Protonation state of MnFe and FeFe cofactors in a ligand binding oxidase revealed by

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1 Supporting Information Protonation state of MnFe and FeFe cofactors in a ligand binding oxidase revealed by X-ray absorption, emission, and vibrational spectroscopy and QM/MM calculations Ramona Kositzki 1, Stefan Mebs 1, Jennifer Marx 2, Julia J. Griese 3, Nils Schuth 1, Martin Högbom 3,4, Volker Schünemann 2, Michael Haumann 1* 1 Freie Universität Berlin, Fachbereich Physik, Berlin, Germany 2 Technische Universität Kaiserslautern, Fachbereich Physik, Kaiserslautern, Germany 3 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden 4 Department of Chemistry, Stanford University, Stanford, California 94305, United States S1

2 Examples of input-files for calculations on model structures. (A) DFT approach for geometry optimization and frequency analysis (Gaussian09) (i) Assignment of anti-ferromagnetic coupling %chk=/path/fefe_oh_h2o_y_a_afc_bt.chk %mem=2000mb %nprocshared=4 #p UB3LYP/TZVP guess=(only,fragment=12) output=wfn FeFe_OH_H2O_Y_a_afc_bt Fe(Fragment=1) Fe(Fragment=2) (ii) Geometry optimization %chk=/path/fefe_oh_h2o_y_b_optf_bt.chk %mem=35000mb %nprocshared=8 #p UB3LYP/TZVP scf=(conver=7,nosymm) guess=read geom=(check,modredundant) #scrf=(solvent=diethylether) opt=(modredundant,tight) freq output=wfn FeFe_OH_H2O_Y_b_optf_bt 0 1 X 9 F X 23 F X 33 F X 45 F X 58 F X 68 F X 80 F X 90 F X 91 F X 92 F X 93 F X 94 F X 95 F X 96 F /path/fefe_oh_h2o_y_b_optf_bt.wfn S2

3 (B) QM/MM approach for geometry optimization and frequency analysis (Gaussian09) (i) Generation of input-file for assignment of anti-ferromagnetic coupling %chk=/path/4hr0_fefe_oh-h2o_y_a_afc-ini.chk %mem=10gb %nprocshared=16 #oniom(ub3lyp/tzvp:uff)=onlyinputfiles geom=connectivity 4HR0_FeFe_OH-H2O_Y_a_afc-ini N-N_ (PDBName=N,ResName=VAL,ResNum=2_B) L C-C_ (PDBName=CA,ResName=VAL,ResNum=2_B) L (ii) Assignment of anti-ferromagnetic coupling %chk=/path/4hr0_fefe_oh-h2o_y_b_afc-fix.chk %mem=40gb %nprocshared=16 # ub3lyp/tzvp geom=connectivity guess=(only,fragment=12) IOp(2/15=1,5/32=2,5/38=1) 4HR0_FeFe_OH-H2O_Y_b_afc-fix Bq-# (Iso= ,Spin=2,ZNuc=7.,QMom=2.044,GFac= ,PDBName=N,R esname=val,resnum=2_b) Bq-# (Iso=12,ZNuc=6.,PDBName=CA,ResName=VAL,ResNum=2_B) (iii) Geometry optimization %chk=/path/4hr0_fefe_oh-h2o_y_c_afc-opt.chk %mem=50gb %nprocshared=16 #opt=tight oniom(ub3lyp/tzvp:uff) guess=read geom=connectivity #scf=(nosymm,maxcycles=1024,conver=5) int=grid=ultrafine 4HR0_FeFe_OH-H2O_Y_c_afc-opt N-N_ (PDBName=N,ResName=VAL,ResNum=2_B) L C-C_ (PDBName=CA,ResName=VAL,ResNum=2_B) L S3

4 (C) Calculation of XAS/XES spectra (ORCA). (i) Single point calculation!uks b3lyp tzvp PAL4 tightscf %cosmo epsilon 4 end %output Print[P_ReducedOrbPopMO_M] 1 end %scf #GuessMode CMatrix FlipSpin 0 FinalMs 0.0 DirectResetFreq 0 TCut 1e-14 Thresh 1e-12 MaxCore 2000 MaxIntMem 2000 CNVZerner true maxiter DIIS Start end DampErr DIISMaxIt 1000 ShiftErr 0.0 # do NOT turn off level shift! Please! LevelShift 0.4 DampFac 0.8 DampMax 0.8 DampMin 0.0 end * xyzfile 0 11 FeFe_OH_H2O_Y_afcM1_b3lyp.xyz (ii) Calculations of XAS/XES spectra!uks b3lyp tzvp PAL4 tightscf moread noiter %moinp "FeFe_OH_H2O_Y_afcM1_b3lyp.gbw" %cosmo epsilon 4 end * xyzfile 0 1 FeFe_OH_H2O_Y_afcM1_b3lyp.xyz %xes MaxNVirt 800 # Number of empty MOs that accept electrons DoQuad true # Switch for the calculation of magnetic dipole and electric quadrupole transition moments DoXAS true # Do absorbtion CoreOrb 0,1,0,1 # List of core orbitals in which a hole is generated OrbOp 0,0,1,1 # The operators for each CoreOrb (0: spin up, 1: spin down) Normalize false # if set to true, most intensive feature will be set to 1 (default true) End S4

