Heterotrimeric NADH-oxidizing methylenetetrahydrofolate reductase from the acetogenic bacterium Acetobacterium woodii

Abstract

The 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 from Acetobacterium woodii and 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 of A. woodii catalyzes 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.

Importance: Energy conservation in the acetogenic bacterium Acetobacterium woodii involves ferredoxin reduction followed by a chemiosmotic mechanism involving Na(+)-translocating ferredoxin oxidation and a Na(+)-dependent F1Fo ATP synthase. All redox enzymes of the pathway have been characterized except the methylenetetrahydrofolate reductase (MTHFR). Here we report the purification of the MTHFR of A. 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 of A. woodii has now been characterized, we can propose a quantitative bioenergetic scheme for acetogenesis from H2 plus CO2 in the model acetogen A. woodii.

FIG 1

Purification of the methylene-THF reductase of A. woodii. Samples from the different purification steps were separated by SDS-PAGE, and proteins were stained with Coomassie Brilliant Blue. Lane 1, cell extract; lane 2, cytoplasm; lane 3, pooled fractions from Q-Sepharose; lane 4, pooled fractions from phenyl-Sepharose; lane 5, pooled fractions from Sephacryl S300; lane 6, pooled fractions from blue-Sepharose. A 10-μg volume of protein was applied to each lane.

FIG 2

MetF, MetV, and RnfC2 build a stable methylene-THF reductase complex that catalyzes NADH:3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) oxidoreductase activity. (A) The purified MTHFR (10 μg) was separated by native PAGE and stained with Coomassie Brilliant Blue. (B) NADH:MTT oxidoreductase activity was determined by incubation of the gel in 10 mM KH₂PO₄ (pH 7.5) with MTT tetrazolium dye and NADH.

FIG 3

UV light/visible light spectrum of the purified methylene-THF reductase. The spectrum of the enzyme (0.6 mg/ml) was recorded in 50 mM potassium phosphate buffer (pH 7) containing 2 mM DTE.

FIG 4

NADH dependence of methylene-THF reductase activity. The methylene-THF-dependent oxidation of NADH was measured in 50 mM potassium phosphate buffer (pH 7) containing 2 mM DTE and 0.25 mM methylene-THF with 10 μM FMN and various amounts of NADH. The activity followed Michaelis-Menten kinetics with a V_max (maximal enzyme velocity) of 212 U/mg and a K_m (Michaelis constant) of 19 μM. Curve fitting and determination of the kinetic parameters K_m and V_max were performed using the GraphPad Prism program (version 4.03) and the Michaelis-Menten equation [Y = (V_max × X)/(K_m + X)].

FIG 5

A second protein is required for MTHFR-catalyzed oxidation of methyl-THF. The methyl-THF-dependent reduction of NAD⁺ was measured in 50 mM potassium phosphate buffer (pH 7) containing 2 mM DTE, 1 mM NAD⁺, 10 μM FMN, and 0.2 mM methyl-THF in the presence of the purified MTHFR (13 μg) (A), the sample containing the enriched MTHF-DH (0.75 μg) (B), or the MTHFR and the enriched MTHF-DH (C). CH₃-THF, methyl-THF.

FIG 6

Genetic organization of methylene-THF reductase complexes. Hdr, heterodisulfide reductase; Mvh, methyl viologen reducing hydrogenase; anabolic/catabolic, physiological roles of the MTHFR in the corresponding organism; CH₃-R(+), the organisms are reported to grow on methylated substrates; CH₃-R(-), the organisms are not reported to grow on methylated substrates.

FIG 7

Model of the methylene-THF reductase complex of A. woodii. Hexagons represent flavins; cubes represent iron-sulfur clusters.

FIG 8

Bioenergetics of acetogenesis from H₂ plus CO₂ in A. woodii. For explanations, see the text. HDCR, H₂-dependent CO₂ reductase.