The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C–H Bond Cleavage and Oxygen Atom Transfer Reactions

Abstract

AbbreviationsIAA, iodoacetamide, FDH, formate dehydrogenase, Moco, molybdenum cofactor, MGD, molybdopterin guanine dinucleotide, MV, methyl viologen, SeCys, selenocysteine, XAS, X-ray absorption spectroscopy 3 AbstractFormate dehydrogenases (FDHs) are capable to perform the reversible oxidation of formate and are enzymes of high interest for fuel cell applications and for the production of reduced carbon compounds as energy source from CO 2 . Metalcontaining FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids for catalysis has not been studied in detail, due to the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes.Here, the roles of these active-site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization and the histidine elevates the pK a of the active-site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable to reduce nitrate to nitrite. We propose a mechanistic scheme, which combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. 4A specific enzyme system of increasing interest is formate dehydrogenase (FDH), being involved in the reversible conversion of CO 2 in biological systems (1,2 Desulfovibrio gigas (FdhAB) have shown that the metal ion in the oxidized enzyme is coordinated by four sulfur ligands of two dithiolene groups of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor, a selenocycteine (SeCys), and by a sixth ligand, which is now established as a sulfido group (5-9) ( Fig. 1). After reduction with formate, the structure of E. coli FdhF showed that the SeCys ligand was displaced from the Mo ion (5). In the second coordination sphere, a highly conserved histidine and arginine are present in all FDH enzymes described so far. Experimental evidence in which these residues are replaced by other amino acids is lacking, due 5 to the high oxygen sensitivity of the E. coli and D. gigas enzymes and the lack of suitable overexpression systems.Closely related to FDHs are periplasmic nitrate reductases (e.g. Cupriavidus necator NapA), which similarly belong to the DMSO reductase family of molybdoenzymes.Periplasmic nitrate reductases comprise an identical Mo coordination sphere containing six sulfur ligands in the oxidized state (including a Cys) and show a remarkable structural homology to FDHs (10-12) (Fig. 1). A conserved threonine and methionine are found close to the Mo ion in periplasmic nitrate reductases ( Fi...

Creating a Low‐Potential Redox Polymer for Efficient Electroenzymatic CO2 Reduction

“…[28] To confirm the selectivity of formate production by the Mo-FDH/Cc-PAA bioelectrode,b ulk electrolysis was performed with an applied potential of À0.66 Vv s. SHE for 90 min, using 50 mm NaHCO 3 as the CO 2 source.W e employed as econdary enzymatic assay to quantify the amount of formate,w hereby NAD-dependent FDH (under aerobic conditions to deactivate Mo-FDH) was added after bulk electrolysis to reduce NAD + by the oxidation of formate that had been produced by Mo-FDH (NAD + and NADdependent FDH were only included after bulk electrolysis had been terminated;F igure S6). To provide additional evidence for the production of formate,N MR analysis was carried out following the bulk electrolysis of isotopically labeled NaH 13 CO 3 .F igure 3s hows 13 CNMR spectra of as odium formate reference (a), the electrolyte before bulk electrolysis (b), and the electrolyte containing [ 13 C]formate after 90 min of bulk electrolysis (c). No unassigned 13 Cpeaks were observed, indicating that formate was the only major product resulting from CO 2 reduction by the Mo-FDH/Cc-PA Ab ioelectrodes.F urthermore,t he total charge passed during bulk electrolysis was integrated to calculate the theoretical number of moles of formate produced, giving aF aradaic efficiencyof9 9AE 5% (Table 1a nd Figure S7).…”

“…To provide additional evidence for the production of formate,N MR analysis was carried out following the bulk electrolysis of isotopically labeled NaH 13 CO 3 .F igure 3s hows 13 CNMR spectra of as odium formate reference (a), the electrolyte before bulk electrolysis (b), and the electrolyte containing [ 13 C]formate after 90 min of bulk electrolysis (c). No unassigned 13 Cpeaks were observed, indicating that formate was the only major product resulting from CO 2 reduction by the Mo-FDH/Cc-PA Ab ioelectrodes.F urthermore,t he total charge passed during bulk electrolysis was integrated to calculate the theoretical number of moles of formate produced, giving aF aradaic efficiencyof9 9AE 5% (Table 1a nd Figure S7).…”

Strategies for Bioelectrochemical CO2 Reduction

“…[14] Formate dehydrogenase (FDH) is an enzyme capable of the interconversion of formate and CO 2 at the low redox potentialo fÀ0.42 Vv s. SHE. [15] Both metal-dependent and NADH-dependentF DHs have been identified;m etal-dependent FDHs typicallyu tilize Mo or Wt oc atalyzef ormate oxidation or CO 2 reduction, whileN ADH-dependent FDHs do not have metal ions or other permanent redoxc enters, instead using NADH directly. [10] The well-characterized NADH-dependentF DH from Candida boidinii is commerciallya vailable and has been used in many bioelectrosynthetic devices for CO 2 reduction.…”

Reversing Conventional Reactivity of Mixed Oxo/Alkyl Rare‐Earth Complexes: Non‐Redox Oxygen Atom Transfer

“…[1][2][3][4][5] Tr aditionally,m etal-mediated OATr eactions are predominantly carried out in two sequential steps,n amely,o xidative oxygenation of low-valent metal species followed by reductive deoxygenation of high-valent metal oxo species.A lternatively,t he metal-mediated OATr eactions can also be driven by the oxidation/reduction of ligands (Scheme 1A). [1][2][3][4][5] Tr aditionally,m etal-mediated OATr eactions are predominantly carried out in two sequential steps,n amely,o xidative oxygenation of low-valent metal species followed by reductive deoxygenation of high-valent metal oxo species.A lternatively,t he metal-mediated OATr eactions can also be driven by the oxidation/reduction of ligands (Scheme 1A).…”