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www.elsevier.nl / locate / jinorgbio Journal of Inorganic Biochemistry 80 (2000) 247–256 Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase Aaron L. Schwartz b , Emine Yikilmaz a,b , Carrie K. Vance b ,1 , Surekha Vathyam b , Ronald L. Koder a , Anne-Frances Miller a,b b a Department of Chemistry, University of Kentucky, Lexington, KY 40506 -0055, USA Departments of Chemistry and Biophysics, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA Received 11 October 1999; received in revised form 7 March 2000; accepted 17 March 2000 Abstract We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461–467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn 21 , Fe 31 or Fe 21 are bound, based on EPR and NMR spectroscopy. However, the mutant’s Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD’s activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn 21 , Fe 31 and 1 H in the presence of Fe 21 distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe 21 SOD and Fe 21 (Mn)SOD by 15 N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe 21 in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower Em in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH 2 coordinated to the metal ion. 2000 Elsevier Science S.A. All rights reserved. Keywords: Superoxide dismutase; Redox potential; Glutamine; Metal ion specificity 1. Introduction Many Fe- and Mn-containing superoxide dismutases (FeSODs and MnSODs) have virtually identical over-all structures and active sites [1,2], homologous amino acid sequences [3,4] and the ability to bind the non-native metal ion, Mn or Fe, respectively. Nonetheless, most are active only with their native metal ion bound [5,6]. Since both Fe and Mn can mediate superoxide dismutation and both the Fe- and Mn-specific proteins ((Fe)SODs and (Mn)SODs) can support this activity, the defect resides in the interaction or match between the protein and the metal ion. We have shown that the inactivity of Fe-substituted (Mn)SOD (Fe(Mn)SOD) can be explained by its anomalously low Em , which is consistent with the (Mn)SOD protein apply- E-mail address: [email protected] (A.-F. Miller). Present address: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA. 1 ing Em tuning appropriate to Mn 31 / Mn 21 to bound Fe . Thus, the SODs are an excellent system for understanding redox tuning. Since there are relatively few conserved amino acid differences between (Fe)SODs and (Mn)SODs, it may be possible to identify specific amino acids responsible for Em tuning. The most striking conserved amino acid differences between Fe- and Mn-SODs are a Gln at position 69 of FeSOD 2 and Gly at the corresponding position (77) in MnSOD, a Gln at position 146 of MnSOD and Ala at the corresponding position (141) in FeSOD, Tyr at position 76 of FeSOD versus Phe84 of MnSOD, and Asp147 of MnSODs versus Gly142 of most FeSODs. FeSOD in which the Tyr76 was mutated to Phe did not acquire 2 Numbering of E. coli FeSOD is used for (Fe)SOD and that of E. coli MnSOD is used for (Mn)SOD. Positions 69, 76 and 141 of FeSOD correspond to positions 77, 84 and 146 of MnSOD. 0162-0134 / 00 / $ – see front matter 2000 Elsevier Science S.A. All rights reserved. PII: S0162-0134( 00 )00086-6 248 A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 Fig. 1. Cartoons of the active sites of FeSOD (left) and MnSOD (right) from E. coli showing the active site Gln and its hydrogen bonding 21 21 ˚ that partners. In Fe SOD the distance from Gln’s N to Fe is 5.1 A, 21 ˚ from Gln’s O to Fe is 6.1 A, in MnSOD the distance from Gln’s N and ˚ respectively, and in Fe 31 (Mn)SOD the O to Mn are 4.6 and 5.7 A, 31 ˚ Coordinates used distance from Gln’s N and O to Fe are 4.4 and 5.5 A. are those of Refs. [2,15]. Mn-supported activity, instead its Fe-supported activity increased . Thus, this residue alone is not a crucial determinant of metal ion catalytic activity. The Gln conserved at position 69 of FeSOD is clearly analogous to the Gln at position 146 of MnSOD as their side chains occupy similar positions in the active site and its hydrogen bond network . The conserved active site Gln is believed to be central to the active site hydrogen bonding network as it hydrogen bonds with both the universally conserved Tyr34 responsible for the pK that governs activity and substrate specificity [10–14] and coordinated solvent (Fig. 1) [2,15]. The strength and polarity of the hydrogen bond with coordinated solvent in effect alters the degree to which the latter is protonated which in turn can have a very large effect on the Em of the metal ion. Indeed, Mn 31 / Mn 21 is calculated to have a 1.3-V higher Em when the coordinated solvent is protonated