Substrate-dependent oxidative inactivation of a W-dependent formate dehydrogenase involving selenocysteine displacement

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards innovative strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be better understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the oxygen sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. This work presents a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a different species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyse the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate.


Supplementary Discussion: DFT calculations of the potential influence of SeCys displacement on the W(V) cofactor g-values
As the g-values of W(V) species can range in a very broad domain and to help detection of potentially new W(V) EPR signals, a quantum chemistry study was performed on several W(V) cofactor models to evaluate the potential influence of SeCys displacement and the binding of oxygenated species on the W(V) g-tensor.Eight structural models (numbered 1 to 8) (Figure S5 and S6) were built based on the structure of the formate and oxygen-treated DvFdhAB (Reox_120min).These models have in common the coordination of W(V) ion by the four sulfur atoms of the two pterins, a η 2 (O-O) or water ligand and a displaced SeCys, while they differ in the nature of the exogenous ligand (sulfur or oxygen), the protonation state of the cofactor and possible H-bonds between a η 2 (O-O) or water ligand and sulfur and/or selenium atoms (Figure S5 and S6).Optimized geometry consistent with protein surrounding constraints could be obtained only for structural models 1-3 and 7.The g-values of W(V) species were calculated for these models.This gives values ranging from about g = 2.1 to 2.6 (Table S5) for models 1-3, much higher than the values observed experimentally for the W(V)F and W(V)D species and calculated for models with bound SeCys ligand. 1 Moreover, the g-values calculated for model 7 are much lower than g=2.00 and does not fit with those of W(V)F and W(V)D species.Thus, in complement to models investigated in previous work, 1 these DFT calculations provide additional support to show that the experimentally detected W(V)F and W(V)D species associated to active DvFdh does not correspond to species with non-coordinated SeCys.DvFdhAB co-crystallized with 10 mM of sodium formate; plate well opened and crystals exposed to atmospheric O 2 , for 12 min, while still in the original drop (which contains 10 mM of sodium formate), and then flash cooled in liquid nitrogen (asisolated/Oxidized).Reox_120min PDB_ID: 8RC9 DvFdhAB co-crystallized with 10 mM of sodium formate; plate well opened and the crystals were exposed to atmospheric O 2 , for 120 min, while still in the original drop (which contains 10 mM of sodium formate), and then flash cooled in liquid nitrogen (W-O=O…SeCys form).Reox_ND_NoFormate PDB_ID: 8RCA DvFdhAB co-crystallized with 10 mM of sodium formate; the crystals were transferred from the original drop to a New Drop (oxygenated), mimicking the mother liquor but without sodium formate, and were exposed to atmospheric O 2 , for 60 min, and then flash cooled in liquid nitrogen (as-isolated/Oxidized).Reox_ND_Formate PDB_ID: 8RCB DvFdhAB co-crystallized with 10 mM of sodium formate; the crystals were transferred from the original drop to a New Drop (oxygenated), mimicking the mother liquor containing 10 mM of sodium formate, and were exposed to atmospheric O 2 , for 34 min, and then flash cooled in liquid nitrogen (W-O=O…SeCys form).HP_CO 2 PDB_ID: 8RCC DvFdhAB aerobically crystallized without sodium formate; the crystals were pressurised with 48 bar of CO 2 , and then flash cooled in liquid nitrogen, under 200 bar helium (W-O=O…SeCys form).
Table S 2-Distances between the W ion, Se atom of SeCys and the (di)oxygen ligand atom(s) for the Control_Red , Reox_120min, Reox_ND_NoFormate, Reox_ND_Formate and HP_CO 2 structures (with respective crystallographic resolutions) and of the optimized W V structural models (Figure S12).

Bond length and distance (in Å)
W             (a) The relative intensity of the 1H NMR formate peak (calculated as the intensity ratio between the formate peak and that of DSS) is plotted as a function of time for the activity assays with formate concentrations of 1 mM (red circle), 10 mM (green circle), 50 mM (yellow circle); and 10 mM in the absence of atmospheric oxygen (grey triangle), the reference (which is calculated in the same way as before, but using the samples prepared in the absence of enzyme) is shown (black diamond).(b -e) Representative 1D 1H spectra of each condition tested (1, 10 and 50 mM formate and 10 mM formate in the absence of atmospheric oxygen, respectively) (black: reference 1H spectrum; magenta: 1st 1H spectrum; blue: last 1H spectrum).Above each spectra the peaks of FMT (formate), Tris (buffer) and DSS are indicated.The spectra corresponding to the 1st and last acquisition points are shifted to the right for better analysis.The C872A variant corresponds to the active form of FdhAB, equivalent to the form obtained when pretreating the enzyme with DTT, as reported in 5 .With PMS + DCPIP as artificial electron acceptors in anaerobic conditions (dark red, glove box) and in aerobic conditions (green).Data are presented as mean values ± s.d.(n = at least 3 assay technical replicates).No DTT was used in the assays.The higher error observed for the aerobic assay of the C872A variant is due to the decreasing activity of the enzyme in these conditions.Anaerobic reduction with formate (black trace; spin intensity reference, 100%) followed by oxygen treatment (red trace; spin intensity, 2%), then degassing and anaerobic reduction with dithionite (green trace; spin intensity, 105%) or formate (blue trace; spin intensity, 55%), and subsequent reduction of formate treated sample with dithionite (cyan trace; spin intensity, 87%).EPR conditions: Temperature, 15 K; microwave power 1 mW at 9.479 GHz, modulation amplitude 1 mT at 100 kHz.

