Crystallographic and kinetic analyses of the FdsBG subcomplex of the cytosolic formate dehydrogenase FdsABG from Cupriavidus necator .

Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 2985121R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1083-351X (Electronic) Linking ISSN: 00219258 NLM ISO Abbreviation: J Biol Chem Subsets: MEDLINE

Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology
Original Publication: Baltimore, MD : American Society for Biochemistry and Molecular Biology

Bacterial Proteins/*chemistry ; Cupriavidus necator/*enzymology ; Formate Dehydrogenases/*chemistry ; Iron-Sulfur Proteins/*chemistry ; Multienzyme Complexes/*chemistry; Catalytic Domain ; Crystallography, X-Ray ; Flavin Mononucleotide/chemistry ; Kinetics ; NAD/chemistry

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD ⁺ -dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe 4 S 4 in FdsB and Fe 2 S 2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH ^• , not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH ^- , initially formed via NADH-mediated reduction, to the Fe 2 S 2 cluster. This Fe 2 S 2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re -face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD ⁺ binding in proper NADH dehydrogenases.
(© 2020 Young et al.)

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Keywords: FdsABG; carbon assimilation; electron transfer; enzyme kinetics; enzyme structure; flavin mononucleotide (FMN); formate dehydrogenase; nicotinamide adenine dinucleotide (NADH); protein crystallization

PDB 6HLI; 6HL2; 6HL3; 5XFA; 5XF9; 2FUG

0 (Bacterial Proteins)
0 (Iron-Sulfur Proteins)
0 (Multienzyme Complexes)
0U46U6E8UK (NAD)
7N464URE7E (Flavin Mononucleotide)
EC 1.17.1.9 (Formate Dehydrogenases)

Date Created: 20200407 Date Completed: 20201223 Latest Revision: 20230106

20231215

PMC7212643

10.1074/jbc.RA120.013264

32249211