than when it is not . This would be more than sufficient to account for the 0.46 V difference between the Em s of FeSOD and Fe(Mn)SOD (Table 1) . Thus, we would like to learn whether the hydrogen bonding between Gln and coordinated solvent is different in (Mn)SOD and (Fe)SOD, with each of Fe and Mn bound. The effects of the active site Gln may indeed be different in the two categories of proteins since it originates from different directions and different elements of the SOD structure in the Fe- and Mn-specific SODs. When the FeSOD of Mycobacterium tuberculosis, which has a His in the place of the MnSOD-characteristic Gln 3 , was mutated to place Gln or Glu there, the former mutant preferentially bound Fe and the latter preferentially bound Mn. No activities were reported . Nonetheless, it is interesting that Gln at position 146 does not enforce Mn-binding. Cambialistic 4 SOD from Porphyromonas gingivalis has also been mutated to incorporate the Gln characteristic of MnSOD, instead of its native Gln characteristic of FeSOD . This resulted in lower-than-WT activity with Fe bound but higher-than-WT activity with Mn bound, demonstrating that Gln at position 146 does increase Mn-supported activity. The authors noted that the Mn 31 optical spectrum was stronger in the mutant, suggesting the possibility that the mutant stabilizes more Mn 31 , and has a lower Em , consistent with the hypothesis of Vance and Miller that greater Mn-supported activity results when the protein depresses the Em more . Here we report the complementary mutant in which a (Mn)SOD protein is mutated to contain Gln at the position characteristic of FeSODs. We have used EPR spectra of Fe 31 or Mn 21 to compare their coordination geometries and strengths of zero-field splittings in different WT and mutant protein environments. In addition we have characterized the active site Gln’s interactions with the metal ion via direct observation of that amino acid by NMR spectroscopy, in which the line widths and positions of paramagnetically shifted resonances reflect the metal ion’s electronic state and geometry (via the magnetic susceptibility tensor) and the degree to which unpaired electron spin density is delocalized (via hyperfine coupling). Comparison of the NMR signals of analogous residues in two different proteins thus reveals 3 M. tuberculosis has a Gly at the position of the FeSOD-characteristic Gln. 4 Active with either Mn or Fe. Table 1 Activity of G69Q / Q146A-(Mn)SOD containing either Fe or Mn Fe-SOD Fe(Mn)SOD Fe-G77Q,Q146A-(Mn)SOD Mn-G77Q,Q146A-(Mn)SOD Mn-SOD Activity a Per metal ion b Fe content c 6500 0 360 3400 6800 6800 (100%) (0%) 450 (6.5%) 4900 (71%) 6900 (100%) 0.95 0.86 0.81 0.01 0 Mn content d e ND ND 0.70 0.98 Em 220 mV f 2240 mV f a Measured using the xanthine oxidase assay at pH 7.8  which has a detection limit corresponding to less than 0.1% of native SOD activity and an uncertainty of |2% of the measured activity. b Activity on a per Fe or Mn basis (as a percentage of the appropriate WT activity). c Fe content was measured using a ferrozine assay  and is expressed on a per subunit basis. The uncertainty is estimated to be 0.03 per subunit. d Mn content was measured by EPR after acidification of the sample, and is expressed on a per subunit basis. The detection limit is less than 1 mM Mn or 0.005 / subunit and the uncertainty is approximately 3%. e None detected. f Versus NHE, from Ref. . A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 differences in their interactions with and position relative to the metal ion, while comparison of general features of the paramagnetically shifted 1 H NMR spectrum provides insight into the state of the metal ion. 2. Methods FeSOD, MnSOD and Fe(Mn)SOD were prepared as in Refs. [7,12,19,20], respectively. The E. coli gene for MnSOD (sodA) was cloned into the overexpression vector pET24d (Novagen) using standard techniques and mutagenized by PCR using the megaprimer method . Both strands of the mutant gene were completely sequenced to confirm the presence of the desired Gly77 to Gln and Gln146 to Ala mutations, and the absence of any others. The mutant gene was overexpressed in E. coli QC774 / DE3 and the protein was purified using the same protocol as for MnSOD [19,20]. Fe content was measured using the ferrozine assay of Carter , and Mn was quantified by EPR at low temperature after acid hydrolysis of the samples to release all Mn as Mn 21 and addition of glycerol to 50% (v / v). Selective 15 N labeling of Gln side chains was accomplished as in Ref. , using the E. coli Asn auxotrophic strain JK120 . EPR spectra were collected on a Bruker 300 EMX calibrated at g952 using DPPH and at g956 using myoglobin in 100 mM