Long-distance charge redistribution upon Cu , Zn-superoxide dismutase reduction : significance for dismutase function

Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is a ubiquitous enzyme with an essential role in antioxidant defense. To better understand structural factors at the origin of the highly efficient superoxide dismutation mechanism, we analyzed the consequence of copper reduction on the electronic properties of the backbone and individual amino acids by using electrochemistry coupled to Fourier transform infrared spectroscopy. Comparison of data recorded with bovine erythrocyte and recombinant chloroplastic Cu,Zn-SOD from Lycopersicon esculentum, expressed as a functional tetramer in Escherichia coli and (14)N- or fully (15)N-labeled, demonstrated that the infrared changes were dominated by reorganizations of peptide bonds and histidine copper ligands. Two main infrared modes of histidine side chain, markers of metal coordination, were identified by using Cu- and Zn-methylimidazole models: the nu(C(4)C(5))at 1605-1594 cm(-1) or approximately 1586 cm(-1) for Ntau or Npi coordination, and the nu(C(5)Ntau) at approximately 1113-1088 cm(-1). These modes, also identified in Cu,Zn-SOD by using (15)N labeling, showed that the electronic properties of the histidine Ntau ligands of copper are mostly affected upon copper reduction. A striking conclusion of this work is that peptide groups from loops and beta-sheet largely participate in charge redistribution upon copper reduction, and in contrast, electronic properties of polar and charged amino acids of the superoxide access channel remain unaffected. This is notably shown for the strictly conserved Arg-143 by site-directed mutagenesis on chloroplastic Cu,Zn-SOD. Charge compensation by the peptide backbone and preserved electronic properties of the superoxide access channel and docking site upon copper reduction may be the determinant factors for the high reaction kinetics of superoxide with both reduced and oxidized Cu,Zn-SOD.


