We have had a long standing interest in the mechanism of

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1 Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase Xiaodong Zhang, Swarnalatha Y. Reddy, and Thomas C. Bruice* Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA Contributed by Thomas C. Bruice, November 16, 2006 (sent for review November 15, 2006) At neutral ph, oxidation of CH 3 OH 3 CH 2 O by an o-quinone requires general-base catalysis and the reaction is endothermic. The active-site CO 2 groups of Glu-171 and Asp-297 (Glu-171 CO 2 and Asp-297 CO 2 ) have been considered as the required general base catalysts in the bacterial o-quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o-pqq when MeOH is hydrogen bonded to Glu-171 CO 2 and the crystal water (Wat1) is hydrogen bonded to Asp-297 CO 2 is G 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated G when MeOH is hydrogen bonded to Asp-297 CO 2 is >50 kcal/mol. The Asp-297 CO 2 Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDHPQQWat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297 CO 2 bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297 CO 2, we find that MeOH returns to be hydrogen bonded to Glu-171 CO 2 and Asp-297 CO 2 coordinates to Ca 2 during 3 ns simulation. The Asp-297 CO 2 Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation G 1.1 kcal/mol for Asp-297 CO 2 general-base catalysis of Wat1 hydration of the immediate CH 2 AO product 3 CH 2 (OH) 2. By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH 2 (OH) 2 is exothermic. hydride transfer molecular dynamics pyrroloquinoline quinone We have had a long standing interest in the mechanism of o-quinone cofactor and model reactions (for examples, see refs. 1 4). More recently, our attention has been directed to the employment of computational methods, particularly in the mechanism of the oxidation of methanol and glucose by the appropriate quinoprotein enzymes (5 11). The present study concerns bacterial quinoprotein methanol dehydrogenase (MDH) catalysis of the oxidation of CH 3 OH 3 CH 2 O by the pyrroloquinoline quinone (PQQ) cofactor. MDH is a hetrotetramer with two heavy and two light subunits. The catalytic chemistry is located in the heavy subunits. The crystal structure (PDB code 1G72; ref. 10) of the reduced PQQ bound to MDH presents Wat1 hydrogen bonding to the CO 2 group of Asp-297 (Asp-297 CO 2 ) and CO 2 group of Glu-171 (Glu- 171 CO 2 ) associated with Ca 2. Attempts to crystallize MDH with MeOH substrate at the active site have not yet been successful (10, 12, 13). The hydride transfer mechanism was shown to be correct by x-ray crystallography (10). During 3-ns molecular dynamics (MD) simulations (6, 7, 9) with MeOH at the active site, it was found that MeOH became hydrogen bonded to Glu-171 CO 2, with the C H of MeOH adjacent to the quinone C5 of PQQ and Wat1 hydrogen bonded to Asp-297 CO 2. This MD derived structure is not in accord with the following: (i) the suggestion that Asp-297 CO 2 serves as the general-base catalyst (14 17); and (ii) that the oxidation takes place, not by hydride transfer, but by an addition elimination reaction (18, 19) (Scheme 1, in which the general base may be Glu-171 CO 2 or Asp-297 CO 2 ). The MD studies (6, 7, 9) showed that Asp-297 CO 2 is not in position to act as a generalbase catalyst for the oxidation of MeOH by MDH. The mechanism of Scheme 2 was proposed. In the present study, we describe the roles played by Glu-171 CO 2 and Asp-297 CO 2 in the overall enzymatic reaction: CH 3 OH o-pqq H 2 O 3 (HO) 2 CH 2 PQQH 2. Results and Discussions Evidence Against Asp-297 CO 2 Being the General Base in the Oxidation of CH 3 OH 3 CH 2 O. In the 3-ns MD simulations (6, 7, 9) of MDHPQQMeOHWat1, Wat1 hydrogen bonded to Asp-297 CO 2 