Supplementary Material s85

Supplementary material

Figure S1. Reference UV-visible spectra of PMC resting state, compound I and compound II. Complete conversion into compound I (dashed line) was obtained from native PMC (solid line) (4 µM heme catalase) in 0.1 M Tris-maleate buffer (pH 6.9), by reaction with 0.42 mM peracetate (final concentration), from 42 mM peracetate stock solution in 0.1 M Tris-maleate pH 5.1 (containing 60 nM heme catalase to eliminate contaminating hydrogen peroxide). Complete conversion into compound II (dotted line) was obtained from compound I preparation by further addition of 10 mM ascorbate and 0.1 mM ferrocyanide (final concentrations). The corrected spectrum of LpH II was deduced from 0.30 × S(6.2) – 0.17 × S(7.9) ≈ 0.4078 × S(C-II) + 0.3420 × S(RE) – 0.0707 × S(C-I), that is S(LpH II) ≈ 0.30 × S(6.2) – 0.17 × S(7.9) – 0.3420 × S(RE) + 0.0707 × S(C-I). Similarly, the corrected spectrum of HpH II was deduced from S(7.9) – S(6.2) ≈ 0.5361 × S(C-II) – 0.5669 × S(RE) + 0.3361 × S(C-I), that is S(HpH II) ≈ S(7.9) – S(6.2) + 0.5669 × S(RE) – 0.3361 × S(C-I). The optimized coefficients have been obtained by a standard least-squares procedure over the (600-800) nm range, the tails of both HpH II and LpH II spectra are expected to be nearly indistinguishable.

Figure S2. Mössbauer spectra of as-isolated 57Fe catalase (PMC) from P. mirabilis (2.2 mM catalase heme in 0.1 M Tris-HCl buffer, 8 % (v/v), pH = 7.5) at 4.2 K, in a magnetic field of (A) 50 mT, (B) 7.0 T applied parallel to the g-beam. The experimental spectra were fitted (solid curves) with the parameters published elsewhere (Horner et al., J. Inorg. Biochem. 2006, 100, 477-479). PMC and MLC (Maeda et al., Biochim. Biophys. Acta, 1973, 303, 230-236) have very close quadrupole splitting values (DEQ = 0.98(4) mm/s and 0.88 respectively) and isomer shift values (d/Fe = 0.45(2) mm/s and 0.44 mm/s, respectively). The isomer shift value can be computed for a model of the native PMC active site (S=5/2) which we geometry-optimized (model 1 minus the apical oxygen atom). The electronic density at the iron nucleus is r(Fe) = 25.5787 (a.u.)-3. With the following correlation expression derived for FeIII ions and here established with ADF precision 6, d/Fe (mm/s) = 7.514 – 0.2774 r(Fe), we obtain a predicted isomer shift of 0.42 mm/s that compares well with the experimental value (d/Fe = 0.45(2) mm/s).

Figure S3. Mössbauer spectrum of compound I in 57Fe catalase from P. mirabilis at 40 K in a magnetic field of 3T applied parallel to the g-beam. Compound I (2.6 mM catalase heme) was obtained by addition of 14 mM peracetic acid, in 0.1 M Tris-maleate buffer, 8 % glycerol (v/v), final pH = 5.8, as described in Materials and Methods. The experimental spectrum was simulated (solid curve) with the parameters set of Table 2.

Figure S4. Mössbauer spectrum of compound II in 57Fe catalase from Proteus mirabilis at 150 K. The spectrum of P. mirabilis catalase compound II (1.1 mM final catalase heme concentration, in 0.3 M Tris-maleate buffer, 8% (v/v) glycerol, obtained using four successive, but alternative, additions of peracetic acid and ascorbic acid up to final concentrations of 20 and 110 mM, and further acidified by citric acid until a final pH of 5.3 ) was recorded at 150 K in a magnetic field of 7.0 T applied parallel to the g-beam. The experimental spectrum were fitted (solid curve) with the parameters set of Table 1. The solid curves above the experimental spectra show the contribution of each iron site ((a) : FeIV from LpH II, (b) : FeIII from native enzyme, (c) FeIV from compound I).