Introduction
Superoxide dismutases (SOD) play a determinant role in protection against the toxic effects of oxidative stress by the scavenging of superoxide radicals and their conversion into oxygen and hydrogen peroxide (1). Four different classes of SOD have been distinguished depending on the metal at the active center, Mn, Fe, Cu and Zn, or Ni (2)(3)(4). Cu,Zn-SOD are ubiquitous. They appear as homodimers in the cytosol, the nucleus and peroxisome of eucaryotic cells and the inter membrane space of mitochondria. They exist as homotetramers in the chloroplast of higher plants and as monomers, in the periplasm of some gram negative bacteria (3).
The monomer fold and the active site are highly conserved within Cu,Zn-SOD according to structural data (5)(6)(7)(8)(9)(10) and high homologies of protein sequences (11)(12)(13). The monomer consists in a βbarrel and the active site defined by two loops (IV and VII) at the surface of the barrel. The active site comprises the redox active Cu with square planar coordination to four histidines (Scheme 1A), one of each is a bridging ligand of Cu and Zn. The other Zn ligands are two histidines and a monodentate aspartate (5,7,8). A channel across the protein leads to the Cu, towards a fifth axial coordination position, occupied by a water molecule in the oxidized enzyme, which is available for inhibitors and possibly for the superoxide substrate (Scheme 1B, [14][15][16]. SOD and particularly Cu,Zn-SOD are characterized by a very high reaction rate (2x10 9 M -1 s -1 ), near the diffusion rate of superoxide which is pH independent between 4.8 and 9.7 (3,17).
Experimental data demonstrated the role of a positive electrostatic gradient formed by charged residues on the superoxide access channel, including a strictly conserved arginine (Arg141, bovine erythrocyte SOD numbering -corresponds to Arg143 for plants and human Cu,Zn-SOD) to steer highly efficiently the anionic superoxide towards the buried Cu (18)(19)(20). Up to now, the high reaction rate prevented the elucidation of reaction intermediates in superoxide dismutation. Another specificity of these enzymes is their equivalent efficiency for both reactions of superoxide oxidation and reduction (21,22). These reactions are coupled with the reduction and oxidation of the Cu, by guest on October 5, 2017 http://www.jbc.org/ Downloaded from respectively. Structural factors at the origin of this specific and efficient dismutase mechanisms are not fully understood. Thorough analyses at the molecular level of structural changes that accompany Cu reduction have been undertaken to detail the active site properties at the origin of the efficient superoxide dismutation and of the protonation events associated to its reduction into hydrogen peroxide (3,5,10,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). A main result concerns the rupture of the interaction between reduced Cu(I) and the His63 bridging ligand, as deduced from NMR (23,24, see also 10,25), EXAFS (26,27) and Raman spectroscopy (28)(29)(30) for Cu,Zn-SOD in solution. Three-dimensional crystallographic structures of reduced Cu,Zn-SOD presented heterogeneity of the active center (16,(31)(32)(33), but a recent high-resolution structure provided convincing evidence for three coordinated Cu(I) (34), in agreement with other spectroscopic data.
This rupture of the Cu-His61 bond upon Cu reduction was the base of the generally accepted reaction mechanism of Tainer et al. (35), in which His61 provides one of the protons necessary for superoxide reduction into hydrogen peroxide at Cu(I). Indeed, NMR data paralleled the loss of Cu,Zn-SOD activity at high pH with His61 deprotonation (36). Also, coordination of His61 to Zn would determine the large pH independence of Cu,Zn-SOD activity (37).
Binding of superoxide as a ligand of Cu(II) and Cu(I) is debated, so as inner or outer sphere electron transfer mechanisms to reduce (oxidize) superoxide (35,38), and the specific role of Arg141 as a docking site for superoxide in reduced Cu,Zn-SOD was proposed (16,38,39).
Experimental data demonstrated thus a crucial role for Arg141 and for charged and polar residues building an electrostatic field in the superoxide access channel for the efficient function of Cu,Zn-SOD (18-20, 38-41) as a dismutase. Since this electrostatic field was predicted from calculations to be contributed not only by charged side chains but also by the Cu and polypeptide backbone (42), we decided to monitor directly the changes in electronic properties of highly conserved amino acids and of peptide bonds upon Cu reduction using Fourier transform infrared (FTIR) spectroscopy. A former study comparing second derivatives of infrared absorption spectra of oxidized or reduced Cu,Zn-SOD by guest on October 5, 2017 http://www.jbc.org/ Downloaded from concluded that conformational changes occur at the level of loop and β-sheet structures upon copper reduction (43). In this work, we used electrochemically-induced FTIR difference spectroscopy (44, 45, for reviews on the technique) to detect, on the same sample, minute perturbations of the electronic properties or hydrogen bonding interactions of both amino acid side chains and of the polypeptide backbone.
We analyzed the redox-induced infrared changes detected on dimeric bovine erythrocyte (BeSOD) and tetrameric chloroplastic (ChSOD) Cu,Zn-SOD of Lycopersicon esculentum, which differ at the level of charged and polar residues of the superoxide access channel (3,12,42,46).
Recombinant ChSOD was expressed in E. coli and 15 N labeled to assign with confidence the vibrational modes from histidine Cu ligands, peptide groups, or conserved amino acid side chains. Site directed mutants at the strictly conserved Arg143 and Thr137 (plant numbering), located in the superoxide access channel were performed to precise their contribution to the electrostatic field reorganization upon Cu reduction in ChSOD.

Samples preparation
BeSOD was purchased from Sigma. Gel electrophoresis on different batches of samples showed that the commercial preparation was sufficiently pure and could be used directly for the FTIR spectroscopic studies (Supplementary Figure 1A). The BeSOD concentration was estimated by the absorbance at 680 nm using ε 680 = 300 M -1 cm -1 and the specific activity determined spectrophotometrically, by the measure of the inhibition of cytochrome c reduction by xanthine oxidase, as described in (47 to ≈10 µL using a microcon 10 (Millipore) system. This sample was diluted again in 400 µL deuterated buffer, incubated for two weeks at 10°C for extensive 1 H/ 2 H exchange, and washed again by concentration / dilution steps in 2 H 2 O-buffer before analysis by FTIR spectroscopy.

Expression of chloroplastic Cu,Zn-SOD
The cDNA gene of ChSOD from Lycopersicon esculentum was kindly provided by Dr. Perl-Treves (46) on the pGEM2 plasmid (Promega). A pair of restriction-enzyme sites (NdeI at the 5' end and BamHI at the 3' end, bold faced characters) were incorporated flanking the cDNA gene of ChSOD by PCR amplification using the oligonucleotide primers : The 480 bp PCR amplified fragment was purified, digested by NdeI and BamHI, and cloned into the NdeI/BamHI-digested pet11-a expression plasmid from Stratagene (Stratagene, La Jolla, USA) using the E. coli strain DH5α. The resulting plasmid pChSOD-1 was used to transform E. coli BL21 plys DE3 strain, allowing IPTG induced expression of the recombinant ChSOD. All digestions, ligations, transformations and other manipulations were performed using standard protocols (48). The DNA sequence was verified by sequencing.
The pChSOD-1 transformed E. coli BL21 plys DE3 cells were grown in minimal M9 medium in the presence of thiamin, ampicillin and chloramphenicol at 10 µg/mL, 100 µg/mL and 25 µg/mL respectively. Induction was triggered by the addition of IPTG at 1 mM/L when the cultures reached an optically density (A 600 ) of 0.5. The cells were harvested 4 hours after induction.
For the production of uniformly 15 N labeled ChSOD, the same supplemented M9 medium was used except that 15