did not exchange with a solvent water, nor exchange position with MeOH hydrogen bonded to Glu-171 CO 2.MD simulations, starting with Wat1 hydrogen bonded to Glu-171 CO 2 and MeOH hydrogen bonded to Asp-297 CO 2, show that Wat1 and MeOH exchange hydrogen bonding positions after tens of ps. Also, MD simulations without the presence of MeOH have been carried out to determine whether the Wat1 hydrogen bonded to Asp-297 CO 2 exchanges with the solvent water. During 0 5 ns, there was no exchange with Wat1 and the distances of O(Wat1) to O5(PQQ) and O(Wat1) to OD1(Asp- 297) are Å and Å, respectively [see supporting information (SI) Table 1 and SI Fig. 7]. Thus, the Asp-297 CO 2 Wat1 structure is very stable. Starting without the presence of Wat1 in the active site and MeOH hydrogen bonded to Asp-297 CO 2, as suggested by electron-nuclear double resonance (ENDOR) studies (17), we found that MeOH moves to hydrogen bond to Glu-171 CO 2 after 2.2 ns simulation whereas Asp-297 CO 2 becomes coordinated to Ca 2 after 1.8 ns time (Fig. 1). On the basis of these MD simulation studies, we conclude that the very stable Asp-297 CO 2 Wat1 complex has some role in the enzymatic reaction. During 3 ns MD simulations of MDHPQQMeOH complex, the structures, in which Asp-297 CO 2 is hydrogen bonded to MeOH and C H of MeOH is adjacent to C5 of PQQ, are present in 0.2% of the simulation. It can be seen from Fig. 2A that the reaction barrier for CH 3 OH 3 CH 2 O oxidation involving Asp- 297 CO 2 as the general-base is 50 kcal/mol. This computation means that the reaction with Asp-297 CO 2 as the general base could not occur. The very large G for Asp-297 CO 2 general base catalysis is related to the distance of 3.7 Å between CB (MeOH) and C5 (PQQ). Glu-171 CO 2 General Base Catalyst. The energies of the ground state (MDHPQQMeOHWat1, Fig. 3), transition state (TS), and immediate product (MDHPQQ(C5H) OACH 2 Wat1) Author contributions: X.Z., S.Y.R., and T.C.B. designed research; X.Z. and S.Y.R. performed research; X.Z. analyzed data; and X.Z., S.Y.R., and T.C.B. wrote the paper. The authors declare no conflict of interest. Abbreviations: QM/MM, quantum mechanics and molecular mechanics; SCC-DFTB, selfconsistent-charge density-functional tight-binding; MD, molecular dynamics; TS, transition state; PQQ, pyrroloquinoline quinone; MDH, methanol dehydrogenase; MeOH, methanol; ENDOR, electron-nuclear double resonance. *To whom correspondence should be addressed. tcbruice@chem.ucsb.edu. This article contains supporting information online at /DC by The National Academy of Sciences of the USA PNAS January 16, 2007 vol. 104 no

2 Scheme 1. were determined by two-dimensional QM/MM potential energy surface (Fig. 2B). The calculated potential energy barrier of Scheme 2 is E 11.5 kcal/mol by QM/MM. This calculated free energy barrier is comparable with the calibrated value (11.1 kcal/mol) determined by single point calculation at the B3LYP/ 6-31G(d,p)//MM level [B3LYP/6-31G(d,p) as implemented in Gamess-US (20)]. Thus, the present QM/MM (i.e., SCC- DFTB/MM) is reliable for the present system. Normal mode analysis shows that the corrections to the barrier height of zero point energy [(ZPE) ], the contributions of entropy (TS ), and the thermal vibrational energy (E vib ) are 2.7 kcal/mol, 2.6 kcal/mol, and 0.7 kcal/mol, respectively. Thus, the calculated free energy barrier for this oxidation reaction is G E E vib (ZPE) TS kcal/mol (Fig. 4), which is in reasonable agreement with 8.5 kcal/mol determined from the experimental Gibbs energy barrier (21) in the wild-type MDH. Computational findings based on the present QM/MM computations and MD simulations, as well as our previous MD simulations (6, 7, 9), lead to the conclusion that only Glu-171 CO 2 could be the general base catalyst