, 109, 295. 462 J. Am. Chem. Soc., Vol. 120, No. 3, 1998 Vance and Miller