Figure S5. Comparison between the visible absorption spectra of compound II samples used for EXAFS and Mössbauer measurements. The absorption spectra were measured at 5°C after about 5-10 fold dilution of compounds II in distillated water and normalised on the spectrum of the Mössbauer compound II sample. For the Mössbauer sample, PMC compound II (1.4 mM heme 57Fe catalase), was obtained by addition of peracetic acid and ascorbic acid up to the final concentrations of 20 mM and 110 mM respectively in 0.3 M Tris-maleate buffer, 16% (v/v) glycerol, final pH 7.9. For the EXAFS sample, PMC compound II (1.2 mM heme 56Fe catalase), was obtained by addition of peracetic acid and ascorbic acid up to the final concentrations of 16 mM and 143 mM respectively in 0.3 M Tris-maleate buffer, 16% (v/v) glycerol, final pH 8.0. Spectra of EXAFS (black line) and Mössbauer (gray line) compound II samples.

Figure S6. Experimental EXAFS of PMC compound II from P. mirabilis at pH 8.0 with results of the EXAFS analysis considering two distinct iron-oxo contributions. The experimental EXAFS data of PMC compound II from P. mirabilis (dashed curve) compared to the theoretical curve of fit (solid), together with the detail of the n-body signals used (solid) and the residual curve (spaced dashed), are reported in the upper part. The theoretical signals coming from the two Fe-O contributions are dashed. The Fourier transforms of the experimental curve (dotted) and of the total theoretical curve (solid) are reported in the lower

part.

Figure S7. Projection of the EXAFS minimization function, considered for fit 2, on the (R-Fe=O, R-Fe-OH) plane, i.e. contour plot (regions enclosed by squares correspond to the 95% confidence interval). The function presents two clearly separated minima in connection with the two combinations of these values, indicating that two distinct distances are present. The shape of the two internal contour regions (internal axes reasonably parallel to the main reference axes) indicates that the two distances are only moderately correlated.

Figure S8. Linear correlation between the computed quadrupole splitting DEQ and the computed electronic density at the iron nucleus r(Fe), by using all six FeIV=O models of compound II at high pH and four possible FeIV-OH models of compound II at low pH (cf. Table S4). By using the six FeIV=O models of compound II at high pH (models 1 and 3 plus variants: see Table 4), we obtained the following linear correlation : DEQ (mm/s) ≈ 1.7979r(Fe) – 46.10. For the Fe-O(R) species, the equivalent correlation is given by DEQ (mm/s) ≈ 1.3413 r(Fe) – 33.38.

Table S1.

