Heterotrimeric NADH-Oxidizing Methylenetetrahydrofolate Reductase from the Acetogenic Bacterium Acetobacterium woodii

ABSTRACTThe methylenetetrahydrofolate reductase (MTHFR) of acetogenic bacteria catalyzes the reduction of methylene-THF, which is highly exergonic with NADH as the reductant. Therefore, the enzyme was suggested to be involved in energy conservation by reducing ferredoxin via electron bifurcation, followed by Na+translocation by the Rnf complex. The enzyme was purified fromAcetobacterium woodiiand shown to have an unprecedented subunit composition containing the three subunits RnfC2, MetF, and MetV. The stable complex contained 2 flavin mononucleotides (FMN), 23.5 ± 1.2 Fe and 24.5 ± 1.5 S, which fits well to the predicted six [4Fe4S] clusters in MetV and RnfC2. The enzyme catalyzed NADH:methylviologen and NADH:ferricyanide oxidoreductase activity but also methylene-tetrahydrofolate (THF) reduction with NADH as the reductant. The NADH:methylene-THF reductase activity was high (248 U/mg) and not stimulated by ferredoxin. Furthermore, reduction of ferredoxin, alone or in the presence of methylene-THF and NADH, was never observed. MetF or MetVF was not able to catalyze the methylene-THF-dependent oxidation of NADH, but MetVF could reduce methylene-THF using methyl viologen as the electron donor. The purified MTHFR complex did not catalyze the reverse reaction, the endergonic oxidation of methyl-THF with NAD+as the acceptor, and this reaction could not be driven by reduced ferredoxin. However, addition of protein fractions made the oxidation of methyl-THF to methylene-THF coupled to NAD+reduction possible. Our data demonstrate that the MTHFR ofA. woodiicatalyzes methylene-THF reduction according to the following reaction: NADH + methylene-THF → methyl-THF + NAD+. The differences in the subunit compositions of MTHFRs of bacteria are discussed in the light of their different functions.IMPORTANCEEnergy conservation in the acetogenic bacteriumAcetobacterium woodiiinvolves ferredoxin reduction followed by a chemiosmotic mechanism involving Na+-translocating ferredoxin oxidation and a Na+-dependent F1FoATP synthase. All redox enzymes of the pathway have been characterized except the methylenetetrahydrofolate reductase (MTHFR). Here we report the purification of the MTHFR ofA. woodii, which has an unprecedented heterotrimeric structure. The enzyme reduces methylene-THF with NADH. Ferredoxin did not stimulate the reaction; neither was it oxidized or reduced with NADH. Since the last enzyme with a potential role in energy metabolism ofA. woodiihas now been characterized, we can propose a quantitative bioenergetic scheme for acetogenesis from H2plus CO2in the model acetogenA. woodii.

Flavin Mononucleotide-Binding Flavoprotein Family in the Domain Archaea

ABSTRACT The protein (AfpA, for archaeoflavoprotein) encoded by AF1518 in the genome of Archaeoglobus fulgidus was produced in Escherichia coli and characterized. AfpA was found to be a homodimer with a native molecular mass of 43 kDa and containing two noncovalently bound flavin mononucleotides (FMNs). The cell extract of A. fulgidus catalyzed the CO-dependent reduction of AfpA that was stimulated by the addition of ferredoxin. Ferredoxin was found to be a direct electron donor to purified AfpA, whereas rubredoxin was unable to substitute. Neither NADH nor NADPH was an electron donor. Ferricyanide, 2,6-dichlorophenolindophenol, several quinones, ferric citrate, bovine cytochrome c, and O2 accepted electrons from reduced AfpA, whereas coenzyme F420 did not. The rate of cytochrome c reduction was enhanced in the presence of O2 suggesting that superoxide is a product of the interaction of reduced AfpA with O2. Although AF1518 was previously annotated as encoding a decarboxylase involved in coenzyme A biosynthesis, the results establish that AfpA is an electron carrier protein with ferredoxin as the physiological electron donor. The genomes of several diverse Archaea contained afpA homologs clustered with open reading frames annotated as homologs of genes encoding reductases involved in the oxidative stress response of anaerobes from the domain Bacteria. A potential role for AfpA in coupling electron flow from ferredoxin to the putative reductases is discussed. A search of the databases suggests that AfpA is the prototype of a previously unrecognized flavoprotein family unique to the domain Archaea for which the name archaeoflavoprotein is proposed.