Site directed mutagenesis of ChSOD
The site directed mutants were obtained by two steps of PCR using the oligo nucleotides 1 and PCR were performed with oligos 1 and 4 and with oligos 2 and 3. The PCR products from this first step were purified, mixed at a 1 to 1 ratio and used to perform the second PCR step to amplify the whole gene using the initial oligonucleotides 1 and 2.

ChSOD purification.
Typically, the cells from 8 liters culture were harvested by centrifugation for 10 min at 7000 g.
The cells suspended in K-phosphate 10 mM at pH 7.8, KCl 8 mM were passed twice through a French press (at 14 MPa). The suspension of soluble proteins was obtained after centrifugation at 5000 g for 10 min at 4 °C followed by centrifugation at 200000 g for 1 hour at 4 °C to discard unbroken cells and membranes. This cell-free extract was loaded onto a DEAE column (80 mL) equilibrated with K-

Sample preparation for FTIR spectroscopy
For spectro-electrochemitry, the Cu,Zn-SOD concentration was comprised between 0.8 and The potential was applied to the electrochemical cell using an EG&G (model 362) potentiostat, triggered by the FTIR spectrometer. The redox titration of the Cu,Zn-SOD Cu center was performed by monitoring the potential-dependent UV-Vis absorption spectra, using the same electrochemical cell and conditions as for FTIR. UV-Vis spectra were recorded with a Varian Cary 50 spectrophotometer. The data were fitted with a Nernst curve using the Erithacus software Grafit.

Infrared spectroscopy
FTIR spectra were recorded at 4 cm -1 resolution, with a Bruker 66 SX spectrometer equipped with a KBr beam splitter and nitrogen-cooled MCT-A detector. The absorption maximum of the sample was optimized at 0.85 -0.9 a.u. at 1645 cm -1 . Single beam spectra recorded before and after a change of the applied redox potential were subtracted to calculate the redox-induced difference spectrum. Typically, 300 scans were accumulated for each spectrum recorded for one electrochemical cycle. The results from 10 to 40 independent redox cycles were averaged to improve the signal to noise. could also be obtained by direct electrochemistry at the PATS-3 modified electrode but its reoxidation necessitated the presence of redox mediators. The redox dependence of the UV-Vis absorption of BeSOD was analyzed in the 500 to -400 mV range, as shown in Figure 1 consecutive electrochemical cycles could be performed to record the FTIR difference spectra associated with copper reduction or oxidation ( Figure 2).

FTIR difference spectra associated to Cu,Zn-SOD reduction
The FTIR difference spectrum associated with copper reduction is shown in Figure 2 Fixation of fluoride and chloride at an anion binding site, involving Arg143 in the superoxide access channel of Cu,Zn-SOD was reported notably by NMR (59,60). FTIR difference spectra obtained using 100 mM NaF, KCl, KBr, or NaClO 4 as supporting electrolytes presented however only very small differences (not shown) and all spectra discussed below were recorded with 100 mM KBr.
The largest bands of the FTIR difference spectra of Figure  IR signals from peptide groups may superimpose with those from amino acid side chains, for arginine, histidine or aspartate and glutamate (65)(66)(67). Also, spectra of Figure

Effect of H 2 O/ 2 H 2 O exchange
The reduced -minus -oxidized spectra recorded with BeSOD in H 2 O (thin line) and in 2  The vibrational changes of peptide groups of the β-barrel upon Cu reduction may be mediated by the three histidine Cu ligands that are anchored in the barrel (5)(6)(7)(8). We therefore analyzed the contribution from histidine Cu ligands in the redox-induced FTIR spectra recorded with BeSOD, using IR markers of histidine -metal coordination identified in spectra of metal -methylimidazole models.