for the oxidation of CH 3 OH 3 CH 2 O. immediate product MDHPQQ(C5H) OACH 2 Wat1 is close to that of the TS. At this point, the reaction is very endothermic. Overall Reaction. The oxidation of CH 3 OH 3 CH 2 O to provide the MDHPQQ(C5H) OACH 2 Wat1 (Fig. 6) is calculated to be endothermic by E 7.1 kcal/mol (Fig. 4). Normal mode analysis shows that the correction of (ZPE), TS, and E vib are 2.0 kcal/mol, 1.6 kcal/mol, and 0.5 kcal/mol, respectively. Therefore, the formation of MDHPQQ(C5H) OACH 2 Wat1 is calculated to be endothermic by G E E vib (ZPE) TS kcal/mol (Fig. 4). The reaction of H 2 O OACH 2 3 H 2 C(OH) 2 is very facile. We propose that Asp-297 CO 2 catalyzes Wat1 hydration of the CH 2 AO immediate product. In the MDHPQQ(C5H) OACH 2 Wat1 structure (Fig. 6), the distance of O (Wat1) from the OACH 2 carbon is 3.56 Å, and the angle of approach of O(Wat1) to the carbonyl carbon plane of OACH 2 is 81. This is the required geometry (NAC) (24, 25) for the hydration reaction. QM/MM computations show that the following reaction Glu-171 CO 2 HOACH 2 Wat1 O 2 C Asp Glu-171 CO 2 Transition State Involving Glu-171 CO 2 as General Base in the Oxidation of CH 3 OH 3 CH 2 O. The structure (Fig. 5) of the transition state (TS) in Scheme 2 was determined by adiabatic mapping at the QM/MM level, which is almost the same as that (SI Table 1) determined by the conjugate peak refinement (CPR) (22) method. Normal mode analysis confirms the nature of the TS (Fig. 5) with only one imaginary frequency of 170i cm 1.Inthe TS (Fig. 5), the distances of HG1 (MeOH) OE1 (Glu-171) and HG1 (MeOH) OG (MeOH) are 1.01 Å and 1.62 Å, respectively; the bond lengths of CB (MeOH) HB1 (MeOH) and HB1 (MeOH) C5 (PQQ) are 1.53 Å and 1.22 Å, respectively. This structure indicates an explosive transition state in which proton and hydride are essentially transferred and MeOH has been almost completely converted to formaldehyde (OACH 2 ). Following the Hammond postulate (23), the free energy of the Scheme 2. Fig. 1. MD simulations. (A) Time-dependent variation of the distances between hydroxyl oxygen of MeOH and the carboxylate oxygen of Glu-171 CO 2 and Asp-297 CO 2, respectively. (B) The coordination of Ca 2 with O7A (PQQ) and the carboxylate oxygen (OD1) of Asp-297 CO 2, respectively, at the ground state of MDHPQQMeOH when Wat1 is replaced by MeOH Zhang et al.

3 Fig. 4. The potential surface at the SCC-DFTB/M level (in kcal/mol) for the oxidation reaction of MeOH catalyzed by MDH (solid line) and the hydration of OACH 2 (dotted line). After the departure of CH 2 (OH) 2, rearrangement (Scheme 3) to provide the cofactor hydroquinone is exothermic by 4.1 kcal/mol at the SCCDFTB/MM level. Thus, the overall reaction, MeOH o-quinone Wat1 3 CH 2 (OH) 2 semiquinone, is favorable. Fig. 2. Contour plot of the potential energy profiles using SCC-DFTB/MM and two-dimensional reaction coordinates y r CB(MeOH)-HB1(MeOH) r HB1(MeOH)- C5(PQQ) and x r OG(MeOH)-HG1(MeOH) r HG1(MeOH)-OD1(Asp-297) (A), and y r CB(MeOH)- HB1(MeOH) r HB1(MeOH)-C5(PQQ) and x r OG(MeOH)-HG1(MeOH) r HG1(MeOH)-OE1(Glu-171) (B) involving Asp-297 CO 2 general base in MDHPQQMeOH complex and Glu-171 CO 2 general base catalyst in MDHPQQMeOHWat1 complex, respectively. The position of the TS is marked by an asterisk. HOCH 2 OH HO 2 C Asp-297 is associated with a very small (i.e., 1.1 kcal/mol) free energy barrier (Fig. 4). The hydration reaction is calculated to be exothermic by G 8.7 kcal/mol at the QM/MM level (Fig. 4). Role of Ca 2 Ion in the Oxidation of CH 3 OH 3 CH 2 O with Glu-171 CO 2 as the General Base. In the QM/MM ground state structure (Fig. 3), Ca 2 is at a distance of 2.45 Å from the quinone carbonyl oxygen O5 of PQQ. The late TS places almost a complete negative charge at C5-O of the cofactor and Glu-171 