5 Table S1: EXAFS simulation parameters. a cofactor N [per metal ion] / R [Å] / 2σ 2 x10 3 [Å 2 ] R F [%] Fe Fe-N/O Fe-C Fe-Fe/Mn FeFe 2.2 * / 1.94 / 5 # 2.0 & / 2.93 / 5 # 1.0 & / 3.49 / 2.8 * / 2.09 / 5 # 1.0 & / 2.45 / 5 # 5.0 & / 4.43 / 5 # MnFe + FeFe 2.4 * / 1.96 / 12 # 2.6 * / 2.10 / 12 # 1.0 & / 2.41 / 12 # MnFe 2.2 * / 1.94 / 3 # 2.8 * / 2.09 / 3 # 1.0 & / 2.31 / 3 # 2.0 & / 3.02 / 7 # 1.0 & / 3.49 / & / 4.48 / 7 # 2.0 & / 3.05 / 4 # 1.0 & / 3.50 / & / 4.50 / 4 # Mn Mn-N/O Mn-C Mn-Fe MnFe 1.7 * / 1.90 / 6 # 3.3 * / 2.12 / 6 # 1.0 & / 2.26 / 6 # 2.0 & / 2.94 / 8 # 1.0 & / 3.51 / & / 4.62 / 8 # a Data correspond to EXAFS spectra at Mn or Fe K-edges in Fig. 5. N = coordination number, R = interatomic distance, 2σ 2 = Debye-Waller factor, R F = fit error sum calculated for reduced distances of 1-4 Å. * Coordination numbers were coupled to yield a sum of 5 for the first two N/O ligand shells (so that together with the third N/O shell a value of N = 6 resulted), # 2σ 2 values were coupled to yield the same value for the indicated coordination shells, & N-values that were fixed in the fit procedure. The coupling of Debye-Waller factors for the N/O shells is justified by using N = 1 for the longest bond (for which a relatively larger 2σ 2 value was expected) and N-values of about 2-3 for the shorter bonds (so that 2σ 2 accounted for the expected bond length variations within a given shell). We note that only insignificant changes in fit qualities or bond lengths were observed for using integer N-values closest to the fitted fractional N-values for the first two coordination shells. S 0 2 was 0.85 for Mn and Fe. S5

6 Table S2: Representative cofactor coordinates from DFT and QM/MM. a (A) DFT: FeFe (µoh/h 2 O) Fe C Fe C O C O H C H C H C H C H C O H O H C H C H C H H H H H H H H H H O N O N C C C C C C H C H C H H H H H H O H O H C H C H C H H O H C H C H C H C N C N C C C C H C H S6

7 C H C H H H H H H H H H H O H H H O H H O H O (B) DFT: MnFe (µoh/h 2 O) Mn C Fe C O C O H C H C H C H C H C O H O H C H C H C H H H H H H H H H H O N O N C C C C C C H C H C H H H H H H O H O H C H C H S7

8 C H H O H C H C H C H C N C N C C C C H C H C H C H H H H H H H H H H O H H H O H H O H O (C) QM/MM (ONIOM high layer): FeFe (µoh/h 2 O) Fe C Fe C O C O H C H C H C H C H C O H O H C H C H C H H H H H H H H H H O N O N C C C C S8

9 C C H C H H H H H H H H O H O H C O C C C C H C H C H C H C H C N H N H C H C H C H C H H H H H H O H H H O H H O H O (D) QM/MM (ONIOM high layer): MnFe (µoh/h 2 O) Mn C Fe C O C O H C H C H C H C H C O H O H C H C H C H H H H S9

10 H H H H H H O N O N C C C C C C H C H H H H H H H H O H O H C O C C C C H C H C H C H C H C N H N H C H C H C H C H H H H H H O H H H O H H O H O a Coordinates represent geometry-optimized structures. Respective structures for other protonation states were derived by removal of one or two protons (and respective charge adjustment), followed by geometry optimization. S10

11 Figure S1: R2lox model structures for QM/MM and DFT. (A) Whole-protein structure for QM/MM calculations using the ONIOM approach. The QM (DFT) high-layer core (balland-stick representation) comprised the cofactor and amino acids similar to structure (b) in (B) and the MM low-layer the protein crystal structure (ribbon representation) according to PDB IDs 4XB9 (FeFe) or 5DCS (MnFe). (B) Cofactor structures for DFT calculations with increasing size (a, 93 atoms; b, 109 atoms; c, 188 atoms). The shown structures correspond to the FeFe cofactor in the H 2 O/µOH protonation state with Y175 being a hydrogen-bond donor to E202, similar models were used for the MnFe cofactor; alternative models comprised deprotonated water species at Mn/Fe1 and in the bridging position and alternative orientations of the phenolic group of Y175 (shown on top in (b) and (c), see Fig. S2). S11

