1. Mo-aminoacid Complexes as Analogs for Mo- based Enzymes: A DFT Approach S. Sabiah 26.06.08

2. An Overview Introduction –Importance of Molybdo-enzymes Focus of Present Study Methodology-DFT Mo-Aminoacid complexes and their anionic analogs Relative Redox Energy of the systems Mo-Substrate intermediates Conclusions

3. Mo-based Enzymes in Biology 3 3 R. L. McNaughton, S. Mondal, V. N. Nemykin, P. Basu, M. L. Kirk, Inorg. Chem. 2005, 44, 8216  Reduction of DMSO to DMS  Oxidation of sulfite to sulfate  Oxidation of xanthine to uric acid  Indirectly involve in the global cycling of sulphur, nitrogen and carbon

4.  XO has gained attention regarding its active site structure and catalytic mechanism is still the subject of controversy  EXAFS studies of oxidized [Mo(VI)] and reduced [Mo(IV)] core structures  Unique sulfido (=S), oxo (=O) liands with hydroxyl (OH) and dithiopterin cofactor (dpt)  Such Mo(VI) core structures (cis Mo=S, =O) are rare  Recent theoretical report on the catalytic mechanism of XO, reveals that the glutamic aminoacid in the surrounding environment plays crucial role Xanthine Oxidase Amano, T.; Ochi, N.; Sato, H.; Sakaki, S. J. Am. Chem. Soc.; 2007, 129, 8131 4 4

5. [Mo 3 (    O) 2 (  -O 2 CCH 3 ) 6 (H 2 O) 3 ] 2+ Annu. Rep. Prog. Chem., Sect. A: Inorg. Chem., 2007, 103, 159 - 169 Molybdocene complex-Antitumor activity Mo-oxo complex-architectures 5 5  But still Mo(VI) complexes with aminoacid are scant in literature.

6. Focus of Present Study 6 6 Try to optimize the hypothesized Mo(VI) aminoacid complexes with sulfido (=S) and oxo (=O) ligands Mo(VI)SO(A)(dt), (dt is dithiolene unit as model for dithiopterin cofactor (S-CH=CH-S) Study the one electron reduced species Estimate the relative redox energies by Born-Heber cycle As a model substrate of xanthine, the catalytic oxidation of pyrimidin-4-ol (Py-4) to pyrimidin-2,4-diol (Py-2,4) Optimize the possible Mo(V) and Mo(IV)-substrate intermediates

7. 7 7 N. Allinger, Encyclopedia of Computational Chemistry, 1997, 1-5, 1320-1325 The energy, E, is a function of the atomic positions, R, of all the atoms in the system, is calculated as a sum of bonded, terms Ebonded, which describe the bonds, angles and bond rotations in a molecule, and a sum of external or nonbonded terms, Enon-bonded The E bonded term is a sum of three terms: which correspond to three types of atom movement: Computational Basics Hψ = E ψ Schroedinger eqn

8. DFT calculations, including geometry optimization and NBO analysis, were performed using a hybrid functional B3LYP as implemented in Gaussian 03.13 The LANL2DZ basis set and effective core potential were used for molybdenum. When the optimization was tried for calculation with mixed basis set with LANL2DZ for Mo and 6-31G(d) for C, N, O and S, resulted in convergence failure. Hence the calculations have been carried out only with LANL2DZ for all Mo analogs However, for the simple organic substrates, pyrimidin-4-ol (Py-4) and pyrimidin-2,4-diol (Py-2,4) the usual 6-31G(d) basis sets were employed Calculation for vibrational frequencies was performed alongside each geometry optimization to ensure the stability of the ground state as denoted by the absence of imaginary frequencies. Methodology Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. 1988, 37, 785. 8 8

9. 9 9 1 trial 84 th trial Geometry Optimization of Mo(OMe) 6 Inorg. Chem. 2001, 40, 3815

10. Total electron density mapSimulated IR spectrum 10

11. 11 Mo-Aminoacid Complexes 1. As a first step for the optimization of aminoacid complexes, a structurally known neutral Mo(VI) complex, [MoO2(L-CysOMe)2], 1 with substituted cystein aminoacid was considered and optimized with B3LYP/DFT calculation. Chemdraw representation of [Mo(O 2 )(L-Cys-OMe) 2 ], 1 Buchanan, I.; Minelli, M. Ashby, M. T.; King, T. J.; Enemark, J. H.; Garner, C. D. Inorg. Chem., 1984, 23, 495 Preliminary Studies