K 2 HPO 4 . EPR spectra of Fe 31 were collected at 9.47 GHz at 70 K, with 80 mW nominal microwave power, 2 G modulation amplitude and 3000 G wide scans centered at 2100 G obtained in approximately 20 min. EPR spectra of Mn 21 were collected as above but with a nominal microwave power of 160 mW, 10 G modulation amplitude and 500065000 G scans of 5.5 min each, except for the spectrum of Mn 21 SOD which was collected at 4 K. EPR samples were buffered with 50 mM potassium phosphate and contained 50 mM NaCl but no cryoprotectant. The samples of MnSOD and Mn-mut-SOD were reduced with dithionite but the sample of Mn(Fe)SOD required no reduction. The pH was measured before freezing and after the sample was thawed to confirm that the pH had been stable. 1 H NMR spectra were obtained at 300 MHz for protons on an AMX 300 at 308C as previously described  using the super-WEFT pulse sequence  with a rapid repetition rate to emphasize the rapidly relaxing resonances from the paramagnetic active site. The acquisition time was 25 ms and the water resonance was saturated during the 35-ms delay between pulses, as well as the 15-ms relaxation delay between repetitions. For each NMR sample, 450 ml of 1 mM SOD in 100 mM K 2 HPO 4 pH 7.6 and 50 ml 2 H 2 O were degassed and reduced with 5 ml of 150 mM sodium dithionite in an NMR tube fitted with a Teflon seal, or a standard NMR tube which was then flame sealed. 15 N NMR spectra were collected on a Unity plus 500 at 249 50.655 MHz and 258C using either 60 or 908 pulses followed by observation without 1 H decoupling, or a super-WEFT pulse sequence  with 1 H decoupling during the 0.1-s acquisition time but not the 0.7-s relaxation delay that followed or the 0.6-s delay between the two pulses. Samples of 1 mM SOD in 50 mM potassium phosphate at pH 7.8 or 7.5 with 10 or 25 mM NaCl and 10% 2 H 2 O were oxidized with KMnO 4 and filtered (Mn 31 SOD) or degassed and reduced as above (Fe 21 SOD and Fe 21 (Mn)SOD) or observed without further treatment (apo(Mn)SOD). 3. Results 3.1. G77 Q ,Q146 A-( Mn)SOD expression and activity The mutant protein overexpressed well but the identity and quantity of metal ion incorporated by the protein varied depending on the composition of the growth medium. Therefore, in order to be able to work with protein containing one metal ion only, bound metal ions were removed and replaced by either Fe or Mn using previously developed methods . The resulting preparations of mutant protein containing Fe (Fe-G77Q,Q146A(Mn)SOD or Fe-mut(Mn)SOD), or Mn (MnG77Q,Q146A-(Mn)SOD or Mn-mut(Mn)SOD) were characterized with respect to both Fe and Mn content and SOD activity in the standard assay . Table 1 shows that Mn-mut-(Mn)SOD retained 70% of WT MnSOD activity. This assay employs low O ?2 concentrations and assesses 2 k cat /KM , which is indicative of binding steps as well as chemical steps. Although the mutations affect residues central to the active site, the retention of 70% of activity suggests that the active site remains intact. Fe-mut(Mn)SOD displayed small but significant activity, corresponding upon correction for substoichiometric Fe incorporation to 7% of WT FeSOD activity. Although the decrease in Mn-supported activity to 70% is virtually insignificant, the increase in Fe-supported activity from 0 to 7% is dramatic. To learn what changes in the active site may be responsible for mut-(Mn)SOD’s significantly increased Fe-supported activity, we have begun characterizing the mutant’s active site and comparing it with those of (Fe)SOD and (Mn)SOD, with each of Fe and Mn bound. 3.2. Spectroscopy of G77 Q ,Q146 A-( Mn)SOD In order to assess the degree to which the active site is disrupted or deformed by relocating the Gln, we have compared the EPR spectra of Mn 21 SOD and Mn 21 -mut(Mn)SOD. Fig. 2 shows that these spectra are similar in general appearance, with similar features appearing in similar places. Although good simulations are required to support a quantitative comparison, the spectra nonetheless suggest that neither the coordination geometry of Mn 21 , 250 A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 Table 2 pH values of samples and g values from Fe 31 EPR spectra of SODs Sample pH g values FeSOD Fe-G77Q,Q146A-(Mn)SOD Fe(Mn)SOD 7.65 7.4 7.53 4.81 5.00 5.00 4.02 3.86 a 3.84 3.69 3.43 3.46 a Due to the overlapping negative portion of the non-active site rhombic Fe signal, this g value was obtained by extrapolating the negative portion of SOD’s signal to lower fields and noting the intersection with the best baseline (see Fig. 2). parison. The EPR spectrum of Fe 31 -mut-(Mn)SOD is more similar to that of Fe 31 (Mn)SOD than that of Fe 31 SOD, based on g values (Table 2) and appearance, neglecting