Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation

Abstract

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

ED Fig. 1. Phylogenetic tree of functionally characterized reductive dehalogenases (RdhAs).

Those in bold have been purified and characterised in vitro. Those in italics have been identified from crude lysate assays via native PAGE or knock out/knock in. Those in plain text have implied substrate range based on transcriptional regulation. The sequence accession codes are as follows, AAD44542 = Desulfitobacterium dehalogenans ATCC 51507 CprA, P81594 = Desulfitobacterium hafniense DCB-2 CprA1, AAQ54585 = Desulfitobacterium hafniense PCP-1 CprA5, AAL84925 = Desulfitobacterium chlororespirans CprA, WP_019849102 = Desulfitobacterium sp. PCE-1, AAK06764 = Desulfitobacterium hafniense PCP-1 CprA3, AAO60101 = Desulfitobacterium hafniense PCE-S PceA, O68252 = Dehalospirillum multivorans PceA, AAF73916 = Dehalococcoides mccartyi TceA, YP_181066 = Dehalococcoides mccartyi 195 PceA, AAW80323 = Desulfitobacterium hafniense Y51 PceA, CAD28790 = Dehalobacter restrictus PceA, CAD28792 = Desulfitobacterium hafniense TCE1 PceA, YP_001214307 = Dehalococcoides mccartyi BAV1 BvcA, YP_003330719 = Dehalococcoides mccartyi VS VcrA, ACF24863 = Dehalococcoides sp. MB MbrA, YP_307261 = Dehalococcoides mccartyi CBDB1 CbrA, BAE45338 = Desulfitobacterium sp. KBC1 PrdA, BAE45337 = Desulfitobacterium sp. KBC1 CprA, ACH87594 = Dehalobacter sp. WL DcaA, CAJ75430 = Desulfitobacterium dichloroeliminans LMG P-21439 DcaA, AFQ20272 = Dehalobacter sp. CF CfrA , AFQ20273 = Dehalobacter sp. DCA DcrA , AAG46187 = Desulfitobacterium sp. PCE1 CprA, YP_002457196 = Desulfitobacterium hafniense DCB-2 RdhA3 , YP_001475501 = Shewanella sediminis HAW-EB3 PceA, WP_015585978 = Comamonas sp. 7D-2 BhbA, WP_008597722 = Nitratireductor pacificus RdhA_NP

ED Fig. 2. EPR spectroscopic analysis of reduced RdhA_NP (79μM).

Left panel X-band EPR spectra of RdhA_NP (a) as isolated, 150 μM (b) reduced using EDTA, deazaflavin and light, 79 μM. Right panel (c) X-band (~9.39 GHz) continuous wave EPR spectrum of reduced RdhA_NP recorded at 10 K showing a 2x[4Fe-4S]¹⁺ signal indicative of two magnetically interacting 4Fe-4S clusters due to features marked ⇢ ; g values marked for rhombic [4Fe-4S]¹⁺ signal. (d) X-band continuous wave EPR spectrum of reduced RdhA_NP at 40 K. (e) X-band continuous wave EPR spectrum of reduced RdhA_NP at 80 K showing small base-on cob(II)alamin signal with g values marked. (f) Subtraction of (e) from (d) reveals a second axial [4Fe-4S]¹⁺ signal, with g values as marked, demonstrating that we can observe EPR signals from two different 4Fe-4S clusters within RdhA_NP. Experimental parameters, microwave power 0.5 mW, field modulation frequency 100 KHz, field modulation amplitude 7 G, temperatures as given.

ED Fig. 3. Crystal structure of RdhA_NP.