IR markers of imidazole -metal interactions
The methylimidazole (MeIm) IR modes have been precisely described (72)(73)(74)(75)(76) and are reported in Table I Table I). As detailed below, these modes are also sensitive to metal coordination.
Literature data on Raman spectroscopy show that a number of vibrational modes of histidine side chains are sensitive to metal coordination. This was precisely described on His-Cu and His-Zn models of known structure, as well as on peptides modeling the Cu binding site of the prion protein, or the Zn/Cu binding site of amyloid β-peptides (28,(77)(78)(79). The ν(C 4 C 5 ) mode occurs at 1590-1580 cm -1 for histidine coordinated with Nπ to Cu or Zn (Scheme 2-d) and at higher frequency, 1606-1594 cm -1 , for histidine coordinated with Nτ (28, 77-79) (Scheme 2 and Table I upon MeIm coordination to hydrated Zn.

IR modes of histidine Cu-ligands in BeSOD
Comparison of the redox-induced FTIR difference spectra obtained with BeSOD with the spectra of model compounds supported the assignment of signals at 1112(+) and 1097(-) cm -1 in  (82,84). This band presented some structure and probably corresponded to more than one residue. In BeSOD, two histidines, His118 and His46 2 , are coordinated through the Nτ to Cu. The spectrum showed that Cu reduction induced a significant upshift by 15 cm -1 of this mode, that may be assigned to a lesser electronegative character of the histidine ring for reduced BeSOD. Contribution of the bridging His61 ligand for oxidized BeSOD was not expected at 1097 cm -1 but rather near 1045 cm - The signals at 1608 and 1592 cm -1 (reduced BeSOD,

Redox-induced infrared changes in chloroplastic SOD
BeSOD and ChSOD have 68% of sequence homology. As compared to BeSOD, ChSOD lacks some charged residues in the superoxide access channel and is functional as a tetramer. Comparison of Further, the positive band at 1111 cm -1 was downshifted to 1102 (-9) cm -1 and the 1095 cm -1 signal to 1086 cm -1 (-10 cm -1 ). These shifts are in agreement with the ≈ 7 cm -1 downshift observed for the histidine ν(CNτ) upon 15 N-labeling (80,(82)(83)(84). The bands at 1111 and 1102 cm -1 for reduced ChSOD and at 1095 and 1086 cm -1 for oxidized ChSOD presented some structure corresponding to more than one chemical group. Finally, a small band at 1225 cm -1 , downshifted by 5 cm -1 upon 15 Nlabeling may also be due to histidine ring vibration (84).

Identification of arginine and threonine side-chain IR modes
Arginine 143 : The side-chain of the strictly conserved Arg143 is located in the superoxide access channel of Cu,Zn-SOD, at ≈ 6 Å of the active site Cu (5,10,34,35). Its positive charge is thought to play determinant role in superoxide attraction and to greatly enhance its reaction kinetics with Cu (18, 19,41). To identify the IR side-chain modes of Arg143, we performed the Arg143Gln mutation on recombinant ChSOD, using the heterologous expression system described above. In this mutant, the activity towards superoxide scavenging was largely impaired, with about 10 % of the activity of WT ChSOD (380 U.mg -1 ). This result is in line with literature data on Arg143 mutants with neutral side chains of human Cu,Zn-SOD (Ile and Ala, 18, 19,41).
Comparison of the reduced -minus -oxidized FTIR spectra recorded with WT (thin line) and Arg143Gln mutant (bold line) is shown in Figure 6-A. The spectra present overall similar shape with a large number of conserved bands. Reproducible changes are displayed more precisely in the mutantminus -WT difference spectrum of Figure 6-B. They may be due to the side-chain modes of Arg, Gln or to other amino acids perturbed by the mutation.
The signals at 1687, 1671 and 1661 cm -1 in Figure 6-B are in the frequency range of the arginine ν as (C 2 N 3 H 5 ) and glutamine or peptide ν(C=O) IR modes. 15 N-labeling of WT ChSOD showed however that no IR mode sensitive to 15  Arg143 were sensitive to Cu reduction. We conclude that while the side chain of Gln is sensitive to the redox state of the Cu, the arginine side-chain in WT Cu,Zn-SOD remains unaffected by the reduction (oxidation) of Cu.
The mutant -minus -WT difference spectrum of Figure 6-B presents small changes at 1648, 1538, 1526, and 1512 cm -1 that correspond to peptide ν(C=O) and ν(CN)+δ(NH) modes. They could arise from a peptide hydrogen bonding network around Arg143, that is perturbed upon Arg143Gln mutation. Finally the bands at 1100 and 1093 cm -1 were assigned to one histidine ligand of the Cu, whose electronic properties are perturbed by the Arg143Gln mutation.
Threonine137 : Thr137 is a strictly conserved amino acid that determines the size of the superoxide access channel together with Arg143 (5,35). Mutation of Thr137 into a Gln was undertaken and the FTIR difference spectra recorded with the WT and Thr137Gln mutant were superimposed in