(a) Optimized coordinates (Å) for Model 1

Fe 0.002622 0.003327 -0.041642

C 2.826826 1.172302 -0.079431

C 4.226404 0.810081 -0.170865

C 5.370721 1.774648 -0.210937

C 4.263122 -0.570432 -0.263318

C 5.397775 -1.464906 -0.431233

C 6.679834 -1.247869 -0.075500

C 2.872328 -1.016001 -0.215928

C 2.481089 -2.347808 -0.271471

C 1.179610 -2.825252 -0.164484

C 0.825269 -4.227800 -0.175285

C 1.775912 -5.363477 -0.403477

C -0.549817 -4.281791 -0.011206

C -1.432609 -5.435807 0.082007

C -1.111357 -6.685528 0.487839

C -1.002726 -2.896345 0.050559

C -2.349985 -2.505973 0.127741

C -2.815930 -1.183961 0.142145

C -4.226095 -0.819641 0.115393

C -5.370691 -1.790526 0.050254

C -4.262120 0.553709 0.108314

C -5.476954 1.438134 0.100866

C -5.961435 1.813952 1.532597

C -7.198769 2.679090 1.457432

C -2.877118 1.008267 0.124568

C -2.487727 2.343242 0.085339

C -1.169483 2.803104 0.064305

C -0.794160 4.213401 0.023484

C -1.744885 5.372466 0.072932

C -2.226069 5.678795 1.506116

C -3.175930 6.857681 1.566374

C 0.575126 4.246500 -0.017265

C 1.492858 5.429898 -0.059015

C 1.019882 2.861166 0.000839

C 2.353676 2.479394 -0.044590

H 5.171471 -2.422368 -0.911176

H 6.998200 -0.344426 0.443077

H 7.446152 -1.994427 -0.283495

H -0.106313 -6.951435 0.821114

H -1.862932 -7.476728 0.505853

H -2.477575 -5.258189 -0.197372

H 1.253255 -6.214734 -0.860487

H 2.593503 -5.070670 -1.076186

H 2.238062 -5.729837 0.528683

H 6.191873 1.374581 -0.821851

H 5.071549 2.741524 -0.635930

H 5.777300 1.971580 0.794596

H 0.929081 6.369421 -0.145420

H 2.107282 5.496560 0.854295

H 2.182673 5.378029 -0.916857

H -1.270292 6.273642 -0.341105

H -2.623768 5.180015 -0.564191

H -2.703561 4.802612 1.968823

H -1.363263 5.919688 2.152491

H -5.280221 2.366563 -0.457635

H -6.299371 0.927827 -0.425533

H -6.172519 0.898969 2.103981

H -5.179363 2.386740 2.049802

H -6.336100 -1.266974 0.010571

H -5.302610 -2.427915 -0.846743

H -5.398070 -2.461673 0.925011

H 3.093402 3.280396 -0.070582

H -3.276682 3.095905 0.052613

H -3.104815 -3.292588 0.128076

H 3.267844 -3.089927 -0.393931

H -9.081108 2.579060 1.415377

H -4.527837 7.614849 2.637202

N 2.027659 0.055205 -0.076719

N 0.060498 -2.037971 0.001626

N -2.019680 -0.062752 0.162277

N -0.054277 2.005715 0.063528

O -3.958851 6.816517 2.692438

O -7.213810 3.885431 1.296833

O -8.351261 1.931449 1.528546

O -3.257051 7.756444 0.748122

O -0.111682 0.005354 -1.687903

C 1.532764 2.530034 4.347833

C -0.878014 2.653290 4.363678

C 1.473255 1.467049 3.444079

C -0.943342 1.586244 3.461735

C 0.238763 0.976908 2.982034

C 0.359264 3.136100 4.814843

H 2.390861 1.006794 3.066140

H -1.907493 1.213216 3.106787

H 2.508136 2.892713 4.686379

H -1.803329 3.114332 4.720519

H 0.409457 3.971519 5.517557

O 0.213606 -0.089963 2.144913

C 2.692155 -2.765318 3.374114

C 0.230965 -2.829271 3.413492

N 1.374145 -2.179582 3.210466

N 0.239913 -4.127563 3.862515

N -0.925374 -2.157093 3.260821

H -0.774638 -1.227578 2.761093

H -1.750463 -2.700194 3.019297

H 1.061683 -4.669280 3.602001

H -0.629242 -4.642199 3.736101

H 1.210141 -1.237052 2.731027

H 3.438012 -1.999859 3.131035

H 2.860636 -3.099718 4.410510

H 2.857635 -3.613103 2.683443

Table S1.