the somewhat larger contribution of rhombic non-active-site (NAS) Fe 31 to the spectrum (Fig. 3). These indicate that the coordination geometry and electronic state of Fe 31 , like those of Mn 21 , are not altered significantly by relocating the Gln side chain in the active site of (Mn)SOD. The EPR signal of Fe 31 -mut-(Mn)SOD is distinctly different from that of Fe 31 SOD however. Fig. 2. EPR comparison of Mn 21 SOD (top), Mn 21 -mut-(Mn)SOD (center) and Mn 21 (Fe)SOD (bottom). Samples were buffered at pH 7.8 with 50 mM potassium phosphate and 50 mM NaCl. the covalency nor the magnitude of zero-field splitting, is changed very much upon relocation of the active site Gln (and replacement of a Gly by an Ala). By contrast, there are a number of clear differences between the spectra of mutant and WT Mn 21 SOD on the one hand, and that of Mn 21 (Fe)SOD on the other. These results are consistent with the fact that mut-MnSOD retains 203 amino acids identical to those of MnSOD and only two identical to Mn(Fe)SOD. Thus the active site Gln’s interaction with Mn 21 does not appear to be strongly sensitive to the origin of the Gln in the amino acid sequence, but is more dependent on the identities of the rest of the active site amino acids. To address possible location-specific interactions with Fe, Fe-mut-(Mn)SOD was compared to FeSOD and Fe(Mn)SOD. EPR was used for the oxidized state comparison and NMR was used for the reduced state com- Fig. 3. EPR comparison of Fe 31 SOD (top), Fe 31 -G77Q,Q146A(Mn)SOD (center) and Fe 31 (Mn)SOD. Sample pH values were 7.65, 7.4 and 7.53, respectively. A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 Thus, variations in the signal appear to predominantly reflect the identity of the rest of the protein, not the different locations of the Gln in the amino acid sequence. The NAS Fe 31 signal indicates that there is approximately a 1:0.4 ratio of active site to NAS Fe 31 . This reflects the mutant protein’s tendency to loose active site Fe when extensive effort was made to remove NAS Fe. However it also indicates that the actual Fe-supported activity on a per active site Fe basis is 1.4 times the activity reported in Table 1 on a per total Fe basis, or 9% of WT activity. Thus Fe-mut-(Mn)SOD appears to have almost 10% WT levels of Fe-supported activity, but certainly more than 7%. Fig. 4 shows that the same general trends hold for the Fe 21 state of Fe 21 -mut-(Mn)SOD, whose NMR spectrum is more similar to that of Fe 21 (Mn)SOD than that of Fe 21 SOD. Subtle differences between Fe 21 -mut(Mn)SOD’s and Fe 21 (Mn)SOD’s spectra between 10 and 15 ppm might be attributable to the mutated amino acids and shifts of conserved features between 22 and 30 ppm, and 215 to 25 ppm may reflect slight conformational changes. However, these distinctions are significantly smaller than the differences between Fe 21 (Mn)SOD and Fe 21 SOD, which result in quite different appearance between 30 and 10 ppm and distinct chemical shifts for the ligand His’ Nd1 protons’ resonances between 30 and 90 ppm. The overall similarity in chemical shift range and line widths in all three spectra nonetheless indicate that Fe 21 ’s electronic structure and geometry are very similar in the three proteins. Since the NMR spectrum is generally similar in Fe 21 SOD and Fe 21 (Mn)SOD , it is not very surprising that it is similar in Fe 21 -mut-(Mn)SOD as well. The spectra above describe three of the four possible metal ion and oxidation states in SOD and indicate that they are not much changed by moving Gln146 to position 77 in (Mn)SOD. This is consistent with Mn-mut(Mn)SOD’s retention of close to full WT activity in the standard assay. However, the acquisition of at least 7% of the FeSOD level of Fe-supported activity indicates that the 251 subtle spectroscopic differences between WT and mut(Mn)SOD reflect electronic or other changes sufficient to support small but significant activity. Since the inactivity of Fe(Mn)SOD can be explained by its anomalously low Em , Fe-mut-(Mn)SOD’s recovery of some activity suggests that its Em is higher than that of Fe(Mn)SOD. The above preliminary characterizations demonstrate that G77Q,Q146A-(Mn)SOD retains basic active site integrity and should be amenable to further study, including redox titrations. 3.3. NMR spectroscopy of the Gln side chain amide in WT Fe 21 ( Mn)SOD The active site Gln hydrogen bonds directly with the coordinated solvent, and may thereby exert a strong effect on the metal ion Em and thus its SOD activity . Therefore, we wish to learn how the Gln’s relation to the metal ion correlates with enzyme activity. We have compared this relation in active Fe 21 SOD with that in inactive Fe 21 (Mn)SOD by NMR. The Gln side chain’s close proximity to the bound metal ion precludes its observation by 1 H NMR, but the low gyromagnetic ratio of 15 N permits direct observation of the side chain amide by 15 N NMR in the presence of Fe 21 [23,30]. 