What is the Advantage and Disadvantage of zinc catalysts

Recent insights on the use of modified Zn-based catalysts in eCO2RR

H. Wang, N. Deng, X. Li, Y. Chen, Y. Tian, B. Cheng and W. Kang, Nanoscale, , 16, DOI: 10./D3NRJ

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CcrA-CEF: binding and reactivity

CEF binding dehydrates almost completely the active site: only one water molecule is found in the close proximity of the metal ions (Figure 4). This molecule (WAT in Figure 2) is interacting with Zn2 (d=3.4(4) Å, Figure 3), H-bonding in addition to CEF carboxylate group (C4 substituent in cephalosporins, Figure 2) and to the C3 substituent. Although not strongly bonding to Zn2 as in the X-ray structure (2.3 Å), WAT is able to maintain a trigonal bi-pyramidal geometry (common of penta-coordinated rather than tetrahedral geometry for Zn2 sphere). The C4 substituent, which is present in all bi-cyclic β-lactams, also H-bonds to backbone N(Asn233) and forms a water-mediated salt bridge with Lys224. The β-lactam carbonyl (O-C8 in Figure 1), mainly H-bonds to Nε(Asn233) (Figure 2, and SI). These minimal binding features are present in all β-lactams (basically present in BcII-CEF adduct either, see below and ref. 47) and are supposed to play a major role in substrate binding. On the other hand, the C3 substituent interacts with Lys224 in loop Cys221-Thr240, which includes several conserved residues among MβL's,4 and C7 substituent binds to Glu62 in loop Ile61-Val67 (see SI). This is by far the most flexible region of the protein, and undergoes major conformational changes upon substrate binding.

The β-lactam C8 carbonyl oxygen strongly H-bonds to Asn233 along this reaction pathway (d9 in Figure 3), whereas it does not bind Zn1 due to the steric orientation of CEF at the catalytic pocket (Zn1 keeps a tetrahedral geometry in R). Furthermore, cefotaxime is found to H-bond to Asn233 for about 90% of the time during the MD trajectory. This interaction is not predicted to be essential in cefotaxime hydrolysis by monozinc BcII,47,48 and mutagenesis studies indicated that Asn233 is not essential for catalysis in CcrA.15 Asn233 fluctuations randomly occur along the MD trajectory when its side-chain flips out of the active site losing the H-bond with the β-lactam, eventually replaced by water molecules coming from the bulk solvent (R in Figure 3). The timescale of such an event is estimated to be few tenths of nanoseconds, and appears to be well sampled within &#;4 ns trajectories. However, it is very unlikely to occur spontaneously during a first principles QM-MM dynamics (&#;70 ps timescale). Based on these observations, this is the only large conformational change not accessible within QM-MM timescale, so that two hydrolytic pathways are explored with the same QM-MM protocol: Path I, where the β-lactam carbonyl does H-bond directly to Asn233, and Path II, in which Asn233 is flipped out, solvated by bulk water molecules (Figure 3). Initial QM-MM equilibrations (&#;20 ps) are performed to check the stability of starting MD configurations for Path I and II. Although, as already stated, this conformation is present for about 10% of the MD simulated time, it points to a non-crucial role of Asn233 for the function, which in turn is consistent with an increase of KM in the N233A, N233L and N233E,15,64 without largely affecting kcat/KM. In this respect, it is worth noting that so far Asn233-Tyr is the only mutation which significant improves the catalytic power in another MβL (BcII) (unpublished results), and it is the only alternative residue found in MβL's at position 233 (namely, in B1 MβL blaB, from E. meningoseptica39), that can perform a similar role. Moreover, a Tyr residue is found in glyoxalase II (a dizinc enzyme belonging to the metallo β-lactamase superfamily65) at the same topological position, and might have a similar binding function.