12. Bond distances (Å)Bond angles ( o ) XRDDFTXRDDFT Mo=O = 1.7141.739, 1.742O-Mo-O'= 108.1108.6 Mo-S = 2.4142.501, 2.513S-Mo-S' =155.6151.2 Mo-N = 2.3752.390, 2.398N-Mo-N' = 80.984.72 S-C = 1.8081.897, 1.897O-Mo-N' = 163.8164.9, 165.1 N-C = 1.4691.490, 1.492O-Mo-N = 86.283.5, 84.5 C-C= 1.5211.542, 1.544O-Mo-S = 101.8105.1, 105.9 C=O = 1.1901.238, 1.239O-Mo-S' = 92.590.9, 91.5 C-O = 1.3361.375, 1.383N-Mo-S = 76.877.3, 77.4 O-CH3 =1.4831.474, 1.479N-Mo-S' = 84.781.3, 81.5 Table 1. Comparison of bond distances (Å) and angles (o) of [Mo(O 2 )(L-Cys-OMe) 2 ] cis-dioxo cis-diamino trans disulfide coordination around Mo 12

13. 2. [Mo(VI)(O)(S)(dt)], 2 and [Mo(VI)(O)(S)(OH)(dt)] -1, 3 have been optimized as the active site models for XO with dithiolene unit (dt, S-CH=CH-S). [Mo(VI)(O)(S)(dt)], 2[Mo(VI)(O)(S)(OH)(dt)] -1, 3 XNO-Active site models 13

14. Mo-L XO, 2 XO-OH, 3 EXAFSThis studyADFEXAFSThis studyADF Mo=S2.17 2.122.182.202.19 Mo=O1.731.711.701.74 1.72 Mo-S2.462.412.322.472.542.43 Mo-S2.462.412.322.472.592.43 Mo-OH--1.981.951.96 Table 2. Comparison of Mo-L distances for XO models 2 and 3 with EXAFS and ADF * * Seisenbaeva, G. A.; Kloo, L.; Werndrup, P.; Kessler, V. G. Inorg. Chem., 2001, 40, 3815 14

15. 3. Having known that DFT theory gives comparable results for complexes 1, 2 & 3, the Mo complexes with aminoacids, [Mo(VI)(S)(O)(A)(dt)], 4-8 [(A= Glycine, 4; Alanine, 5; Valin, 6; Cystein, 7; Histidine, 8 and their reduced analogs, [Mo(VI)(S)(O)(A)(dt)] -1, 9-13 have been considered. The optimized Oh geometry of Mo-gly complex, 4 Optimization of Mo-aminoacid complexes cis-oxo, sulfido cis-amino, carboxylato cis disulfide coordination around Mo stabilization energy is in the range of -3.94 to -5.90 a.u. The addition of aminoacid stabilizes the molecules compared to isolated free atoms 15

16. M-L distances (Å) M-LMo-GlyMo-AlaMo-ValMo- Cys Mo- Hist Mo=S2.24 Mo=O1.73 Mo-S2.53 2.522.532.52 Mo-S2.64 2.652.642.63 Mo-N2.482.462.452.492.46 Mo-O2.03 2.042.06  L-Mo-L (  ) S-Mo-O oxo 104.5 105.2104.4 N-Mo- O acid 71.671.471.171.070.7 S dt -Mo-S dt 80.680.580.680.480.7 S-Mo-S dt 158.4158.7158.6158.7158.9 N-Mo-S dt 84.284.984.784.685.5 O-Mo- O acid 97.596.997.197.696.02 Table 3. Comparison of selected bond parameters of Mo-aminoacid complexes  The Mo=O distances are ~1.73 Å and much deviated from the hydroxyl coordinated molybdenum complexes (Mo-O~ 1.95-1.98 Å)  no covalent interactions between the metal and sulfur or ring nitrogen of Cyst and Hist  cis-dioxo widens up to an angle of ~108  (in complex 1) and in the case of Mo-aminoacid complexes with O-Mo-S up to 105.9 .  The small differences in angles are due to the constraints of the extra chelate ring with bidentate dithiolate and aminoacid ligands 16