CO 2 is essentially protonated. Thus, Ca 2 electrostatic stabilization of the TS exceeds that with the ground state. Thus, Ca 2 plays a catalytic role as in the oxidation of CH 3 OH 3 CH 2 O. This finding is in accord with Ca 2 polarizing O5 of PQQ (11). It is to be expected that the interaction of Ba 2 with the developing negative charge at the TS would be greater than that of Ca 2.By experiment, it is found that replacement of Ca 2 by Ba 2 to provide MDHPQQBa 2 system significantly decreases the G of activation as compared with Ca 2 -MDH (3.4 kcal/mol vs. 8.5 kcal/mol) (21). Fig. 3. The structure of the active site of MDHPQQMeOHWat1 complex at the ground state (A) and the close-up of the reaction region (B) as determined by ABNR energy-minimizing the final structure of our previous 3-ns MD simulations at the SCC-DFTB/MM level. Zhang et al. PNAS January 16, 2007 vol. 104 no

4 Fig. 5. The structure of the transition state (TS) (Scheme 2) at the active site (A) and the close-up of the reaction region (B) as determined by adiabatic mapping at the SCC-DFTB/MM level. Conclusion In order for MeOH to reduce an o-quinone by hydride equivalent transfer at neutral ph, the reaction must be general-base catalyzed (B: HOCH 2 H C5AO 3 BH OACH 2 C5H O ). The only two possible general bases present are Glu-171 CO 2 and Asp-297 CO 2. The following MD observations support Glu-171 CO 2 as the catalytic general base. In the x-ray structure, Glu-171 CO 2 is electrostatically near the Ca 2 whereas Asp-297 CO 2 is hydrogen bonded to Wat1. When a molecule of MeOH was placed in various positions in the active site followed by 3 ns MD simulations, the MeOH was found to be hydrogen bonded to Glu-171 CO 2. During 3 ns MD simulations on MDHPQQMeOHWat1, Wat1 is hydrogen bonded to Asp-297 CO 2 and not replaced by MeOH. Indeed, in the absence of MeOH, Wat1 does not exchange with the solvent water (TIP3P model) during 5-ns MD simulations. From the present QM/MM calculations, the free energy barrier (G ) for the reaction in which MeOH is hydrogen bonded to Glu-171 CO 2 is 10.7 kcal/mol compared with the experimental value of 8.5 kcal/mol. The transition state for the Glu-171 CO 2 general base hydride equivalent transfer is late with the proton and hydride almost completely transferred and OACH 2 almost completely formed. In accord with the Hammond postulate, this first step in the overall reaction is endothermic. Without the presence of Wat1 at the active site and with MeOH hydrogen bonded to Asp-297 CO 2, as suggested by ENDOR studies of Kay et al. (17), the value of G for MeOH reduction of o-quinone is at least 50 kcal/mol. The important function for Asp-297 CO 2 hydrogen bonded to Wat1 is to catalyze the hydration of the OACH 2 direct product. The potential energy barrier for this reaction is calculated to be 1.1 kcal/mol. Most importantly, the exothermic hydration reaction (Eq. 1B) pulls the endothermic hydride transfer reaction (Eq. 1A), such that o-quinone CH 3 OH Wat1 3 hydroquinone H 2 C(OH) 2 is thermodynamically favorable: [1A] [1B] Although the experiment by ENDOR supported Asp-297 CO 2 as the general base, it must be pointed out that enzyme structure is different at the lower temperature (60 to 100 C) as compared with ambient temperature, the decrease in the distances of electrostatic interactions occurs with decrease in temperature (26, 27). In addition, the model calculations including MeOH in place of Wat1 show that the reaction does not occur. Computational Methods MD Simulations. Based on the procedures described in our previous study (9), stochastic boundary MD was carried out on Fig. 6. The structure of the intermediate MDHPQQ(C5H) OACH 2 Wat1 at the active site as determined by the SCC-DFTB/MM ABNR energy-minimization method. Scheme Zhang et al.