15 NH 3 is incorporated directly into the side chain of Gln by E. coli while direct incorporation into Asn is prevented by the use of an auxotrophic strain. Most other amino acid positions derive their N indirectly from Gln. Thus, in an 15 N NMR spectrum of protein isolated from E. coli / JK120 grown in the presence of 15 NH 3 and all the amino acids except Gln in 14 N form, only the side chains of Gln appear . The side chain N of Gln69 of Fe 21 SOD has been observed at 158 ppm, strongly shifted from the 113- and 114-ppm resonances of Gln side chains distant from Fe 21 . When MnSOD was labeled with 15 N in the side chains of its 6 Glns, there was little leak into the side chains of Asn, since only five strong pairs of resonances plus two Fig. 4. NMR comparison of Fe 21 SOD (top), Fe 21 -G77Q,Q146A-(Mn)SOD (center) and Fe 21 (Mn)SOD. 252 A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 Fig. 5. HSQC of [ 15 N]Gln-labeled (Mn)SOD. NMR spectra were collected at 258C on a Unity plus spectrometer at 500 MHz for 1 H and 50 MHz for 15 N. 1 H– 15 N HSQCs  were collected with sweep widths of 1650 Hz for 15 N and 6500 Hz for 1 H with GARP decoupling of 15 N during acquisition. Data were processed using NMRPipe . Chemical shifts were referenced to water at 4.78 ppm for 1 H and external 15 NH 4 Cl (2.9 mM) in 1 M HCl at 208C at 24.93 ppm . weak ones were observed (Fig. 5). Nor was there any detectable label leak into the backbone based on the complete absence of any backbone resonances (between 7 1 15 and 9 H ppm and 108 and 126 N ppm, ), or resonances lacking partners at the same 15 N chemical 5 shift . The selectivity of the labeling signifies that all strong 15 N resonances observed can be safely ascribed to Gln side chain amides. 31 The HSQC of Mn SOD in Fig. 5 is not expected to contain signals from Gln146’s side chain 15 N as the latter ˚ from the bound Mn 31  and subject to severe is 4.6 A paramagnetic relaxation. The other 5 Gln side chain Ns are ˚ or further from the Mn based on the crystal structure 14 A of MnSOD , consistent with the five pairs of strong resonances observed. These are poorly resolved in the 15 N 1D spectrum. However, direct detection of 15 N permits observation of more rapidly relaxed 15 N nuclei. Indeed, an additional resonance is observed at 192 ppm in the 15 N spectrum of Fe 21 (Mn)SOD (Fig. 6). This was not present in either Mn 31 SOD or apo(Mn)SOD, presumably due to the stronger paramagnetic relaxation and lesser paramag- netic shifting conferred by Mn 31 in the former case , and the absence of paramagnetic shifting in the latter case. Thus, the extreme chemical shift reflects proximity to, and possible hyperfine interaction with Fe 21 . ˚ Gln146 is the only Gln with its side chain within 14 A ˚ from Fe 31 . The of Fe, and its N is only 4.4 A assignment of the 192 ppm signal to Gln146 is strengthened by this signal’s accelerated 1 / T 1 relaxation rate, evinced by its resistance to saturation in a rapidrepetition rate super-WEFT spectrum  that partially saturates the resonances of the other Gln side chain 15 Ns (Fig. 7).6 Like Gln69 of Fe 21 SOD , Gln146 of Fe 21 (Mn)SOD is strongly affected by Fe 21 . However, the effect is clearly different as Gln146’s 15 N is paramagnetically shifted by |79 ppm, almost twice as much as Gln69’s (shifted by |45 ppm). This difference could reflect differences in the electronic state of Fe 21 , its interactions with its ligands in general, its interactions with Gln in particular or the geometry of the active site. The gross similarity of the 1 H NMR spectra of Fe 21 SOD and Fe 21 (Mn)SOD indicate similar electronic structures for the Fe 21 in the two 5 Since side chain amides have two Hs bound to a single N, their HSQC resonances are characterized by two H resonances with identical 15 N chemical shifts, whereas back bone amides consist of a single H bound to an N and so occur as independent resonances that do not share a chemical shift with any other except fortuitously. 6 The 192-ppm signal also appears broader than the other Gln side chain N signals, indicating that it has faster 1 / T 2 relaxation, but this is not easily quantified due to the insensitivity of the spectra and the overlap of the diamagnetic 15 N signals. A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 Fig. 6. 15 N NMR spectra of Fe 21 SOD (top), Fe 21 (Mn)SOD (second), apo(Mn)SOD (third) and Mn 31 SOD (bottom). 