Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

Abstract

Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO 2 reduction to formate. The tungstencontaining FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe−4S] 1+ clusters, and full reduction requires Ti(III)−citrate. The redox potentials of the four [4Fe−4S] 1+ centers range between −250 and −530 mV. Two distinct W V signals were detected, W D V and W F V, which differ in only the g 2 -value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of W VI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionitereduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formatereduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate W V state of FdhAB.

The Mechanism of Metal-Containing Formate Dehydrogenases Revisited: The Formation of Bicarbonate as Product Intermediate Provides Evidence for an Oxygen Atom Transfer Mechanism

“…Often, EPR studies are considered as an argument for the oxidation state-dependent active site structure of the enzyme. However, the Mo V active site constitutes an intermediate state after product release and one electron oxidation in which the amino acid ligand is quite likely to rebind again to the molybdenum atom which otherwise would be coordinatively unsaturated [ 18 , 49 ]. In previous studies, several groups proposed a hydride transfer mechanism with the formate being bound within the second coordination sphere of the active site metal [ 15 , 50 ].…”

Electrochemical Kinetics Support a Second Coordination Sphere Mechanism in Metal‐Based Formate Dehydrogenase

“…A variation on this mechanism, proposed by Mota and co‐workers using DFT computations, [9] suggests movement of the proteic ligand from the first to the second coordination sphere, via the formation of a S−Se or S−S bond, before complete decoordination in the course of catalysis. However, these mechanisms are not supported by the recent structural data obtained by some of us, which show that the SeCys ligand remains coordinated in all the redox states of the Desulfovibrio vulgaris Hildenborough FdhAB, [4, 10] making it unlikely that decoordination is a requirement for catalysis. Another mechanism avoiding a 7‐coordinated Mo/W was proposed by Niks and co‐workers, who observed the formation of a 6‐S Mo V species coupled to a proton originating from formate upon exposure of FdsABG from Cupriavidus necator to formate.…”

“…Note that the data of Figure 3B are consistent with the results of a recent EPR titration of Dv FdhAB. [10] The data we obtained by modeling the dependence of the catalytic wave shape on formate concentration and pH strongly constrain the possible mechanisms of catalytic formate oxidation. We observed that the two-electron oxidation of the active site is coupled to one deprotonation (as expected from the stoichiometry of the overall catalytic reaction), which occurs in the (IV) to (V) step independently of pH in the case of Bs ForCE1, and in one redox step or the other depending on pH in the case of Dv FdhAB.…”

Electrochemical Kinetics Support a Second Coordination Sphere Mechanism in Metal‐Based Formate Dehydrogenase

“… [8] A variation on this mechanism, proposed by Mota and co‐workers using DFT computations, [9] suggests movement of the proteic ligand from the first to the second coordination sphere, via the formation of a S−Se or S−S bond, before complete decoordination in the course of catalysis. However, these mechanisms are not supported by the recent structural data obtained by some of us, which show that the SeCys ligand remains coordinated in all the redox states of the Desulfovibrio vulgaris Hildenborough FdhAB,[ 4 , 10 ] making it unlikely that decoordination is a requirement for catalysis. Another mechanism avoiding a 7‐coordinated Mo/W was proposed by Niks and co‐workers, who observed the formation of a 6‐S Mo V species coupled to a proton originating from formate upon exposure of FdsABG from Cupriavidus necator to formate.…”

“…Note that the data of Figure 3B are consistent with the results of a recent EPR titration of Dv FdhAB. [10] …”