Linus O.
Johannissen
*a,
David
Leys
ab and
Sam
Hay
*ab
aSYNBIOCHEM, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. E-mail: linus.johannissen@manchester.ac.uk; sam.hay@manchester.ac.uk
bSchool of Chemistry, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK
First published on 6th February 2017
Distinct mechanisms have been proposed for the biological dehalogenation catalyzed by cobalamin-dependent enzymes, with two recent crystallographic studies suggesting different mechanisms based on the observed interaction between the organohalide substrate and cobalamin. In one case, involving an aromatic dibromide substrate in NpRdhA, a novel CoII–Br interaction was observed using EPR, suggesting a mechanism involving a [Co⋯X⋯R] adduct. However, in the case of trichloroethylene in PceA, a significantly longer Co–Cl distance was observed in X-ray crystal structures, suggesting a dissociative electron transfer mechanism. Subsequent DFT models of these reactions have not reproduced these differences in binding modes. Here, we have performed molecular docking and DFT calculations to investigate and compare the interaction between different organohalides and cobalamin in both NpRdhA and PceA. In each case, despite differences in binding in the CoII state, the reaction likely proceeds via formation of a [Co⋯X⋯R] adduct in the CoI state that weakens the breaking carbon–halide bond, suggesting this could be a general mechanism for cobalamin-dependent dehalogenation.
A crystallographic study of PceA by Bommer et al. suggested a dissociative electron transfer (eT) from cobalamin to the substrate trichloroethylene (TCE) during the reaction (Scheme S1 in the ESI†).6 This forms a dichlorovinyl radical, and with free cobalamin in solution this would recombine with the resulting cob(II)alamin forming a Co–C bond.10 However, in the enzyme this recombination is likely sterically hindered, leading to the proposal that the proximal RDase [4Fe–4S] cluster facilitates a second eT, generating a transient carbanion species which is protonated through the active site Tyr. On the other hand, it has been suggested based on electrochemical data, that an outer sphere eT mechanism is unlikely, and that the mechanism probably involves a “more intimate interaction” between cobalamin and substrate.11 Along these lines, a mechanism involving a cobalamin–substrate adduct was proposed by Payne et al. for the debromination of 3,5-dibromo-4-hydroxybenzoic acid (DBHB; Scheme S2, ESI†) by NpRdhA. In this case, a direct CoII–Br interaction was observed by EPR, although only substrate-free and halide-bound X-ray crystal structures of the enzyme were obtained.7 The mechanism was proposed to follow either homo- or heterolytic C–Br bond cleavage. The heterolytic cleavage mechanism requires that the substrate be sufficiently activated after adduct formation (step 2A in Scheme S2, ESI†) to directly deprotonate the active site Tyr (step 2B). If this is not the case, further reduction of the substrate, via reduction of cobalamin by the proximal [4Fe–4S] cluster or directly by eT from the proximal [4Fe–4S] cluster, may be required.
Liao et al. have since employed DFT modelling to investigate the mechanism in both NpRdhA8 and PceA9 for a range of halides and proposed that for both enzymes dehalogenation occurs by a heterolytic C–X cleavage mechanism after reduction of CoII to CoI, via a [Co⋯X⋯R] adduct (with bonding between Co and the transferring halide), and that this is concerted with the proton transfer from the active site Tyr. However, these DFT calculations did not reproduce the different experimentally observed CoII binding modes, and resulted in cobalamin–substrate adducts in their PceA/TCE models (with Co–X distances of 2.79 to 2.96 Å depending on the substrate orientation in the model) as well as NpRdhA/2,6-dibromo-4-methylphenolate (DBMP) (3.16 Å). In this study we employ a computational method that qualitatively reproduces the observed cobalamin–substrate interactions in the CoII oxidation state of both enzymes, and investigate the nature of this interaction, its effect on the dehalogenation mechanism and the factors that affect it.
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Fig. 1 Docked structures of (A) DBHB in NpRdhA and (B) TCE in PceA. Key active site residues and the Co–X distances are labeled and the substrates are rendered in ball and stick mode. |
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Fig. 2 Optimized geometries of the NpRdhA/DBMP (A and C) and PceA/TCE (B and D) active site models in the CoII (A and B) and CoI (C and D) oxidation states. Only polar hydrogens are shown. Equivalent structures for all four enzyme/substrate models are shown in Fig. S2 (ESI†). |
Molecular orbitals, charges and spin densities were obtained from single point calculations on truncated models that include the cobalamin and substrate. Charges were calculated both from natural bonding orbitals analyses and the Merz–Kollman method, which maps point charges onto the electrostatic potential.25,26
To investigate the organohalide–cobalamin interactions in NpRdhA and PceA, DFT calculations were performed on active site models in both the CoI and CoII states. Active site models included the bound PceA substrates TCE and its analogue tribromoethylene (TBE) and the NpRdhA DBHB substrate analogue DBMP and its chlorinated equivalent 2,6-dichloro-4-methylphenolate (DCMP). These models are illustrated in Fig. S1 (ESI†), along with the substrates and the truncated cobalamin model used. In all cases, the substrates remained bound within the active site, with the Co–X distance shorter, and substrate–X bonds longer, in the CoI oxidation state (Fig. 2 and Table 1). The CoII–Br ‘bond’ distance in NpRhA/DBMP is also shorter than the CoII–Cl bond distance in PceA/TCE, consistent with EPR7 and crystallographic6 data for these systems.