(a) Stereo view of an overlay of the RdhA_NP cobalamin-binding domain (in blue), the N-terminal non-functional cobalamin binding domain (in light blue) and the human vitamin B12-processing enzyme CblC (in green). The cobalamin cofactor is bound in a similar base-off manner by both RdhA_NP and CblC. The RdhA_NP non-functional cobalamin binding domain does contain an irregular water filled cavity corresponding to the cofactor binding site, but no longer contains any of the residues implied in cofactor binding. (b) Representation of the RdhA_NP cofactors in space filling spheres. The second [4Fe-4S] cluster is in van der Waals contact with the corrin ring, the B pyrrol d-amide corrin moiety is lined along one side of the cluster and forms a hydrogen bond to one of the S-ions. (c) Detailed view of the RdhA_NP 5^th ligand binding site. Residues in close contact with the chloride ligand are shown in atom colored sticks (color coded as in Fig 2). The 2F_o-F_c map is contoured at 1 sigma and shown as a blue mesh. The Tyr426-Lys488 distance is 2.7 Å. (d) Model of the RdhA_NP-substrate complex (color coded as in the main manuscript, Fig 2e) with the F_oF_c-omit map for an iodide-soaked crystal (to 3.5 Å; contoured at 6 sigma) depicted by a green mesh. Difference density can be clearly seen at positions corresponding to those predicted to accommodate the bromide atoms of the substrate. (e) Putative structure of an organometallic substrate-cobalamin intermediate within the RdhA_NP active site. The substrate was positioned to minimize close contacts with active site residues. Severe clashes can be observed between the substrate and the corrin ring (highlighted by red lines).

ED Fig. 4. Additional EPR spectroscopic data.

Left panel Identification of contaminating EPR signals in aerobically purified RdhA_NP (150μM). (a) X-band continuous wave EPR spectrum showing a [3Fe-4S]¹⁺ cluster signal isolated by subtraction of the spectrum recorded at 20 K from that recorded at 12 K. (b) X-band continuous wave EPR spectrum showing cob(III)alamin-O₂^●— (superoxide) signal isolated by subtracting the spectrum recorded at 20 K from that recorded at 30 K followed by subtraction of a proportion of the [3Fe-4S]¹⁺ signal. Neither of these signals quantitates to more than 4% of the total EPR signal in the protein as isolated. Experimental parameters, microwave power 0.5 mW, field modulation frequency 100 KHz, field modulation amplitude 5 G, temperatures as given. Right panel. Binding of 3,5-dichloro-4-hydroxybenzoate to 150μM RdhA_NP leads to a spectrum exhibiting multiple A_┴^Co and A_||^Co splitting of the spectrum and no apparent chlorine superhyperfine coupling (A_||^Cl) (spectrum d). This suggests relatively disordered binding and possibly substrate movement within the active site on the nanosecond time scale even at the cryogenic temperatures employed in the EPR experiment. Such disorder and dynamics may explain the poor efficacy of this substrate relative to the dibromo- analogue (see main Fig. 1E) in addition to precluding analysis of the EPR spectrum.

ED Fig. 5. Structure of the RdhA_NP active site DTF model.

a) Chemical structures used for DFT calculations and b) overlay of the optimized active site models in the Co(II) (pink carbons) and Co(I) (green carbons; Br is shown as discrete sphere) oxidation states (right panel). The Co and the 3 atoms indicated with arrows (a) were fixed during optimisation. Selected parameters are given in Extended data Table 2 and Cartesian coordinates of the optimised structures are included in the Supplementary material. The Cbl-Br models comprise the ‘trimmed’ cobalamin with a single axial Br ligand and contain 91 atoms (40 heavy atoms). The full active site model contains 148 atoms (66 heavy atoms).

ED Fig. 6. Characterisation of RdhA_NP mutants.

a) UV-Vis spectra of RdhA_NP variants normalised using the A₂₈₀ absorbance. Mutants Y426F (107 μM), K488Q (100 μM) and R552L (95 μM) have a similar profile as the wild type enzyme (WT, 176 μM) purified from B. megaterium. RdhA_NP WT purified from E. coli (lacking the corrinoid cofactor; 250 μM) is shown for comparison. b-c-d) Continuous wave X-band EPR at 30K for RdhA_NP mutants. (b) R552L mutant (75 μM), (c) K488Q mutant (65 μM) and (d) Y426F mutant (65 μM). Each shows the presence of cob(II)alamin which is base on in R552L and base off in the other two mutants. Experimental parameters: microwave power 0.5 mW, modulation frequency 100 KHz, modulation amplitude 7G.

ED Fig. 7. Mechanistic proposal for biological reductive dehalogenation.

