Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal‐Dependent Formate Dehydrogenase

Abstract

Biological carbon dioxide (CO 2 ) reduction is an important step by which organisms form valuable energyricher molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO 2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO 2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a noncompetitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.

A Minimal Light‐Driven System to Study the Enzymatic CO2Reduction of Formate Dehydrogenase

“…In panel (A), the positive absorption at 2342 cm −1 reflects the formation of the released CO 2 from the sacrificial electron donor EDTA. Simultaneously, free azide (negative band at 2049 cm −1 , dashed red line) is presumably decomposed into N 2 upon irradiation, though this band might also partially reflect non‐competitive azide binding to Rc FDH WT observed previously [29,42] . The more relevant second negative absorption at 2031 cm −1 (orange dashed line) is assigned to a competitively inhibiting azide species at the bis‐MGD active site.…”

“…Simultaneously, free azide (negative band at 2049 cm À 1 , dashed red line) is presumably decomposed into N 2 upon irradiation, though this band might also partially reflect non-competitive azide binding to RcFDH WT observed previously. [29,42] The more relevant second negative absorption at 2031 cm À 1 (orange dashed line) is assigned to a competitively inhibiting azide species at the bis-MGD active site. (B) The peak integrals of both azide species were calculated from band fitting of the absorbance spectra before and after irradiation.…”

“…[39] Moreover, the specific frequency of this mode depends on the polarity of the environment of the azide anion. [29,40,41] The inhibitory nature of azide on CO 2 reduction was assessed by inhibition kinetics on RcFDH WT performed using methyl viologen as an electron acceptor. Interestingly, similar to formate oxidation, CO 2 reduction by RcFDH WT exhibited a mixed (competitive-non-competitive) inhibition profile for azide, with a K i of 58 � 3 μM and an αK i of 361 � 5 μM (Figure S8, Table S1).…”

“…RcFDH protein was heterologously expressed in E. coli as described elsewhere. [9,26,29] Purification includes Ni nitrilotriacetic acid-based (Ni-NTA) affinity chromatography and size exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 pg column, which was performed aerobically in the presence of 10 mM NaN 3 at 4 °C. Prior to and immediately after SEC, RcFDH was concentrated with ultracentrifugation devices (Vivaspin 20, 30 kDa MWCO; Sartorius AG, Göttingen, Germany).…”

“…[27] Moreover, recent studies revealed that azide, which is isoelectronic to CO 2 and charged as formate, inhibits the catalytic oxidation reaction. [28,29] Herein, one inhibitory azide anion binds between a highly conserved Arg587 and the inorganic sulfur ligand [30] of the bis-MGD cofactor, thereby shielding the active site. [26,29] Thus, by monitoring the specific IR absorption of this inhibitory azide molecule, we could further confirm the competitive inhibition mode under CO 2 reduction conditions.…”