DISCLOSING THE ENZYMATIC REACTION MECHANISM FOR THE REDUCTION OF CARBON DIOXIDE

Only a couple of enzymes in nature are able to metabolise carbon dioxide. Here, structural data together with biochemical analysis unveil new features of the catalytic mechanism of the tungsten-dependent formate dehydrogenase, which is able to reduce carbon dioxide to formate.

STRUCTURAL BIOLOGY

52 ESRF

The chemical transformation of carbon dioxide (CO2) into valuable products is increasingly important as atmospheric levels continue to rise because of human activity. However, chemical conversion requires high energy inputs, has low selectivity, low catalytic efficiencies, and often needs rare-metal catalysts. Mo- and W-formate dehydrogenases (Fdhs) are unique prokaryote enzymes that catalyse the reversible reduction of CO2 to formate [1], with potential applications in green gas sequestration and fuel production [2]. However, the catalytic mechanism of these enzymes is still unclear, thus, there is an urgent need for detailed structural information on Fdhs in different catalytic/redox states.

This work reports an expression system to produce recombinant W/Sec-FdhAB from Desulfovibrio vulgaris Hildenborough in the holoform. This enzyme is responsible for CO2 reduction in D. vulgaris [3] and is the main Fdh expressed in this organism if tungsten is available [4], revealing its physiological preference over Mo-containing isoenzymes. The crystal structures of FdhAB in oxidised and

reduced forms were determined from X-ray data collected at beamlines ID29 and ID30B at up to 1.9 Å resolution. FdhAB is a soluble heterodimer comprising a [W(MGD)2, SH, Sec] cofactor and four [4Fe4S] clusters responsible for electron transfer to and from the active site (Figure 38). The active site of the oxidised form presents the W hexacoordinated by four sulfur ligands from 2 MGD cofactors, a SeCys and a sulfido group. In the second coordination sphere, highly conserved His193 and Arg441 residues are also proposed to play a role in catalysis (Figure 39a). His193 establishes a π interaction with the Se from Sec192, and Arg441 hydrogen-bonds a glycerol molecule. The W redox state IV in formate- reduced crystals was confirmed by electron paramagnetic resonance (EPR) spectroscopy on a sample prepared with crystals spread in cryoprotectant solution.

The comparison of the oxidised and reduced crystal structures allows the identification of conformational changes that disclose new features of the reaction mechanism. In the formate-reduced form, the Sec192 loop and part

Fig. 38: Architecture of D. vulgaris FdhAB.

a) Overall structure of the heterodimer

(α-subunit, cyan, β-subunit, blue

marine). b) The metal cofactors, tungsten ion

in blue, sulfide group in yellow, selenium

from selenocysteine in orange, two MGD

(molybdopterin-guanine dinucleotide) and four

[4Fe-4S] centres.