Our proposed RdhA_NP mechanism is fundamentally different from those for other B12-containing enzymes,, but also differs from the hydrolytic dehalogenases, which use an S_N2 mechanism whereby either an activated water molecule or a catalytic Asp residue attacks the substrate carbon. The latter contain a specific halogen binding site that is believed to contribute to stabilization of the transition state and to facilitate departure of the halide leaving group. In contrast, we propose the reductive dehalogenase uses the cobalamin cofactor to attack the substrate halogen atom itself, leading to C-halogen bond breakage concomitant with protonation of the leaving group. Distinct variations upon this theme could occur within the RdhA family: aliphatic organohalide reductases could operate via heterolytic C-halogen bond cleavage concomitant with halogen-Co(III) bond formation and substrate protonation (panel a). Those acting on (unactivated) aromatic organohalides are likely to operate via a radical substrate intermediate using homolytic C-halogen bond cleavage (panel b), while certain reductive dehalogenases have been shown to catalyse vicinal reduction or dihalo-elimination, and we propose formation of the Co-halogen bond occurs concomitant with leaving of the vicinal halogen atom (panel c).

Fig. 1. Characterisation of RdhA_NP as an ortho-dibromophenol reductase.

(a) Cartoon representation of the domain structure of the two reductive dehalogenase enzyme classes. (b) UV-Vis spectrum of 79 μM RdhA_NP as purified under aerobic conditions and following reduction using deazaflavin and EDTA. The Co(I) concentration estimated using ε=26 000 M^-1 cm^-1 suggest 0.8 Co per RdhA_NP. (c) Relative activity with organohalide substrates using methyl viologen as electron donor. Results are shown as mean ± s.e.m; n=2. Highest activities (above the dotted line) were obtained when substrates resembled the 2,6-dihalophenol structure shown as inset. (d) HPLC product profiles obtained when using spinach ferredoxin:NADP⁺ oxidoreductase and ferredoxin to couple NADPH oxidation to RdhA_NP 3,5-dibromo-4-hydroxybenzoic acid reductase activity. (e) Steady state kinetic profile obtained with 3,5-dibromo-4-hydroxybenzoic acid or 3,5-dichloro-4-hydroxybenzoic acid and reduced methyl viologen as substrates. Data points are shown as mean ± s.d; n=4.

Fig. 2. Crystal structure of RdhA_NP in the resting state.

(a) Overall structure of RdhA_NP color coded according to domain structure as in Fig 1a and presented in two orientations. (b) Detailed view of the cobalamin binding pocket. Key residues are shown with carbons colored as in panel A. Direct polar interactions between cobalamin and the enzyme are indicated by black dashed lines. Residues that are conserved in an alignment of functionally characterized RdhAs are underlined. (c) Solvent accessible surface of RdhA_NP color coded as in panel A, the orientation is similar to that of right hand panel 1a. (d) Detailed view of the 2x[4Fe-4S] cluster binding region, representation as in panel B. (e) Detailed view of the RdhANP active site with a docked 3,5-dibromo-4-hydroxybenzoic acid substrate. Representation as in panel B, a transparent surface indicates the hydrophobic cavity that serves to bind the non-cobalt ligating bromine.

Fig. 3. EPR spectroscopy of RdhA_NP reveals a direct halogen-cobalamin interaction.

X-band (~9.39 GHz) continuous wave (CW) of (a) 150 μM RdhA_NP in non-halide containing buffer with g values marked. (b) RdhA_NP plus 200 mM NaCl, with a ‘quartet’ arising from chloride (³⁵Cl) superhyperfine coupling indicated and g values marked (c) RdhA_NP plus 25 mM KBr with g values marked. (d) RdhA_NP plus 20 mM 3,5-dibromo-4-hydroxybenzoic acid with an example of the overlapped quartets arising from superhyperfine coupling to ⁷⁹Br and ⁸¹Br indicated and g values marked. Key: Cobalt hyperfine coupling is indicated in blue (showing the eight expected for an I = 7/2 ⁵⁹Co), halogen superhyperfine coupling (where present) is also indicated. For spectra C and D that exhibit overlapped A_┴^Co and A_┴^Br only A_||^Co is indicated. For spectrum parameters, see Extended data Table 3. * indicates a superposition of a [3Fe-4S]⁺ spectrum, the spectrum of a cob(III)alamin-superoxide species and an unassigned organic radical signal which together comprise less than 5% of the cob(II)alamin spectrum (see Extended data Fig. 4).

Fig. 4. RdhA_NP proposed mechanism.

Our data suggest that the RdhA_NP mechanism involves formation of a bromide-Cob(II)alamin complex either via heterolytic cleavage of the carbon-bromide substrate bond (in blue box) or homolytic cleavage of the carbon-bromide substrate bond (in green box). For clarity, only the 2,6-dibromophenol moiety of the substrate is shown.