Model | R(Co–X)/Å | R(Ca–X)/Å | R(C–Hb)/Å | |
---|---|---|---|---|
a C refers to the C bound to the transferring X. b H refers to the proton transferred from the active site Tyr. c Active site model with neutralised Arg552 residue. | ||||
PceA/TCE | CoII | 3.83 | 1.72 | 2.81 |
CoI | 2.77 | 1.79 | 2.65 | |
PceA/TBE | CoII | 2.62 | 1.92 | 2.45 |
CoI | 2.57 | 2.17 | 2.15 | |
NpRdha/DBMP | CoII | 3.60 | 1.93 | 2.48 |
CoI | 2.84 | 2.02 | 2.33 | |
CoII![]() |
3.39 | 1.93 | 2.52 | |
CoI![]() |
2.96 | 1.98 | 2.47 | |
NpRdha/DCMP | CoII | 3.70 | 1.77 | 2.51 |
CoI | 3.10 | 1.78 | 2.37 |
There is no transfer of electron density between molecular orbitals (MOs) from the substrate and cobalamin for PceA/TCE, while inspection of these MOs confirms that an adduct is formed in the other three models, although the specifics of the interaction differs between the two enzymes (Fig. 3 and Fig. S3, ESI†). For the NpRdhA models, electron density is transferred from the substrate into the singly occupied CoII dz2 orbital, so that the lowest unoccupied MOs (LUMOs) consist of orbitals form the highest occupied MO (HOMO) of the substrate, leading to elevated charges and spin densities on DBMP and DCMP (Table 2). For PceA/TBE, the substrate HOMO is delocalised with several bonding orbitals of the cobalamin (Fig. S3, ESI†), so that the LUMO is formed by delocalisation of the CoII dz2 orbital with a C–Br anti-bonding orbital. For PceA/TCE there is no delocalisation of orbitals from the substrate and cobalamin, and no transfer of electron density between substrate and cobalamin. Thus, a cob(II)alamin–halide adduct is formed in each model except that for PceA/TCE.
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Fig. 3 LUMOs for the CoII optimised active site models: (A) NpRdhA/DBMP, (B) NpRdhA/DCMP, (C) PceA/TBE and (D) PceaA/TCE. |
Model | Δ chargea | Spin densityb | |
---|---|---|---|
a Difference in charge relative to the isolated substrate (−1 for DBMP and DCMP, 0 for TBE and TCE); charges shown are from natural bond orbital analysis (and the Merz–Kollman method in parentheses). b Spin densities of the CoII (singlet radical) models. | |||
PceA/TCE | CoII | 0.006 (−0.033) | 0.002 |
CoI | −0.153 (−0.112) | N/A | |
PceA/TBE | CoII | 0.179 (0.198) | 0.078 |
CoI | −0.334 (−0.386) | N/A | |
NpRdhA/DBMP | CoII | 0.499 (0.522) | 0.490 |
CoI | 0.003 (0.068) | N/A | |
NpRdhA/DCMP | CoII | 0.977 (0.961) | 0.989 |
CoI | 0.018 (0.077) | N/A |
Despite the differences in the substrate–cobalamin interactions in the (formally) CoII state, the interactions after reduction to CoI are very similar (Fig. 4). In each case, there is transfer of electron density from the Co dz2 orbital into the C–X antibonding orbital for the breaking C–X bond, as previously proposed by Cooper et al.27 This has the effect of activating the substrate towards C–X bond cleavage by stretching the C–X bond and concomitantly decreasing the active site Tyr proton-substrate carbon distance (C–H in Table 1). This effect is strongest for PceA, where a significant amount of charge is transferred onto TCE and TBE, and very little charge transfer is observed for NpRdhA (Table 2). Thus, while these DFT models are consistent with the different binding modes observed experimentally for NpRhA/DBMP and PceA/TCE in the CoII states, they suggest that reduction to CoI leads to very similar [Co⋯X⋯R] adducts (Fig. 4), and that the strongest CoI interaction (based on Co–X distance and degree of charge/spin transfer) corresponds to the weakest CoII interaction.