12 Figure S2: Cofactor structures with protonation and hydrogen-bonding variations. Similar cofactor structures were used for DFT and QM/MM calculations. (a-f) Structures with different protonation states and proton orientations of the water species at Mn/Fe1 and of the bridging oxygen species; shown structures represent the FeFe cofactor, similar structures were used for the MnFe cofactor. (g-k) Structures with alternative orientations of the phenolic group of Y175; (g, h) FeFe cofactor, (i, k) MnFe cofactor. S12

13 Figure S3: ctv (pre-edge absorption) and vtc (Kß satellite emission) spectra for H-bonding variations. Shown are calculated Mn or Fe ctv (top) and vtc (bottom) spectra for the H 2 O/µOH protonation state of the MnFe and FeFe cofactors from the B3LYP/TZVP (DFT) and QM/MM (ONIOM) approaches (afc coupling) for the absence of Y175 in the model structures and for two orientations of hydrogen bonding of the Y175 phenolic proton (to the water ligand at Mn/Fe1 or the E202 carboxylic group) (see Fig. S2 for respective structures). Spectra were shifted on the energy scale similar to data in Figs. S7 and S8. Note the largely increased high-energy maximum in the ctv spectra for Y175 being a H-bond donor to the water ligand, which was not observed in the experimental data (Fig. 7), thereby favoring Y175 to function as a (weak) H-bond donor to E202 and a (weak) H-bond acceptor of the water ligand. S13

14 Figure S4: ctv and vtc spectra for electronic coupling variations from DFT. Data are for the H 2 O/µOH protonation state (Y175 H-bond donor to E202) for ferromagnetic (fc) or antiferromagnetic (afc) coupling of the Mn/Fe1(III) and Fe2(III) ions. (A) Spectra from B3LYP/TZVP DFT calculations on MnFe and FeFe cofactor model structures (Fig. S1). (B) Spectra for the MnFe cofactor in the QM/MM whole-protein model (Fig. S1). Spectra in (B) were derived from single-point DFT calculations on the high-layer core of the QM/MM optimized model structure. S14

15 Figure S5: Metal and ligand contributions to target MOs for ctv transitions for four cofactor protonation states at Mn and Fe K-edges. Protonation states and ligand species are defined as in Fig. 7. S15

16 Figure S6: Metal and ligand contributions to source MOs for vtc transitions for four cofactor protonation states. Protonation states and ligand species are defined as in Fig. 8. S16

17 Figure S7: Charge (A) and spin (B) distributions in MnFe and FeFe cofactors. MnFe, black; FeFe, grey. M1, M2 = metal ion in site 1 or 2, His1, His2 = histidine ligand at M1 or M2, FA = fatty acid ligand. The insets show the MnFe FeFe difference values. S17

18 Figure S8: Inter-molecular contacts in MnFe and FeFe structures from DFT. Data correspond to model size (c) in Fig. S1-B. Shown bonding patterns and secondary inter-molecular contacts stem from atoms-in-molecules (AIM) topology analysis; small red dots denote bondcritical-points. Color code: white, Mn and Fe; blue, N; black, C; grey, H; red, O. Note weak H-bonding interactions between backbone C-H groups and the water ligand at Mn/Fe1. S18

19 Figure S9: Calculated NRVS spectra for model variations. (A) PDOS spectra for the H 2 O/µOH state from DFT for increasing model size compared to spectra from QM/MM (Fig. S1, B3LYP/TZVP in both approaches). (B) PDOS spectra for the H 2 O/µOH state for different hydrogen-bonding situations of Y175 (Fig. S2) from DFT. (C) PDOS spectra for the H 2 O/µOH state from QM/MM for ferromagnetic or anti-ferromagnetic coupling of the Mn1 and Fe2 ions. S19

20 QM / MM DFT Figure S10: Calculated NRVS spectra for four cofactor protonation states. QM/MM: ONIOM approach on the whole-protein structure (Fig. S1, A and B-b) using the B3LYP/CEP31g functional/basis-set combination and ferromagnetic coupling of the Mn/Fe1 and Fe2 ions. DFT: DFT approach on the cofactor structure with Y175 being a hydrogen-bond donor to E202 (Fig. S2; a, b, d, e) using the B3LYP/TZVP functional/basis-set combination and antiferromagnetic coupling of the Mn/Fe1 and Fe2 ions. Note similar intense high-frequency vibrational bands for both theory levels for the three less protonated MnFe and FeFe cofactor structures, which are absent or show lower intensities in the calculated spectra of the H 2 O/µOH state and in the experimental spectra (Fig. 11). S20

21 Figure S11: Experimental NRVS spectra for Mn/Fe loaded R2lox. The MnFe + FeFe spectrum represents a mixture of FeFe and MnFe cofactor sites (see Fig. 2). Subtraction of 35 % of the FeFe spectrum (Fig. 11) from the MnFe + FeFe spectrum yielded the pure Fe spectrum of the MnFe cofactor shown in Fig. 11. S21

22 Figure S12: Calculated NRVS spectra for theory level variations. PDOS spectra were calculated using the QM/MM approach and the indicated functional/basis-set combinations. Respective difference spectra are shown on the bottom. (+/-Y175, Tyr175 was included or not in the DFT high-layer in the ONIOM calculations). S22