5 MDHPQQWat1 for 5 ns to verify the stabilization of the crystal water (Wat1) in the absence of substrate methanol (MeOH); 3-ns MD simulations were performed on MDHPQQMeOH, with Wat1 removed from the active site and MeOH hydrogen bonded to Asp-297 CO 2 to investigate the validity of the model as suggested by ENDOR studies (17). QM/MM Setup. The self-consistent-charge density-functional tightbinding [SCC-DFTB (28, 29)] approach implemented in CHARMM (30) (version 31b1) was used as the QM method. The QM region included the cofactor PQQ, the substrate MeOH, and (i) the side chains of Glu-171 CO 2 and Arg-324, in case Glu-171 CO 2 acts as the general base in the MDHPQQMeOHWat1 complex; (ii) the side-chain of Asp-297 CO 2, in case Asp-297 CO 2 acts as the general base in MDHPQQMeOH model described by Kay et al. (17). The link atoms were introduced to saturate the valences of the QM boundary atoms in the QM/MM calculations. Ca 2 parameters are not available for SCCDFTB formulism. The stochastic boundary (31) with a 25-Å radius was centered at the cofactor PQQ. Included within the stochastic boundary were the PQQ cofactor with 27 atoms, substrate MeOH with 6 atoms, one Ca 2 ion, 7,043 protein atoms, 105 x-ray crystal water molecules, and 276 TIP3P water molecules. A Poisson- Boltzmann (PB) charge-scaling scheme (32) was used to include the correction of long-range electrostatic interactions in the simulation. PB calculations determined a set of scaling factors, which reduce the partial charges of charged residues in the QM/MM electrostatic potential calculations so as to avoid artifactual structural change. To determine the activation energies for the reactions, adiabatic mapping calculations were carried out by using twodimensional reaction coordinates, which were the antisymmetric stretch involving the donor, the transferring proton, and the acceptor as the reaction coordinate, respectively. The starting structure (Fig. 3) of the PQQ methanol dehydrogenase (MDHPQQMeOHWat1) complex in the water solvent was obtained from the final structure of 3-ns MD simulations (6, 7, 9). The ground state was obtained by QM/MM energy-minimizing the final structure from our previous MD simulation (6, 7, 9) for MDHPQQMeOHWat1 complex by Adopted Basis Newton-Raphson (ABNR) method until the gradient was 0.01 kcal/(molå). The similar procedure was used to obtain the QM/MM starting structure from the snapshot (1.2 ns) of 3-ns MD simulations for MDHPQQMeOH model. The transition state was obtained by using the adiabatic mapping method, and confirmed by normal mode analysis, which provided only one imaginary frequency. The immediate product MDHPQQ(C5H) OACH 2 Wat1 (Scheme 2) was obtained by QM/MM ABNR minimization until the gradient was 0.01 kcal/ (molå). The residues within 16 Å from the cofactor at the ground state (MDHPQQMeOHWat1), immediate product (MDH PQQ(C5H) OACH 2 Wat1), and transition state (TS) were included in normal mode analyses to provide 3N-6 frequencies, which were used to calculate the vibrational contributions of zero-point energies ((ZPE)), entropies (TS), and the thermal vibrational energies (E vib ) to the reaction barrier. Whereas the contributions from transition and rotation motions could be neglected for the enzymatic reactions at the constant temperature according to their corresponding statistical equations (33). Beyond that, residues were fixed in the vibrational calculations, and the vibrational contributions ((ZPE), TS, and E vib ) were estimated with the harmonic approximation at 25 C. Thus, the equation G E (ZPE) TS E vib (34) could be used to evaluate the free energy of activation and the reaction free energy. Some of the calculations were performed at the National Center for Supercomputing Applications (University of Illinois at Urbana Champaign, Urbana, IL). 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