17. Table 4. Comparison of calculated M-L distances for reduced Mo-aminoacid analogs M-LMo-Gly anion Mo-Ala anion Mo-Val anion Mo-Cys anion Mo-Hist anion Mo=S2.36 2.352.36 Mo=O1.73 Mo-S2.54 2.532.52 Mo-S2.69 2.722.66 Mo-N2.492.47 2.562.49 Mo-O2.15 2.14 2.18 Optimization of Mo-aminoacid anionic analogs  Mo-S and Mo=S distances are longer for anionic analogs than their parent structures due to electronic delocalization around sulfur atoms 17

18. Vibrational Analysis The simulated IR spectrum of Mo-Gly complex, 4 IR vibratonsMo- Gly Mo- Ala Mo-ValMo- Cys Mo-Hist -NH 2 symmetric asymmetric bending 3515 3638 1678 3498 3622 1680 3470 3607 1674 3498 3624 1680 3422 3628 1663 -CH=CH symmetric asymmetric 3205 3176 3205 3170 3205 3170 3208 3174 3160 3120 -COO asymmetric 16521644164116571620 -C-O12501248123512081218 CH, CH 2, CH 3 3076 3141 3085 3125 3036, 3051 3101, 3111 3146, 3158 3177 3100 3081 3033 3100 Table 5. selected IR vibrations 18

19. Population Analysis The HOMO and LUMO of Mo-Gly complex, 4 The HOMO consists of <5% Mo p and d atomic wave funcitons Oxo p orbital contributes ~15% The dithiolate sulfur contributes 17-31% and C ~10% The sulfido orbital contribution reduced to ~5-10% compared to parent xanthine oxidase (XO 30%) The reduction in orbital contribution indirectly indicates that these centers can act as oxdising agents for model substrates significant molybdenum d orbital contribution (~50%) to LUMO 19

20. XOXO-OHMo-GlyMo-AlaMo-CystMo-HistMo-Valin Mo (p)3.83 0.79 1.844.284.23 0.953.07 Mo (d)6.42 1.46 3.272.852.89 1.201.97 =O(p) oxo 6.62 3.16 13.3115.6913.24 0.8120.72 -S (p)13.96 27.95 17.2123.4822.28 31.13 17.19 -S (p)13.96 23.75 24.4417.0719.75 23.57 20.11 -C(p)9.90 12.47 13.808.6910.15 10.56 11.04 -C(p)9.90 14.48 9.679.439.42 14.42 8.97 =S(p)30.83 14.15 6.949.878.93 10.84 10.54 From aminoacid N(s)0.291.641.99 1.37 1.19 N(p)0.381.081.16 1.94 0.50 Table 6. Atomic orbital contribution (%) to HOMO  orbital contribution from aminoacid residues is insignificant 20

21. Table 7. Atomic orbital contribution (%) to LUMO XOXO- OH Mo-GlyMo-AlaMo- Cyst Mo- Hist Mo-Valin Mo (p)6.742.812.281.451.341.812.25 Mo (d)41.8656.1441.9647.4247.2754.1442.85 =O(p) oxo 8.260.641.171.922.0341.130.86 -S (p)9.048.083.472.302.341.165.98 -S (p)9.040.903.012.552.776.341.22 -C(p)5.440.251.762.072.220.961.40 -C(p)5.440.680.920.870.930.150.99 =S(p)3.2219.1428.1530.1829.1026.1726.34 From aminoacid N(s)4.841.441.850.010.94 O(p)0.581.791.714.531.31 C(p) nextO 0.551.421.190.702.57 =O(p) C=O 0.371.581.500.352.39  sulfido orbitals contribute ~30% to LUMO which implies that the electronic delocalization is also possible through this sulfur during catalytic oxidation of substrates. 21

22. Qualitative Redox Energy of the Molecules Topol, I. A.; McGrath, C.; Chertova, E.; Dasenbrock, C.; Lacourse, W. R. Eissenstat, M. A.; Burt, S. K.; Henderson, L. E.; Casas-Finet, J. R. Protein Sci., 2001, 10, 1434 22