The putative reaction coordinate dRC, namely the distance between the nucleophilic oxygen O(OH-) and β-lactam carbonyl carbon C8 (Figure 2), is very short during MD simulations (3.4(2) Å) suggesting that this model provides a good picture of the Michaelis complex (R, Figure 3). The catalytic pathway of CEF hydrolysis is studied using hybrid QM-MM simulations, starting from an average-dRC MD snapshot equilibrated by &#;20 ps QM-MM dynamics on the reactant state, R. The free simulation suggests that the coordination sphere of the metal ion is preserved as in the X-ray structure (Figure 3, Scheme 1A) and dRC is similar to the MD value (dRC=3.3(3) Å). dRC is then adiabatically shortened to force the hydrolysis of the β-lactam CEF (products state, P).

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In Path I, the nucleophile OH- approaches the β-lactam C8 maintaining its coordination with metal ions. The whole adduct is not relevantly affected by any other structural changes until dRC=2.0 Å. At dRC=1.8 Å, the Zn2-OH- bond weakens (d2=2.6 Å, Figure 3), while the Zn1-Zn2 distance increases simultaneously (d3=4.0 Å). The metal-ligand bond lengths do not experience significant structural modifications, but WAT molecule definitively detaches from Zn2, being scattered away from the active site (d4=4.4 Å). At the same time, Zn2 weakly binds to N5 (d5=2.3 Å), consequently lengthening the C8-N5 bond in CEF (d7=1.6 Å). At dRC=1.6 Å, the force on the constrained O(OH-)-C8 distance is negative indicating that the transition state has been reached, and can be located reconstructing the free energy profile at dRC&#;1.7 Å. When the constraint on the O(OH-)-C8 distance is released, the system falls freely on a stable intermediate state (INT, Figure 3), where the C8-N5 bond is broken (d7=3.1 Å), and the development of partial negative charge on N5 is stabilized by a stronger N5-Zn2 bond (d5=1.6 Å). The C4-carboxylate group from the substrate becomes a Zn2 ligand at this point. Reorganization of Zn2 coordination sphere induces detachment of WAT from the site. C8 fully acquires sp2 hybridization, and its newly formed carboxyl moiety binds Zn1 in a bi-dentate manner. This is accompanied by the protonation of Asp120, likely promoted to stabilize Zn1 coordination sphere.

The formation of INT requires a large activation free energy (ΔF=30(3) kcal mol-1), which is clearly inconsistent with the fact that cefotaxime is readily hydrolyzed by CcrA with an estimated free energy of &#;17 kcal mol-1. In addition, the presence of an intermediate is not consistent with the fact that the process is likely supposed to be a single step process, as proposed by experimental evidences on similar adducts.13 Finally, in INT the strong N5-Zn2 and O(C8)-Zn1 bonds seem to prevent, due to steric hindrance, the approach of a water molecule from the bulk solvent. This is required to protonate the β-lactam N5 and to release the products. A possible proton donor candidate in INT conformation can be the protonated Asp120, as previously proposed, although this would require a significant rearrangement of the active site. For cefotaxime hydrolysis however this path is already largely energetically unfavorable, but for other substrates it cannot be definitively excluded.

Interestingly, this state closely resembles the crystal structure of L1 MβL from B3 subclass recently solved in complex with hydrolyzed moxalactam.66 3&#;-Elimination of the substrate bypasses N5 protonation producing a stable intermediate by product inhibition. In this conformation, as well as in the INT state, N5 and the β-lactam carboxylate are bound to Zn2, and the hydrolyzed C8 moiety is bound to Zn1. The only difference with the X-ray structure is the presence of a hydroxide-water bridging the Zn2+ ions. This moiety can eventually occupy this position giving a longer sampling time in the QM-MM simulation. It is worth noticing that QM cell including only CEF bi-cyclic core does not allow a possible 3&#;-elimination of C3 acetate as in L1 case, which may make N5 protonation unnecessary. Since many cephalosporins equally do not possess a good 3&#; leaving group this might suggest their presence could favor the formation of stable INT-like intermediate state. Moreover, PM3-MD computational structures reported for the intermediate of hydrolysis of nitrocefin67 have similar features in comparison with INT: the N5 atom is bound to Zn2, and the hydrolyzed C8 carboxylate is bound to Zn1. However, the C4 carboxylate is not interacting with Zn2. So, the INT species is the outcome of a poorly efficient first step of cefotaxime hydrolysis, but based on these independent evidences it further indicates the feasibility of such an intermediate state in MβL-mediated hydrolysis of other substrates.