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Fig. 4 MOs corresponding to the Co dz2 orbital for the CoI optimised active site models: (A) NpRdhA/DBMP (HOMO−2), (B) NpRdhA/DCMP (HOMO−2), (C) PceA/TBE (HOMO) and (D) PceaA/TCE (HOMO). |
Consistent with previous models,8,9 dehalogenation after reduction to CoI proceeds via a concerted mechanism for each model (combining steps 2B and 2C in Scheme S2, ESI†), with a single transition state for both C–X bond cleavage and protonation by the active site Tyr (Table 3 and Fig. S4, S5, ESI†) to yield a CoIII–X species which will be rapidly reduced to the experimentally observed CoII–X species.9 For the PceA reactions, the product is cis-1,2-dihaloethene as shown in Fig. S4 (ESI†), which is the experimentally observed product for dehalogenation of TCE. The preference for this regiochemistry is not surprising given that substrate reduction weakens the cleaved C–X bond the most (Fig. S5, ESI†).
Model | ΔG‡ (ΔE‡)/kJ mol−1 | ν i /cm−1 | R(Co–X)/Å | R(C–X)/Å | R(C–H)/Å |
---|---|---|---|---|---|
a Gibbs free energies and gas phase potential energies in (parentheses) relative to the CoI ground state. b In each case, this vibrational mode corresponds principally to motion of the transferring H, with small contributions from C and X. The eigenvalues for H, C and X, respectively, are as follows. PceA/TCE: 0.907, 0.228, 0.049; PceA/TBE: 0.976, 0.108, 0.014; NpRdhA/DBMP: 0.996, 0.0490, 0.0; NpRdhA/DCMP: 0.994, 0.0490, 0.010. | |||||
PceA/TCE | −11.4 (7.22) | 469.6i | 2.27 | 2.62 | 1.53 |
PceA/TBE | −14.3 (7.98) | 847.3i | 2.40 | 2.65 | 1.47 |
NpRdhA/DBMP | 41.1 (53.3) | 1386.7i | 2.47 | 2.68 | 1.39 |
NpRdhA/DCMP | 49.9 (78.5) | 1412.8i | 2.36 | 2.55 | 1.39 |
For PceA, the gas phase potential energies for the transition states (ΔE‡) are so low that the resulting continuum solvation-corrected Gibbs free energies are negative, suggesting that this reaction is effectively barrierless. Qualitatively, these results are very similar to those of Liao et al., who determined barriers of 30.5 and 2.09 kJ mol−1 for NpRdhA/DBMP8 and PceA/TCE9 (with the TCE orientation most similar to that in this study), respectively. Unsurprisingly, the trend in ΔG‡ is dominated by the gas phase potential energy for the reaction (ΔE‡). The bond dissociation energies (BDEs) for TBE and TCE are much larger than those for the DBMP and DCMP (Table S1, ESI†), but ΔE‡ for both PceA models are much smaller than those for NpRdhaA. This is due to the partial reduction of both PceA substrates prior to C–X bond cleavage, as the substrate BDEs are significantly decreased by reduction (Table S1, ESI†).
One of the key differences between the PceA and NpRdhA active sites, apart from the former being more occluded, is the presence of the additional positively charged residue in the NpRdhA active site, R552. In both enzymes, a charged residue – R305 in PceA and K488 in NpRdhA – stabilizes the deprotonated form of the active site Tyr, and the additional Arg in NpRdhA might serve to enhance binding by stabilising the negatively charged (deprotonated) substrate. However, this additional positive charge adjacent to the substrate is also likely to aid in transfer of electron density from the CoI dz2 orbital into the substrate. To test this hypothesis, a NpRdhA/DBMP model with a neutral R305 analogue was optimized (Fig. S7, ESI†). In this model, the C–X bond is shortened and the Co–X distance increased (Table 1), consistent with a weaker cob(I)alamin–DBMP interaction. In other words, the positive charge on R552 helps activate the substrate towards C–X bond cleavage. Conversely, since the cob(II)alamin–DBMP interaction involves transfer of electron density in the opposite direction, R552 weakens this interaction so that the original model has a longer Co–X bond than the neutral analogue.
The different effect of the enzyme active sites was further analysed by energy minimising an artificial NpRdhA/TCE model (Fig. S8, ESI†). As expected, the positive charge on R552 enhances the degree of eT from cobalamin into TCE in the CoI state, and in this case the C–Cl bond is fully broken to form the dichlorovinyl carbanion. For the neutral R552 analogue, the C–Cl bond is also significantly stretched (2.47 Å) compared to the PceA model, due to the proximity of partial positive charges from the R552 amino groups. NpRdhA enhances the degree of eT relative to PceA because the higher energy LUMOs of DBMP and DCMP compared to TCE or TBE (Table S2, ESI†) makes transfer of electron density into the substrate more difficult.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cp08659d |
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