Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha

Niks, Dimitri; Duvvuru, Jayant; Escalona, Miguel; Hille, Russ

doi:10.1074/jbc.m115.688457

Cited by 88 publications

(177 citation statements)

References 35 publications

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“…This chemical strategy -direct hydride transfer-was also recently suggested by Niks et al 32 The carbon dioxide reduction is suggested to follow the reverse reaction mechanism (Fig. 6.A, red arrows).…”

Section: Reaction Mechanismsupporting

confidence: 70%

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Reduction of Carbon Dioxide by a Molybdenum-Containing Formate Dehydrogenase: A Kinetic and Mechanistic Study

et al. 2016

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Carbon dioxide accumulation is a major concern for the ecosystems, but its abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels. However, the thermodynamic and kinetic stability of the carbon dioxide molecule makes its activation a challenging task. Studying the chemistry used by nature to functionalize carbon dioxide should be helpful for the development of new efficient (bio)catalysts for atmospheric carbon dioxide utilization. In this work, the ability of Desulfovibrio desulfuricans formate dehydrogenase (Dd FDH) to reduce carbon dioxide was kinetically and mechanistically characterized. The Dd FDH is suggested to be purified in an inactive form that has to be activated through a reduction-dependent mechanism. A kinetic model of a hysteretic enzyme is proposed to interpret and predict the progress curves of the Dd FDH-catalyzed reactions (initial lag phase and subsequent faster phase). Once activated, Dd FDH is able to efficiently catalyze, not only the formate oxidation (k cat of 543 s–1, K m of 57.1 μM), but also the carbon dioxide reduction (k cat of 46.6 s–1, K m of 15.7 μM), in an overall reaction that is thermodynamically and kinetically reversible. Noteworthy, both Dd FDH-catalyzed formate oxidation and carbon dioxide reduction are completely inactivated by cyanide. Current FDH reaction mechanistic proposals are discussed and a different mechanism is here suggested: formate oxidation and carbon dioxide reduction are proposed to proceed through hydride transfer and the sulfo group of the oxidized and reduced molybdenum center, Mo6+S and Mo4+-SH, are suggested to be the direct hydride acceptor and donor, respectively.

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Section: Reaction Mechanismsupporting

confidence: 70%

“…32 In this novel proposal, the focus is shifted from the selenocysteine and turned into the sulfo group of the active site, with the formate oxidation/carbon dioxide reduction occurring via hydride transfer (eq. 3, not eq.…”

Section: A New Look At the Reaction Mechanism Of Fdhmentioning

confidence: 99%

Reduction of Carbon Dioxide by a Molybdenum-Containing Formate Dehydrogenase: A Kinetic and Mechanistic Study

et al. 2016

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“…The results shown here are supported by previous results obtained in the crystal structure of E. coli FdhF, which showed that in the reduced state with formate the cysteine ligand was displaced from the active site (5). Further, the mechanism is consistent with the ones proposed by Cerqueira and coworkers by theoretical studies (14,(23)(24)(25), but is inconsistent with a recent report by Nicks et al (26), which proposed formate binding only in the second Mo coordination sphere by studies on…”

Section: Nitrate Reductase Activity Of Fdhsupporting

confidence: 50%

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

et al. 2016

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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...

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“…It is thus likely that the decrease in activity above pH 7.0 as described by pK a2 eff was principally due to ionic strength effects rather than ionization phenomena. The identity of the ionizable group whose protonation results in loss of activity at low pH is not known, although a likely candidate is the Mo(VI)ϭS group of the molybdenum center, which is known to be protonated at pH 7.0 upon reduction of the molybdenum (11).…”

Section: Ph Dependence Of the Reverse Reactionmentioning

confidence: 99%

“…C. necator (formerly known as R. eutropha) strain HF210 was grown as described previously (11). All protein purification steps were performed at 0 -4°C with an Ä KTA FPLC system (GE Healthcare) in a procedure modified from that of Niks et al (11).…”

Section: Protein Preparationmentioning

confidence: 99%

Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha)

Yu¹,

Niks²,

Mulchandani

et al. 2017

Journal of Biological Chemistry

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103

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The ability of the FdsABG formate dehydrogenase from (formerly known as) to catalyze the reverse of the physiological reaction, the reduction of CO to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction with a of 11 ± 0.4 s We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reactions are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO-utilizing enzyme to catalyze the reverse direction to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme and on the basis of the present work, we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalyzing the reversible interconversion of CO and formate under the appropriate experimental conditions.

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Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha

Cited by 88 publications

References 35 publications

Reduction of Carbon Dioxide by a Molybdenum-Containing Formate Dehydrogenase: A Kinetic and Mechanistic Study

Reduction of Carbon Dioxide by a Molybdenum-Containing Formate Dehydrogenase: A Kinetic and Mechanistic Study

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

Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha)

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