1 H coupled spectra were collected at 258C at 50.655 MHz on a Unity plus 500 MHz spectrometer in a 5-mm broadband probe using 908 pulse excitation, 0.125-s acquisition times, no decoupling and 1-s delays, except for Fe 21 (Mn)SOD which was collected with a 608 excitation pulse and 0.3-s acquisition time. A total of 59 498 scans were collected for Fe 21 SOD, 102 048 for Fe 21 (Mn)SOD, 9 917 376 for apo(Mn)SOD and 152 368 for Mn 31 SOD. Thirty-Hz line broadening was applied to all spectra. Fe 21 (Mn)SOD was at pH 7.5 prior to reduction and the other samples were at pH 7.8 prior to reduction or oxidation. The chemical shift of Gln69 in Fe 21 SOD is affected by a pK of 8.5 but only moves by 10 ppm total between pH 6 and 10, and moves very little between pH 7.5 and 7.8. proteins and the comparable chemical shifts of the ligand His’ indicate similar degrees of covalency with respect to the His’ at least . In apparent contrast, the 55% difference between the two Glns’ 15 N chemical shifts 7 is striking, and suggests alteration of a specific relation between Fe 21 and the Gln side chain N. It is most likely that the very different Gln chemical shifts reflect different distances from Fe 21 to N in the (Fe)SOD and (Mn)SOD proteins. Although a structure has not been reported for Fe 21 (Mn)SOD, the N to Fe 31 ˚  vs. 5.1 A ˚ between distance in Fe 31 (Mn)SOD is 4.4 A ˚ in Fe 31 SOD ). N and Fe 21 in Fe 21 SOD (and 5.0 A Since dipolar paramagnetic chemical shifting scales as 1 /r 3 where r is the distance to the metal ion, the different distances to Fe suggest that the chemical shift of Gln146 will be |1.6 times that of Gln69. This accounts for a substantial fraction of the observed ratio of 1.8. Possible 7 34 /((45179) / 2)555% 15 253 21 Fig. 7. N NMR spectra of Fe (Mn)SOD collected using (top) super WEFT regime and (bottom) the 908-observe experiment. The 908-observe 1 spectrum was collected without H decoupling as in Fig. 5 and the super WEFT spectrum  employed a 44.5-ms inversion pulse followed by 15 0.6-s delay during which fast-relaxing N signals fully recovered but most 15 N signals decayed to close to zero magnetization, a 908 observe 1 pulse and 0.125-s acquisition (with H decoupling) followed by a 0.7-s relaxation delay. Thus, slowly relaxing signals are suppressed relative to fast relaxing signals. Thirty-Hz line broadening was applied to both spectra. displacement of Gln146’s side chain relative to Fe in Fe(Mn)SOD upon reduction is expected to increase the distance and decrease the ratio of the inverse cube dis˚ distance increase the ratio drops to tances (e.g. for a 0.1 A 1.5). The balance of the extra chemical shifting could reflect slightly different polar and azimuthal angles for the Gln relative to the Fe 21 susceptibility tensor, and / or a greater hyperfine contribution to paramagnetic chemical shift. The latter and the shorter distance to Fe in Fe(Mn)SOD are both suggestive of stronger hydrogen bonding. Weaker paramagnetic shifting than expected based on Fe–N distances in Fe 21 SOD does not appear to support 21 reversal of the side chain N and O of Gln69 in Fe SOD. If the side chain O instead of the NH 2 were closer to Fe 21 , ˚ (the then the Fe 21 to N distance would become 6.1 A distance to O in the reported crystal structure of Fe 21 SOD ). This distance results in a predicted chemical shift ratio of 2.7, much larger than is observed. Thus, barring cancellation of distance effects by orientational or scalar ones, it appears that the Gln side chain retains the same polarity in the reduced state of FeSOD as in the oxidized state. 254 A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 4. Discussion The active site Gln is one of a few conserved differences between Fe- and Mn-specific SODs. The Gln side chain hydrogen bonds with the coordinated solvent [2,15] thus tuning the degree of protonation of this ligand. This in turn tunes the Em of the metal ion. Thus, the nature of the interaction between the Gln side chain and the active site metal ion is important to understanding the metal ion specificity of SODs . To the extent that the active site Gln side chain can affect the metal ion, it is itself affected by the metal ion. The 15 N chemical shift is indicative of both electron density delocalization onto Gln via the hyperfine contribution, and position relative to Fe via the dipolar contribution. The very different chemical shifts of Gln146 and Gln69 can be substantially explained by the different distances from their Ns to Fe 21 , but may also include different hyperfine contributions to chemical shift as well. Thus, our NMR results extend to solution and the reduced state an important difference between the crystal structures of Fe 31 SOD and Fe 31 (Mn)SOD. The significantly shorter distance between