(b) Optimized coordinates (Å) for Model 1ter

Fe 0.008246 0.020020 -0.034399

C 2.837663 1.188874 -0.124801

C 4.238538 0.826287 -0.190513

C 5.384611 1.789221 -0.227614

C 4.277808 -0.556961 -0.244002

C 5.409286 -1.455964 -0.373174

C 6.706901 -1.225241 -0.077253

C 2.883240 -0.999772 -0.193924

C 2.490340 -2.332613 -0.186891

C 1.188309 -2.805591 -0.072487

C 0.834656 -4.211448 -0.059085

C 1.793565 -5.349383 -0.219093

C -0.542347 -4.264575 0.066902

C -1.432398 -5.412618 0.094202

C -1.144419 -6.691574 0.428656

C -0.992579 -2.873303 0.109254

C -2.337472 -2.481849 0.177988

C -2.807384 -1.161291 0.163973

C -4.219165 -0.802470 0.162672

C -5.360954 -1.779073 0.169016

C -4.260155 0.570182 0.117883

C -5.478388 1.447685 0.119076

C -5.945925 1.843723 1.554344

C -7.193343 2.686031 1.472529

C -2.875025 1.027906 0.088414

C -2.484846 2.364598 0.029782

C -1.163033 2.825376 0.000391

C -0.785857 4.236333 -0.023873

C -1.731339 5.399277 0.047969

C -2.223765 5.673882 1.483713

C -3.154293 6.862378 1.577029

C 0.584181 4.267053 -0.057264

C 1.505716 5.448572 -0.059335

C 1.026857 2.880701 -0.055357

C 2.361764 2.496433 -0.097111

H 5.166505 -2.448420 -0.766535

H 7.050770 -0.291590 0.365247

H 7.457612 -1.996965 -0.250330

H -0.159128 -6.998360 0.782579

H -1.917841 -7.461053 0.387703

H -2.470452 -5.203139 -0.190928

H 1.298048 -6.205606 -0.698903

H 2.653921 -5.065069 -0.840857

H 2.189057 -5.699564 0.748801

H 6.200816 1.398216 -0.852380

H 5.081949 2.763759 -0.633314

H 5.801124 1.968486 0.777991

H 0.947109 6.392930 -0.139029

H 2.101528 5.494724 0.868058

H 2.215158 5.410830 -0.902491

H -1.248194 6.308587 -0.339036

H -2.608238 5.227946 -0.598428

H -2.717464 4.791048 1.916952

H -1.365769 5.874912 2.150424

H -5.296805 2.372193 -0.451083

H -6.309710 0.927518 -0.385163

H -6.132681 0.936304 2.145073

H -5.159514 2.434119 2.044433

H -6.331185 -1.262309 0.126285

H -5.310340 -2.460515 -0.697035

H -5.361068 -2.407870 1.075246

H 3.101970 3.297800 -0.109452

H -3.273835 3.118439 0.004330

H -3.089100 -3.269795 0.227430

H 3.277936 -3.080939 -0.251317

H -9.071398 2.577507 1.509886

H -4.448467 7.649638 2.690117

N 2.042263 0.072773 -0.111251

N 0.067929 -2.019796 0.044746

N -2.019643 -0.040101 0.129010

N -0.049367 2.030405 -0.009628

O -3.891615 6.843096 2.738236

O -7.242652 3.876642 1.216478

O -8.340183 1.936922 1.644702

O -3.268008 7.763294 0.762308

O -0.097526 -0.024906 -1.703163

C 1.439579 2.408559 4.412299

C -0.966591 2.547106 4.340231

C 1.414427 1.407291 3.441825

C -1.001163 1.546208 3.364092

C 0.198924 0.951469 2.867758

C 0.252004 3.000744 4.872590

H 2.341185 0.948517 3.084734

H -1.953143 1.193627 2.958670

H 2.404355 2.739003 4.815134

H -1.906575 2.984953 4.695074

H 0.277254 3.791296 5.628579

O 0.221124 -0.009612 1.967824

Table S1.

