Oxidation-State-Dependent Binding Properties of the Active site in a Mo-Containing Formate Dehydrogenase
W.E. Robinson, A. Bassegoda, E. Reisner, J. Hirst
Journal of the American Chemical Society
2017
vol. 139
p. 9927- 9936

DOI: 10.1021/jacs.7b03958

Description of open data files
Abstract (directly reproduced from the above-named paper and authors):
'Molybdenum-containing formate dehydrogenase H from Escherichia coli (EcFDH-H) is a powerful model system for studies of the reversible reduction of CO2 to formate. However, the mechanism of FDH catalysis is currently under debate, and whether the primary Mo coordination sphere remains saturated or one of the ligands dissociates to allow direct substrate binding during turnover is disputed. Herein, we describe how oxidation-state-dependent changes at the active site alter its inhibitor binding properties. Using protein film electrochemistry, we show that formate oxidation by EcFDH-H is inhibited strongly and competitively by N3–, OCN–, SCN–, NO2–, and NO3–, whereas CO2 reduction is inhibited only weakly and not competitively. During catalysis, the Mo center cycles between the formal Mo(VI)═S and Mo(IV)—SH states, and by modeling chronoamperometry data recorded at different potentials and substrate and inhibitor concentrations, we demonstrate that both formate oxidation and CO2 reduction are inhibited by selective inhibitor binding to the Mo(VI)═S state. The strong dependence of inhibitor-binding affinity on both Mo oxidation state and inhibitor electron-donor strength indicates that inhibitors (and substrates) bind directly to the Mo center. We propose that inhibitors bind to the Mo following dissociation of a selenocysteine ligand to create a vacant coordination site for catalysis and close by considering the implications of our data for the mechanisms of formate oxidation and CO2 reduction.'

Figure 2:
(Panel A) Chronoamperometry trace recorded at −0.1 V vs SHE in 5 mM aqueous sodium formate solution (pH 7, 23.5 °C, electrode rotation rate 2000 rpm). Aliquots of a 15 mM solution of sodium azide (also containing 5 mM formate) were added to adjust the azide concentration. pH and substrate concentration were constant throughout, and the data were corrected for film loss.
(Panel B) Dependence of the normalized current (current observed at the given inhibitor concentration divided by the current observed in the absence of inhibitor) on azide concentration, derived from the data in panel A. The data were fit using the standard dose-effect relationship with a Hill coefficient of 1.

Figure 3:
Dependence of inhibitor IC50 values of substrate concentration for formate oxidation and CO2 reduction. Nitrite was reduced by the electrode by the electrode and thus was ommitted from the CO2 reduction graph. Conditions: 23.5 °C, pH 7, −0.1 V vs SHE (formate), −0.6 V vs SHE (CO2).

Figure 4:
Global fits to data on the inhibition of formate oxidation and CO2 reduction by azide, using equations 1 and 2 from the main text with common parameters.
(Panel A): Dependence of the normalised formate oxidation rate on the azide concentration for three formate concentrations and −0.1 V vs SHE.
(Panel B): Dependence of the normalised CO2 reduction rate on the azide concentration for three CO2 concentrations at −0.6 V vs SHE.
(Panel C): Dependence of the normalised formate oxidation rate on the azide concentration at two potentials in 10 mM formate.
(Panel D): Dependence of the normalised CO2 reduction rate on the azide concentration for five potentials in 8.31 mM CO2
Best fit lines were calculated using Km(CO2) = 2.5 mM, Km(formate) = 0.8 mM, K6 = 2 uM, K5 = 1 M, K4  = 42 mM, E1 = −0.365 V vs SHE, E2 = −0.656 V vs SHE, kcat(CO2)/k0 = 5.13, kcat(formate)/k0 = 0.5.
Conditions: pH 7, 23.5 °C.

Table 1:
Best fit parameters obtained using equations 1 and 2 with Km(formate) = 0.8 mM, Km(CO2) = 2.5 mM, E1 = −0.365 V vs SHE, E2 = −0.656 V vs SHE, kcat(formate)/k0 = 0.5 and kcat(CO2) = 5.13 and were taken from figure S4.

Figure 5:
(Panel A): Dependence of Km(formate) on pH. A line was calculated as an eye guide.
(Panel B): Dependence of K6(azide) on pH. The date were fit using a square scheme with K6(high pH) = 2.82 uM, pKa(1) = 6.3 (inhibitor free state) pKa(2) = 7.3 (for the inhibitor-bound state), K6(low pH) = 0.36 uM.

Figure 6:
Dependence of K6 on ligand electrochemical [A. B. P. Lever, Inorg. Chem. 1990, 29, 1271−1285] and resonance [C. Hansch, A. Leo, R. W. Taft, Chem. Rev. 1991, 91, 165- 195] parameters.

Figure 7:
Reductive activation of electrocatalysis by EcFDH-H. Conditions: pH 7.2, 10 mM CO2, 10 mM formate, 25 mM each MES, TAPS, HEPES, potassium acetate. 23.5 °C, 2000 rpm, scan rate 25 mV/s.

Figure S1:
(Panel A): Chronoamperometry traces showing data used to determine the Km for formate oxidation.
(Panel B): Dependence of the current on formate concentration, fit using the Michaelis Menten equation. with Imax = 243 uA/cm2, and Km(formate) = 0.8 mM.
(Panel C): Chronoamperometry traces showing data used to determine the Km for CO2 reduction (data given as current magnitude).
(Panel D): Dependence of the current magnitude on CO2 concentration, fit using the Michaelis Menten equation with Imax = 74.4 uA/cm2, Km(CO2) = 2.5 mM.
Conditions: pH 7, 25 mM each MES, TAPS, HEPES, potassium acetate, 23.5 °C, 2000 rpm, −0.1 V vs SHE (formate oxidation), −0.6 V vs SHE (CO2 reduction).

Figure S2:
Least squares error values describing the agreement between the data and the fit as a function of E1, E2, kcat(formate)/k0, kcat(CO2)/k0, K6, K5 and K4 for azide inhibition of formate oxidation and CO2 reduction. Values for each parameter tested were set and the LSQE vale was minimised by allowing all other parameters to form a best fit.
Km(formate) and Km(CO2) were fixed at 0.8 M and 2.5 mM, respectively. The fits were performed on the data in Figure 4A-D of the main text.

Figure S3:
Plots of normalised formate oxidation rate vs A: cyanate, C: thiocyanate, E: nitrate, G: nitrite concentration at three formate concentrations each.
PLots of normalised CO2 reduction rate vs B: cyanate, D: thiocyanate, F: nitrate concentration at three CO2 concentrations.
Fits were calculated according to equations 1 and 2 of the main text and parameters given in table 1.
Conditions: pH 7, 25 mM each MES, TAPS, HEPES, potassium acetate, 23.5 °C, 2000 rpm, −0.1 V vs SHE (formate oxidation), −0.6 V vs SHE (CO2 reduction).

Figure S4:
LSQE values between the data and the fit as a function of K6, K5 and K4 for A: cyanate, B: thiocyanate, C: nitrite and D: nitrate inhibition of formate oxidation and CO2 reduction. Values for each parameter were set and the LSQE values minimised by allowing all other parameters to form a best fit. Km(formate) and Km(CO2) were fixed to 0.8 mM and 2.5 mM, respectively and the best fit values for E1 = −0.365 V vs SHE, E2 = −0.656 V vs SHE, kcat(formate)/k0 = 0, kcat(CO2) = 5.13 from figure 4 were also fixed.