Gln and the metal ion in (Mn)SOD was not noted in the crystallographic reports, but such positional differences may be more important than oft-noted differences in the chemical identities of the side chains in the active sites of (Fe)SOD and (Mn)SOD. A shorter N to Fe 21 distance in Fe 21 (Mn)SOD (and more hyperfine shifting) may imply stronger hydrogen bonding with coordinated solvent in Fe 21 (Mn)SOD than in Fe 21 SOD. Thus, the system with the higher Em , FeSOD, appears to have weaker hydrogen bonding between Gln and the coordinated solvent. Similarly, mutation of the active site Gln to Asn in human MnSOD resulted in a longer Mn to amide N distance and a substantially increased Em . The mutant active site incorporates an additional solvent molecule which is presumed to mediate hydrogen bond interactions between the Asn side chain and coordinated solvent . However, this indirect hydrogen bonding is likely weaker than direct hydrogen bonding. Thus, in both E. coli and human SOD, weaker hydrogen bonding is associated with a higher Em relative to MnSOD. It therefore appears that H bonding from Gln destabilizes additional electron density on the metal ion or has the effect of diminishing proton density around the metal ion. The active site Gln is ideally positioned to do this by stabilizing coordinated OH 2 over H 2 O (i.e., by increasing coordinated solvent’s acid dissociation constant). Eq. (1a) depicts proton and electron uptake in FeSOD, with the amino acid ligands shown only in one protonation and oxidation state, for simplicity. Since coordinated solvent is believed to take up a proton upon metal ion reduction in MnSOD (Vance and Miller, unpublished) as well as in FeSOD  (diagonal line in Eq. (1a)), the dissociation constant of the proton acceptor in the reduced state, Kred , is smaller than that in the oxidized state, Kox and it is the difference between Kred and Kox that determines the Em at a given H 1 concentration, (1a) 1 RT Kred 1 [H ] ] ]]]] Em 5 EAH 1 ln , F Kox 1 [H 1 ] (1b) where EAH is the reduction potential relating the protonated states (1a). The larger Kox and the smaller Kred , the lower Em should be. If the difference between the redox tuning of the two different SOD proteins is to be ascribed to different pK values for coordinated solvent, then DE, the Em of a metal ion bound to (Fe)SOD minus its potential when bound to (Mn)SOD, can be written in terms of four pK values: 0.5 V¯ DE( Fe)SOD – ( Mn)SOD 1 Mn (K Fe red 1 [H ]) 3 K ox 5 60 mV log]]]]]] Mn 1 K Fe ox (K red 1 [H ]) (2) where superscripts are used to identify the metal ion specificity of the SOD protein to which a dissociation constant pertains. Eq. (2) incorporates the simplification that Kox 4[H 1 ] for both FeSOD and MnSOD, the difference between the potentials of Fe in the two proteins is 0.46 V (Table 1) , and that of Mn is even larger (Vance Fe Mn and Miller, unpublished). Thus, either K Mn ox .K ox , (K red 1 1 Fe 1 Fe 1 [H ]),(K red 1[H ]) or both. However, K red <[H ] for Fe pH,11  so the relative magnitudes of K Mn red and K red should be much less important than the relative magnitudes Fe of K Mn ox and K ox . Thus, stronger hydrogen bonding from Gln to the coordinated solvent could produce the observed trend in Em values, via its larger effect on the oxidized state. For the oxidized state pK values to fully account for the redox tuning difference between (Fe)SOD and (Mn)SOD, the pK of H 2 O / OH 2 must be |8 pH units lower in (Mn)SOD than in (Fe)SOD, when a given metal ion is bound. An 8 pH unit shift in a pK is somewhat larger than A.L. Schwartz et al. / Journal of Inorganic Biochemistry 80 (2000) 247 – 256 has been reported in proteins so far, but pK shifts of 6 and 5 pH units are known . Therefore, it is possible for this mechanism to contribute a substantial fraction of the Em tuning difference between (Fe)SOD and (Mn)SOD. Since ionization of coordinated solvent has been assigned to a pK of 5.1 in Fe 31 SOD , a pK substantially lower than 5 and as low as 23.2 is predicted for Fe 31 (Mn)SOD. Such a pK is unlikely to be experimentally accessible. Similarly, coordinated solvent’s pK in Fe 21 (Mn)SOD should be very high, consistent with the absence of a pK ascribable to coordinated solvent in the range of pH 6–11 . Finally, the predicted pK values also indicate that proton uptake will always accompany reduction in Fe(Mn)SOD, justifying the application of the above equations to this case. The distance from Gln’s N to the metal ion is a property of the protein, not the result of the metal ion identity, since ˚ are observed in shorter distances of 4.4 and 4.6 A ˚ Fe(Mn)SOD and MnSOD, respectively, than the 5.1-A distance in FeSOD. This is a result of