Supplementary Material Finding molecular dioxygen tunnels in homoprotocatechuate 2,3-dioxygenase: implications for different reactivity of identical subunits Liang Xu 1, 2, Weijie Zhao 3, Xicheng Wang 1, * 1 Department of Engineering Mechanics, State Key Laboratory of Structural Analyses for Industrial Equipment, Dalian University of Technology, Dalian 116023, China. 2 Department of Chemisty, Dalian University of Technology, Dalian 116023, China. 3 School of Chemical Engineering, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China. Computational details Simplified model structure for defining force parameters are shown in Figure S1. For acetate and imidazole, the CHARMM force field parameters (MacKerell, et al. 1998) of His and Glu were used. The lacking parameter set for the 4NC dianion was determined using the program PARATL, a plug-in for the molecular viewer VMD (Humphrey et al. 1996). The molecular geometry of the model structure was optimized at the HF/6-31+G(d,p) level. The electrostatic potential was calculated at the MP2/6-31+G(d,p) level using MK scheme. The restrained electrostatic potential (RESP) method (Bayly et al. 1993) was used to fit the electrostatic potential to get atomic partial charges. All quantum chemical calculations were carried out with the Gaussian 03 software package (Frisch et al. 2004). To achieve an integer charge for the whole His and Glu, additional charges were evenly distributed to the connecting 1
heavy atoms. It should be noted that this procedure described above is crude, but adequate for the purpose of MD simulations presented in this work. The final charges employed in the MD simulations are presented in Table S1. As can be seen, ligand to metal charge transfer (LMCT) could occur. Forces between Fe and its coordinated atoms were modeled through non-bonded interactions, as other study has been used (van den Bosch et al. 2004). The distances from Fe to its coordinated atoms were averaged over the 10-ns MD simulation and compared to the corresponding bond length of the crystal structure (Table S2). Additional CHARMM27 parameters for 4NC dianion used in the MD simulation were generated by PARATL and given in Table S3. References Bayly, CI, Cieplak, P, Cornell, WD, Kollman, PA (1993) A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J Phys Chem 97:10269-10280 Frisch, MJ, Trucks, GW, Schlegel, HB, Scuseria, GE, Robb, MA, Cheeseman, JR, Zakrzewski, VG, Montgomery, JAJr, Stratmann, RE, Burant, JC, Dapprich, S, Millam, JM, Daniels, AD, Kudin, KN, Strain, MC, Farkas,, Tomasi, J, Barone, V, Cossi, M, Cammi, R, Mennucci, B, Pomelli, C, Adamo, C, Clifford, S, chterski, J, Petersson, GA, Ayala, PY, Cui, Q, Morokuma, K, Malick, DK, Rabuck, AD, Raghavachari, K, Foresman, JB, Cioslowski, J, rtiz, JV, Stefanov, BB, Liu, G, Liashenko, A, Piskorz, P, Komaromi, I, Gomperts, R, Martin, RL, Fox, DJ, Keith, T, Al-Laham, MA, Peng, CY, 2
Nanayakkara, A, Gonzalez, C, Challacombe, M, Gill, PMW, Johnson, B, Chen, W, Wong, MW, Gonzalez, C, Pople, JA. (2004) Gaussian 03, Revision C02, Gaussian Inc: Wallingford CT Humphrey, W, Dalke, A, and Schulten, K (1996) VMD Visual Molecular Dynamics J Mol Graph 14:33 38 MacKerell, AD, Bashford, D, Bellott, M, Dunbrack, RL, Evanseck, JD, Field, MJ, Fischer, S, Gao, J, Guo, H, Ha, S, Joseph-McCarthy, D, Kuchnir, L, Kuczera, K, Lau, FTK, Mattos, C, Michnick, S, Ngo, T, Nguyen, DT, Prodhom, B, Reiher, WE, Roux, B, Schlenkrich, M, Smith, JC, Stote, R, Straub, J, Watanabe, M, Wiorkiewicz-Kuczera, J, Yin, D, Karplus, M (1998) All-atom empirical potential for molecular modeling and dynamics Studies of proteins J Phys Chem B 102:3586-3616 van den Bosch, M, Swart, M, van Gunsteren, WF, Canters, GW (2004) Simulation of the substrate cavity dynamics of quercetinase J Mol Biol 344:725-738 3