(c) Optimized coordinates (Å) for Model 2

Fe -0.010036 -0.004900 0.021462

C 2.819180 1.201185 -0.095194

C 4.217616 0.845569 -0.203030

C 5.356181 1.815230 -0.269230

C 4.259688 -0.536683 -0.284092

C 5.399499 -1.427319 -0.459066

C 6.676389 -1.193157 -0.102807

C 2.875932 -0.991132 -0.220788

C 2.493340 -2.324926 -0.257376

C 1.196203 -2.805816 -0.156063

C 0.850110 -4.207584 -0.175538

C 1.812695 -5.338197 -0.365625

C -0.529161 -4.273500 -0.041739

C -1.409802 -5.435477 -0.017415

C -1.091603 -6.692692 0.355171

C -0.996007 -2.899913 0.035449

C -2.341013 -2.530608 0.137190

C -2.813165 -1.214498 0.170258

C -4.221891 -0.858085 0.160552

C -5.359801 -1.836049 0.144724

C -4.269252 0.515376 0.129624

C -5.489621 1.392564 0.108051

C -6.026283 1.714493 1.532330

C -7.094264 2.779649 1.459408

C -2.891748 0.982007 0.128827

C -2.514910 2.319127 0.083580

C -1.200452 2.786849 0.067355

C -0.838350 4.192526 0.024296

C -1.796645 5.346201 0.057652

C -2.295202 5.666407 1.481653

C -3.181836 6.899677 1.515224

C 0.537588 4.239956 -0.015894

C 1.438819 5.435247 -0.073085

C 0.999204 2.865867 0.008603

C 2.337650 2.502266 -0.046851

H 5.181317 -2.384637 -0.941716

H 6.984369 -0.284683 0.413243

H 7.452236 -1.929710 -0.308571

H -0.098808 -6.965089 0.717447

H -1.837912 -7.487630 0.319044

H -2.442863 -5.254737 -0.335007

H 1.323654 -6.175441 -0.881010

H 2.681495 -5.037097 -0.964464

H 2.194643 -5.727917 0.592189

H 6.162018 1.418559 -0.901705

H 5.046921 2.782595 -0.684426

H 5.787889 2.008706 0.725654

H 0.861933 6.366359 -0.152250

H 2.065368 5.510858 0.830763

H 2.116214 5.390970 -0.940684

H -1.319095 6.245289 -0.356613

H -2.665360 5.148923 -0.590756

H -2.839173 4.819703 1.925755

H -1.440305 5.860067 2.153126

H -5.273561 2.341424 -0.405516

H -6.286566 0.901464 -0.472394

H -6.400901 0.804143 2.019849

H -5.207592 2.128585 2.139739

H -6.327916 -1.317897 0.129613

H -5.318039 -2.486454 -0.743994

H -5.351100 -2.491113 1.031319

H 3.067652 3.311055 -0.078609

H -3.308000 3.066304 0.047563

H -3.087699 -3.323785 0.141797

H 3.284634 -3.062888 -0.368282

H -8.969190 3.030833 1.534005

H -4.448614 7.773636 2.605290

N 2.023519 0.077054 -0.087958

N 0.062316 -2.022998 -0.003307

N -2.019868 -0.087714 0.174713

N -0.071712 1.994165 0.071528

O -3.922046 6.946487 2.664092

O -6.857585 3.951417 1.231598

O -8.351915 2.272650 1.630829

O -3.232079 7.758023 0.655222

O -0.121970 0.029006 -1.766383

H 0.366644 0.805687 -2.111527

C 1.407513 2.410826 4.430407

C -1.012383 2.482804 4.357872

C 1.415592 1.435357 3.432438

C -1.016746 1.506342 3.353972

C 0.204589 0.970674 2.885992

C 0.197363 2.941441 4.901373

H 2.353958 1.025352 3.057756

H -1.951984 1.141203 2.930898

H 2.355709 2.760884 4.847752

H -1.960381 2.885229 4.723652

H 0.198816 3.700289 5.683492

O 0.243257 -0.057401 1.994188

C 2.761216 -2.803963 3.492552

C 0.291874 -2.862724 3.467174

N 1.445923 -2.241369 3.219887

N 0.289333 -4.100890 4.027933

N -0.863389 -2.210091 3.240371

H -0.758823 -1.324086 2.702946

H -1.702455 -2.758773 3.075729

H 1.123374 -4.672391 3.933874

H -0.589022 -4.608208 4.078124

H 1.339691 -1.349142 2.696212

H 3.513188 -2.036433 3.278858

H 2.856653 -3.095446 4.549742

H 2.977462 -3.672524 2.846256

Table S1.

(d) Optimized coordinates (Å) for Model 2ter

Fe 0.003446 0.011375 0.045539

C 2.838093 1.198258 -0.115384

C 4.234687 0.827112 -0.185661

C 5.385094 1.785095 -0.219003

C 4.265671 -0.555487 -0.254610

C 5.397885 -1.460037 -0.392631

C 6.675754 -1.241105 -0.026526

C 2.875979 -0.996797 -0.211547

C 2.484141 -2.328821 -0.207563