23. Parameter Mo-GlyMo-CystMo-Hist Gaseous state: E (a.u)-534.49-583.89-798.80 ∆ HOMO-LUMO (Kcal/mol) 45.1845.8043.95 ∆Ganion-mol (Kcal/mol) -1.63-1.9-2.2 Imaginary Freq000 Solution state: ∆ HOMO-LUMO (Kcal/mol) 41.4242.6745.18 ∆Ganion-mol (Kcal/mol) -1.63-2.51 Imaginary Freq011 Table 8. Computational output for Mo-aminoacid analogs of Gly, Cyst and Hist 23

24. SystemsΔE g (A to A - ) g (Kcal/mol) ΔG s (A s to A g ) (Kcal/mol) ΔG s (A s - to A g - ) (Kcal/mol) ΔE s (A to A - ) s (Kcal/mol) Mo-Gly+26.34-1.49-0.22+25.07 Mo-Ala+27.47-0.42+0.17+26.88 Mo-Valin+27.01-0.24+0.12+26.65 Mo-Cyst+17.60-0.04-0.65+18.21 Mo-Hist+19.16-0.47-0.68+19.37 Table 9. Approximate redox energy  molybdenum aminoacid complexes show redox energy in the same range, +18 to +25 Kcal/mol indicating similar oxidising power 24

25.  as an oxygen insertion in a CH bond of five or six membered heterocycles  theoretical studies emphasizing catalytic pathway and optimization of reactive intermediates as models for xantine oxidase have been reported * Oxidation of model substrate by Mo-aminoacid complex *Amano, T.; Ochi, N.; Sato, H.; Sakaki, S. J. Am. Chem. Soc.; 2007, 129, 8131-8138 25

26. The proposed catalytic oxidation of Py-4-ol by Mo-Gly complex Aminoacid is labile OH coordination from water Substrate binding Mo(IV) and Mo(V) intermediates Product released 26

27. [Mo(IV)(SH)O(O-Py-4)(dt)] 2-, 14[Mo(V)SO(O-Py-4)(dt)] 2-, 15 Optimization of Mo-substrate intermediates  Mo is in distorted square pyramidal geometry with τ = 0.02 for 14 and 0.32 for 15  C-O distance (~1.28 Å) is comparable with Mo-imidazole intermediate (1.28 Å)  The Mo-C2 (carbon flanked between both the nitrogens) distance is 3.237 Å (15) which is little longer than the Mo-C distance in imidazole analogue (3.122 Å)   Mo-O-C2 is much deviated (~139  ) from the imidazole (  Mo-O-C2 = 128.8  ) * *Bayse, C. A. Inorg. Chem., 2006, 45, 2199-2202 27

28. All the optimized complexes with aminoacids have Mo(VI) in six coordination with bidentate aminoacid, bidentate dt, monodentate sulfido and oxo ligands Frequency analysis confirms the absence of imaginary frequencies and the stability of these complexes in gaseous state The redox behaviour is primarily molybdenum centered, based on metal orbital contribution towards LUMO The Gibbs free energy change between the anion and free molecule is ~ -2 Kcal/mol (∆G anion-molecule ) which is comparable to that of the free enzyme. The optimized geometry with Mo(V) and Mo(IV) core with substrate reveals that the hydroxyl oxygen from Mo is being inserted in to the C-H bond of the substrate which is consistent with the imidazole report. Conclusions 28

29. Acknowledgements Prof. B. Viswanathan, Head, NCCR, IIT Madras Prof. A. V. Ramaswamy All Faculties Dear Friends

31.  L-Mo-L (  ) 1415 S dt -Mo-S dt 83.982.74 S dt -Mo-O oxo 105.0100.6 O oxo -Mo- S108.8111.9 S-Mo-O82.090.7 O-Mo-S dt 84.680.06 S dt -Mo-O oxo 107.7111.8 S dt -Mo-S143.5136.2 S dt -Mo-S87.689.4 S dt -Mo-O144.5155.2 O oxo -Mo- O110.5102.3 Mo-O-C2144.2139.0 Transition state