Infrared modes of histidine ligands
Infrared studies on protoporphyrin-methylimidazole model compounds and on isolated cytochrome b559 (84) or on the non heme iron of photosystem II coordinated by four histidine (80)(81)(82) showed an histidine IR mode at ≈ 1100 cm -1 characteristic for histidine ligands of the iron. This signal, assigned to a His ν(C 5 Nτ) ring mode by normal mode calculations (86) was also observed for the http://www.jbc.org/ Downloaded from methyl-imidazolate contributes at lower frequency (at 1099 cm -1 ) than the ligand methyl-imidazole (at 1103 cm -1 ) in the protoporphyrin IX-methylimidazole model compounds (84). Such a downshift is also predicted for Nτ methylimidazolate ligands by DFT calculations (86).
In this work, we also show that the ν(C 4 C 5 ) IR mode of histidine side-chains can be used in metalloenzymes, as a marker of coordination to a metal. We identified the ν(C 4 C 5 ) IR mode of histidine ligands of the Cu, on the basis of a comparison with Raman data (28,(77)(78)(79), Several of the modes identified by Raman spectroscopy for oxidized Cu,Zn-SOD were assigned to the His63 bridging ligand (28)(29)(30). In particular, the ν(C 4 C 5 ) mode of bridging His63 imidazolate was assigned at 1567-1555 cm -1 (28)(29)(30)79). No signals were assigned to the other histidine ligands of the copper using UV-Raman. In the present work, we tentatively assigned a small band at 1560 cm -1 to the bridging imidazolate in oxidized ChSOD.
The new low-temperature structure of reduced BeSOD, at 1.15 Å resolution showed that Cu reduction induces a displacement by 1.3 Å of the Cu relative to its position in oxidized BeSOD which together with the rupture of the imidazolate bridge between Cu and Zn increases the distance between Cu and Zn from ~6 Å for oxidized BeSOD to 6.9 Å for reduced BeSOD (34). The movement also involves His118 (Nτ) which maintains the usual distance to the copper. The FTIR data suggest that the electronic properties of the His118 ring are affected by the Cu reduction together with that of His46.

Contribution from peptide amide I and II modes
One striking result of the present FTIR study is that most of the infrared changes detected upon Cu

Contribution of Arg143, polar and charged amino acids in the superoxide access channel
Charged Glu (Glu130 and 131) and Lys (Lys120 and 134) of the superoxide access channel in BeSOD are substituted in ChSOD by uncharged residues (5,7,46). The FTIR difference spectra recorded with BeSOD and ChSOD showed overall similarity, meaning that these residues do not contribute significantly in the spectrum recorded with BeSOD. Mutation of these residues in human  Cu,Zn-SOD (34,35) led to the conclusion that the hydrogen bonding network involving Arg sidechain constrains the guanidium group, ideally oriented as a docking site for superoxide. The FTIR data presented here show that the properties of the Arg143 side chain are maintained whatever the redox state of the copper, which is not the case of the substituted glutamine side chain. The fact that the overall fold of the access channel stabilizes the properties of Arg143 side chain whatever the redox state of the copper may be crucial for the function of Cu,Zn-SOD as a dismutase, with high attractive power towards superoxide of both reduced and oxidized Cu.
In conclusion, the long distance charge redistribution observed at the level of peptide groups upon Cu reduction and the preserved electrostatic properties of the superoxide access channel may constitute a specific mechanism of Cu,Zn-SOD that contrasts to the reorganizations observed for the amino acid side chains near the Fe active site of superoxide reductase (85) and may be a key factor of the superoxide dismutase function.

Acknowledgments
We gratefully acknowledge Perl Treaves for the gift of the pGEM plasmid bearing the cDNA of the     Arg143Gln -minus -WT difference spectrum calculated from spectra of Figure 6-A; C) reducedminus -oxidized FTIR spectra recorded with Thr137Gln mutant (bold line) and WT ChSOD (thin line) in Ches pH 9.3; D) Thr137Gln -minus -WT difference spectrum calculated from spectra of