both the different backbone origins of the Gln in (Fe)SOD and (Mn)SOD, and the positioning of Gln’s side chain by the active site hydrogen bond network. The former is reversed in our G77Q,Q146A mutant, and results in restoration of small but significant Fe-supported activity. Our G77Q,Q146A mutant of MnSOD retains the active site Gln, but at the backbone location characteristic of FeSOD. Thus, it represents a very conservative but informative pair of mutations. While a crystal structure is not yet available, we can begin evaluating the integrity of the mutated active site by spectroscopic means. Subtle effects associated with nuclear movements too small to be unambiguously resolved by protein crystallography, but associated with sufficient distortion of the metal ion electronic structure and geometry to significantly affect activity, can be detected immediately by EPR, NMR and other spectroscopic methods that probe the metal ion electronics directly. Indeed, the altered metal ion coordination geometry observed in Fe(Mn)SOD crystallographically , was first emphasized based on EPR . Moreover the fact that only one of two crystallographic active sites displayed the altered geometry is also explained by the EPR data which showed that the novel coordination geometry is characterized by a pK of 8.4 , very close to the crystallization pH of 8.5 . Our current results demonstrate conferral of low but significant Fe-supported activity in (Mn)SOD upon relocation of the active site Gln. This and the work of Yamakura  are the first experimental validation of the proposal of Parker et al.  that the Gln is a determinant of metal ion specificity. However, since less than 10% WT Fe-supported activity is obtained, the Gln is clearly not the sole determinant of metal ion specificity (also ). Our spectra all demonstrate that the mutant retains basic active site integrity and metal ion binding, indicating that the low activity is unlikely to be due to gross structural 255 factors. However, the fact that the mutant’s active site resembles that of the (Mn)SOD parent more than the (Fe)SOD active site which the mutant’s Gln location emulates indicates that additional amino acids contribute to metal ion electronics and geometry. These residues most likely include the hydrogen bonding partners of Gln since these position and orient the Gln side chain relative to coordinated solvent, and they most likely include the other ‘missing’ determinants of metal ion activity. These residues may be identified by incorporating more amino acid substitutions into our G77Q,Q146A-(Mn)SOD. It may also be possible to correlate the subtle spectroscopic differences between G77Q,Q146A-(Mn)SOD, (Mn)SOD and (Fe)SOD with activity, and thus obtain insight into electronic bases for Em tuning and activity. Since the inactivity of WT Fe(Mn)SOD can be explained by an anomalously low Em  below that required for O ?2 oxidation, we anticipate 2 that Fe-G77Q,Q146A-(Mn)SOD’s small but significant activity will be accompanied by an increase in Em above that of Fe(Mn)SOD. Although the Fe-supported activity of Fe-mut-(Mn)SOD is low compared to WT FeSOD and MnSOD, it is comparable to the activities reported for cambialistic SODs. The SOD from Methylomonas J. has Mn-supported activity of 2300 u / mg protein and Fe-supported activity of 100 u / mg protein after correction for incomplete metal ion incorporation , that of Propionibacterium shermanii has activities of 900 and 700, respectively , Mycobacterium smegmatis SOD has activities of 5000 and 300  and Porphyromonas gingivalis SOD has Mn- and Fesupported activities of 1200 and 1000 . Our Fe-supported activity of at least 450 on a per Fe basis thus falls well within the range of activities considered biologically significant, and our mutation in effect converts MnSOD into a cambialistic SOD. 5. Notation 1D, one-dimensional; DPPH, a,a9-diphenyl-b-picryl hydrazine; DSS, 4,4-dimethyl 4-silapentane sodium sulfonate; EPR, electron paramagnetic resonance spectroscopy; FeSOD, Fe-containing superoxide dismutase; (Fe)SOD, the protein of FeSOD; Fe(Mn)SOD, Fe-containing (Mn)SOD protein; Fe-mut-(Mn)SOD, Fe-containing G77Q,Q146A(Mn)SOD protein; G77Q,Q146A-MnSOD, mutant MnSOD with glycine 77 mutated to glutamine and glutamine 146 mutated to alanine; MnSOD, Mn-containing superoxide dismutase; (Mn)SOD, the protein of MnSOD; Mn(Fe)SOD, Mn-containing (Fe)SOD protein; mutMnSOD, G77Q,Q146A-MnSOD, mutant MnSOD with glycine 77 mutated to glutamine and glutamine 146 mutated to alanine; NAS, non-active site; NHE, normal hydrogen electrode; NMR, nuclear magnetic resonance spectroscopy; PCR, polymerase chain reaction; SDS– 256 A.L. 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