Figure S 3 -
Figure S 3-Solved, unrefined, structure obtained from the DvFdhAB WT crystals co-crystalized with formate and exposed to air for 20 min.DvFdhAB 20 min structure displaying a mixture of states (50%/50%) of both WT as isolated (oxidized) (magenta) (PDB_ID: 6SDR) and Reox_120min (yellow), near the active site (I191-S194).2Fo-Fc maps at 1 σ (blue mesh) and Fo-Fc maps at 3 σ (green and red mesh, respectively for positive and negative densities) are shown.Image produced with Coot 4 .

Figure S 4 -
Figure S 4-Alternative modelling of different ligands for the electron density between W and Se in the DvFdhAB Reox_120min structures.The W ion (grey cross), SeCys Se atom (grey sphere), the two MGD co-factors (yellow lines), sulfido ligand (yellow cross) and water molecules (red sphere) are shown.2Fo-Fc electron density map contoured at 1σ (blue mesh) and Fo-Fc map contoured at 3σ (green and red mesh, respectively for positive and negative density) are shown.Images produced with Coot 4 .(a) Modelling hypothesis with one water molecule at full occupancy.(b) Modelling hypothesis with two water molecules, each at half occupancy.(c) Modelling hypothesis with two sulfido ligands, each at half occupancy.

Figure S 5 -
Figure S 5-Structural models of the W V cofactor used for DFT calculations.Interatomic distances were indicated, in Å, only for models leading to optimized geometry consistent with protein surrounding constraints.

Figure S 6 -
Figure S 6-Optimized geometry of the W V models obtained by DFT calculations.The W ion (cyan), Selenium atom (orange), Sulfur (yellow), carbon (dark grey), oxygen (red), nitrogen (blue) and hydrogen (white) atoms are shown as spheres.

Figure S 9 -
Figure S 9-Control HP experiment with anaerobic DvFdhAB crystals under CO 2 pressure.Anaerobic, under CO 2 pressure, DvFdhAB structure.The W ion (grey cross), SeCys Se atom (grey line), the two MGD co-factors (yellow lines) and sulfido ligand (yellow cross) are shown.2Fo-Fc electron density map contoured at 1σ (blue mesh), Fo-Fc map contoured at 3σ (green and red mesh, respectively for positive and negative density) and anomalous electron density map at 3 σ (violet mesh) are shown.Image produced with Coot 4 .

Figure S 10 -
Figure S 10-Relative activity for CO 2 reduction (red) and formate oxidation (blue) of as-isolated DvFdhAB incubated in anaerobic conditions in the presence of CO 2 .T=0h was considered as 100% of activity.Data are presented as mean values ± s.d.(n = 3 assay technical replicates).

Figure S 11 -
Figure S 11-Relative activity for CO 2 reduction of as-isolated DvFdhAB incubated in aerobic conditions in the presence of CO 2 .T=0h was considered as 100% of activity.Data are presented as mean values ± s.d.(n = 3 assay technical replicates).

Figure S 12 -
Figure S 12-DvFdhAB activity assays by 1H NMR.(a)The relative intensity of the 1H NMR formate peak (calculated as the intensity ratio between the formate peak and that of DSS) is plotted as a function of time for the activity assays with formate concentrations of 1 mM (red circle), 10 mM (green circle), 50 mM (yellow circle); and 10 mM in the absence of atmospheric oxygen (grey triangle), the reference (which is calculated in the same way as before, but using the samples prepared in the absence of enzyme) is shown (black diamond).(b -e) Representative 1D 1H spectra of each condition tested (1, 10 and 50 mM formate and 10 mM formate in the absence of atmospheric oxygen, respectively) (black: reference 1H spectrum; magenta: 1st 1H spectrum; blue: last 1H spectrum).Above each spectra the peaks of FMT (formate), Tris (buffer) and DSS are indicated.The spectra corresponding to the 1st and last acquisition points are shifted to the right for better analysis.

Figure S 13 -Figure S 14 -
Figure S 13-Solved, unrefined, structure (yellow) obtained from the DvFdhAB WT crystals produced with the concentrated solution left from the NMR experiments with formate under air, at a resolution of 2.83 Å. 2Fo-Fc maps at 1 σ (blue mesh), Fo-Fc maps at 3 σ (green and red mesh, respectively for positive and negative densities) and anomalous map peaks at 3 σ (violet mesh) are shown.Images produced with Coot 4 .(a) and (c) DvFdhAB WT "after-NMR" structure, solved using Reox_120min as molecular replacement model (yellow), superposed with the Reox_120min structure (green).(b) and (d) DvFdhAB WT "after-NMR" structure, solved using DvFdhAB WT as-isolated (oxidized) (PDB_ID: 6SDR) as molecular replacement model (yellow).

Table S 1-Description of the procedure used to obtain the different structures of DvFdhAB in this and other works.
DvFdhAB crystallized as-isolated in the presence of O 2 (As-isolated).DvFdhAB co-crystallized with 10 mM of sodium formate and not exposed to O 2 (Reduced).DvFdhAB co-crystallized with 10 mM of sodium formate and not exposed to O 2 (Reduced).