His155 HN His214 N N Fe Glu267 N H H 4 2 3 4 N 2 5 1 3 H 1 2 1 6 3 H 1 4NC Fig. S1 Simplified structural model of the iron(ii) complex for the determination of force field parameters. The labels at the ligands map the acetate and imidazole model compounds of the amino acids in the original structure. The 4NC binds as dianion. 4
Fig. S2 Structure superposition of subunits and Fe-bound ligands after minimization and equilibration. Subunit A-D are shown in red, green, yellow and blue, respectively. Active site residues are shown in black CPK representation. The white van der Waals spheres demonstrate example tunnels. 5
Fig. S3 ptimal tunnel profiles for subunit A D of 2,3-HPCD dioxygenase with 4NC, extending from the position of Fe to the bulk solvent. nly the result of the second 10-ns MD simulation is given here. 6
Fig. S4 Energy profiles for 2 tunnels in subunit A D, relative to the solvent (8.2 kj/mol). nly the result of the second 10-ns MD simulation is given here. 7
Fig. S5 Electrostatic potentials of subunit A, B, C and D, with electron clouds in red for negative and blue for positive. Active site residues are shown in CPK representation. The yellow van der Waals spheres demonstrate the corresponding optimal tunnels. Electrostatic potentials were calculated and images rendered by using Accelrys Discovery Studio v21. 8
Table S1 Charge distribution of the active site Charge for delta-his and Glu from CHARMM27 parameter file is shown for comparison. His155/ Calculated CHARMM Calculated CHARMM Glu267 His214 charge charge charge charge 4NC N -0.47-0.47 N -0.47-0.47 C1-0.13 HN 0.31 0.31 HN 0.31 0.31 C2 0.22 CA 0.07 0.07 CA 0.07 0.07 C3 0.47 HA 0.09 0.09 HA 0.09 0.09 C4-0.5 CB -0.10-0.09 CB -0.19-0.18 C5 0.09 HB1 0.09 0.09 HB1 0.09 0.09 C6-0.45 HB2 0.09 0.09 HB2 0.09 0.09 H1 0.12 ND1-0.29-0.36 CG -0.48-0.28 H2 0.23 HD1 0.31 0.32 HG1 0.12 0.09 H3 0.20 CG -0.02-0.05 HG2 0.12 0.09 1-0.73 CE1 0.22 0.25 CD 0.99 0.62 2-0.88 HE1 0.13 0.13 E1-0.78-0.76 N1 0.82 NE2-0.50-0.7 E2-0.78-0.76 3-0.55 CD2 0.01 0.22 C 0.51 0.51 4-0.55 HD2 0.10 0.10-0.51-0.51 C 0.51 0.51-0.51-0.51 Fe 1.38 Total -1.00 9
Table S2 Non-bonded distances (Å) between Fe and its coordinated atoms in the active site of the X-ray (2IGA) and the corresponding MD averaged (20 ns) structures (subunit A, B, C and D) Standard deviations of the MD values are given in parentheses. Non-bonded distances X-ray (A) MD (A) X-ray (B) MD (B) NE2(His155) Fe NE2(His214) Fe E1/2(Glu267) Fe 2.16 2.39(±0.11) 2.22 2.41(±0.12) 2.29 2.39(±0.11) 2.26 2.42(±0.12) 2.06 2.04(±0.05) 2.01 2.05(±0.05) 1(4NC) Fe 2.18(±0.07) 2.19(±0.08) 2(4NC) Fe 2.14(±0.06) 2.14(±0.07) 11(XXP) Fe 2.25 13(XXP) Fe 1.97 7(XX3) Fe 2.16 8(XX3) Fe 2.26 13(XX3) Fe 2.22 Non-bonded distances X-ray (C) MD (C) X-ray (D) MD (D) NE2(His155) Fe NE2(His214) Fe E1/2(Glu267) Fe 2.23 2.39(±0.12) 2.23 2.42(±0.14) 2.29 2.41(±0.12) 2.22 2.44(±0.14) 2.09 2.05(±0.06) 1.97 2.06(±0.06) 1(4NC) Fe 2.18(±0.08) 2.23(±0.09) 2(4NC) Fe 2.13(±0.06) 2.14(±0.06) 7(XX2) Fe 2.16 8(XX2) Fe 2.24 7(XX3) Fe 2.24 8(XX3) Fe 2.31 13(XX3) Fe 2.09 10
Table S3 Additional CHARMM27 parameters for 4NC dianion used in the MD simulation Atom numbering is the same as given in Fig S1. Atom C1 C2 C3 C4 C5 C6 H1 Atom Type CA CA CA CA CA CA HP Atom H2 H3 1 2 N1 3 4 Atom Type HP HP H1 H1 N N N EB bond =K b (b-b 0 ) 2 K [kcal/mol/ Å 2 ] b [Å] b 0 CA-N 441.770 1.3525 N-N 519.140 1.2463 Eangle=Kθ(θ-θ 0 ) 2 2 Kθ [kcal/mol/ rad ] θ 0 [degree] CA CA N 49.749 120.55 CA N N 68.289 120.78 N N N 74.624 118.45 E =K dihedral χ(1 + cos(n(χ) - δ)) Kχ[kcal/mol] n δ[degree] H1 CA CA H1 1.0355 2 180.00 CA CA CA N 0.4093 2 180.00 HP CA CA N 0.413 2 180.00 CA CA N N 2.5694 2 180.00 Parameters for nonbonded interaction terms atom ε[kcal/mol] R /2 [Å] min N -0.120 1.824 N -0.170 1.661 11