In Path II, the reaction proceeds similarly to Path I until dRC=2.2 Å (the only noticeable events up to that stage are the increase of the C8-N5 bond (d7=1.4 Å, Figure 3, Scheme 1A), and the stronger bond between Zn2 and WAT, d5=2.5 Å). At dRC=2.0 Å, the force on RC nullifies, indicating formation of the TS through a cascade of almost simultaneous events (Figure 3).&#; (i) OH- moves out from the Zn1-Zn2 plane attacking C8 in a apical position with respect to Zn1, while the Zn1-OH- bond is being weakened (d1=2.2 Å). (ii) The OH--Zn2 bond is lost upon nucleophilic attack, and the Zn1-Zn2 distance increases to 3.8 Å. A hydroxide simultaneously bonded to Zn1 and Zn2 is in fact expected to be a poor nucleophile, so it is reasonable that OH- attacks while detaching from at least one Zn2+ ion. As a consequence, the Zn2 coordination number changes from 5 to 4. (iii) WAT ligand, previously weakly bonded at the apical position of the Zn2 bi-pyramidal polyhedron, moves towards Zn1 to give rise to a tetrahedral geometry. The WAT-Zn2 distance (d5) gradually decreases thus lowering the WAT pKa. (iv) The Zn2-bound WAT comes closer to the β-lactam ring, consequently increasing the C8-N5 distance. The partial negative charge on N5 together with the enhanced nucleophilicity of WAT produces a proton shuttle from WAT to the N5 atom that finally triggers the C8-N5 bond cleavage (d7=2.6 Å). (v) Deprotonated water (Figure 3, WAT &#; OH-&#;) binds Zn1 completing the tetrahedral coordination sphere of Zn2, and perfectly replacing the position of the OH- nucleophile in R state. Zn1 at this point switches to a penta-coordinated bi-pyramidal coordination from the initial tetrahedral one. (vi) Upon nucleophilic attack and C8-N5 bond breaking, C8 acquires an sp2 hybridization. (vii) The O(C8)-Asn233 H-bond is formed again: the partially hydrolyzed substrate gets farther from the metal center upon TS formation (mainly due to OH- switching on Zn1 apical position), being able to H-bond to Asn233.

When the constraint of RC is released, the system evolves onto the products state (P, Figure 3), where OH-&#; reorients H-bonding to Asp120 and bridging the Zn1 and Zn2 ions as in R state. The CEF substrate is completely hydrolyzed, the β-lactam ring is open and C8 acquires planar sp2 hybridization, and it is finally detached from the metal center losing the OH--Zn1 bond. The O(C8)-Asn233 H-bond is stronger than in TS, indicating that the flexibility and electrostatic properties of this residue play a role for binding as well as for the product release of degraded cephalosporins. As soon as the product is going to be expelled from the active site, water molecules start to solvate the metal site first interacting with Zn1 (meanwhile returned to a tetrahedral geometry), and eventually coordinating Zn2 as in the unbound structure.42 The calculated estimated ΔF=18(2) kcal mol-1 is consistent with (i) experimental evidences that suggest a single-step, efficient reaction for cephalosporin hydrolysis68 with (ii) a free energy of activation of about 17 kcal mol-1 for similar reactions,49 and with (iii) the fact the uncatalyzed reaction in water solution has an estimated barrier of 48(3) kcal mol-1.48

This also suggests that Asn233 has been selected by evolution as the best compromise for its involvement in tuning the binding, catalysis, and product release processes. Asn233 is in fact able to interact with the β-lactam carbonyl moiety accommodating the substrate in an optimal position for the nucleophilic attack, but at the same time it can temporarily lose this interaction allowing the attack on the carbonyl along with the reorganization of the metal center, finally facilitating release of the inactivated β-lactam from the active site.

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