A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD: A TOOL FOR REDOX REGULATION IN AN ANAEROBE

Abstract

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F₄₂₀ (F₄₂₀H₂), a stronger reductant with a mid-point redox potential (E'₀) of -360 mV; E'₀ of NAD(P)H is -320 mV. Because F₄₂₀ is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F₄₂₀H₂ to thioredoxin via protein-bound flavin; K_m values for thioredoxin and F₄₂₀H₂ were 6.3 and 28.6 μm, respectively. The E'₀ of DFTR-bound flavin was approximately -389 mV, making electron transfer from NAD(P)H or F₄₂₀H₂ to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F₄₂₀/F₄₂₀H₂ pair could be as low as -425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F₄₂₀-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F₄₂₀ systems involved in microbial pathogenesis and antibiotic production.

Keywords: Methanocaldococcus jannaschii; archaea; coenzyme F420; deazaflavin; electron transfer; evolution; flavin-gated; methanogen; redox regulation; thioredoxin reductase.

FIGURE 1.

Structural and spectroscopic characteristics of recombinant Mj-DFTR. A, UV-visible spectrum of purified recombinant Mj-DFTR. A solution of the protein (10 μg) in 1 ml of solution containing 100 mm potassium phosphate buffer, pH 7, was analyzed in a cuvette with 1-cm light path. Prior to analysis, the protein was reconstituted with FAD to provide full incorporation of the coenzyme (A₂₈₀/A_{460 nm} = 4). B, SDS-PAGE profile. A 12% polyacrylamide gel was used. M, unstained protein ladder, broad Range (New England Biolabs, Ipswich, MA), with molecular masses of 250, 150, 100, 80, 60, 50, 40, 30, 25, 20, 15, and 10 kDa; selected bands have been marked to the left of the image. Two micrograms of Mj-DFTR in a solution containing 100 mm potassium phosphate buffer, pH 6.8, was analyzed.

FIGURE 2.

UV-visible spectra of Mj-DFTR incubated with various reductants. Oxidized Mj-DFTR (30 μm) was incubated anaerobically in a solution containing 100 mm potassium phosphate buffer, pH 7, with one of the following compounds at the indicated final concentrations: F₄₂₀H₂, 0.1 mm; dithionite, 1 mm; NADPH, 0.1 mm. Oxidized Mj-DFTR-bound flavin showed typical flavin absorption peaks at 380 and 460 nm (32). F₄₂₀ showed absorbance maxima at 420 nm (32).

FIGURE 3.

Electron flow in the deazaflavin-dependent thioredoxin reductase system of M. jannaschii. F₄₂₀H₂-generating systems: F₄₂₀-reducing hydrogenase (Frh) (93); Hmd-Mtd cycle, H₂-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd) + F₄₂₀H₂-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) (94). Inset, structures of coenzyme F₄₂₀ and F₄₂₀H₂. *, the Trx targets and Trx controlled systems were identified in proteomics study (20).

FIGURE 4.

Effects of temperature and pH on the activity of Mj-DFTR. Concentrations of Mj-Trx1 and F₄₂₀H₂ in the activity assay were 10 and 50 μm, respectively. A, temperature study. Activities at 10 different temperatures ranging from 25 to 90 °C were measured. Inset, re-plot of the temperature data according to the Arrhenius equation, k = A e^−Ea/(RT), where k, A, E_a, R, and T are rate constant, frequency factor, energy of activation (kJ mol⁻¹), universal gas constant (8.314 J K⁻¹ mol −¹), and temperature (K), respectively. The value of the energy of activation was calculated from the slope (− E_a/R) of the linear segment of the curve (50–90 °C). B, pH Study. Assays were conducted at 11 different pH values ranging from 4 to 9 with 0.5-unit increment in buffers with constant ionic strength (32).

FIGURE 5.

Kinetic analysis of Mj-DFTR reaction. Each panel provides specific activities of the enzyme at various concentrations of a substrate as indicated: A, Mj-Trx1; B, Mj-Trx2; C, F₄₂₀H₂; D, DTNB. Assay at each concentration of the varied substrate was performed in triplicate. Each solid curve represents the best fit of the respective data to the Henri-Michaelis-Menten's hyperbola v = V_max[S]/K_m/[S]. ±, standard deviation.

FIGURE 6.

Amino acid sequence alignment of Mj-DFTR and other low molecular weight flavin-containing TrxRs. The alignment was performed by the use of PROMALS3D (92) with the three-dimensional structures listed below as guides. % identities and % similarities with Mj-DFTR: Ta-TrxR, 36, 50; Tm-NTR, 37, 53; Ec-NTR, 33, 50; Cp-TrxR, 27, 43. Abbreviations (organism, enzyme, NCBI accession number, PDB code) are as follows: Mj-DFTR (M. jannaschii, DFTR, Q58931); Ta-TrxR (T. acidophilum, non-NAD(P)H-dependent TrxR, WP_010901395, 3CTY); Tm-NTR (T. maritima, NADH-NADPH dual specificity NTR, AAD35951.1); Ec-NTR (E. coli, NTR, WP_001460710, 1CL0); Cp-TrxR (C. pasteurianum, ferredoxin-TrxR, WP_015617437.1). Conserved amino acids are in bold. Consensus predicted secondary structures: red h, α-helix; blue e, β-strand. Gray shading, the redox active CXXC motif. Black bar, conserved motifs as found in low molecular weight NTR and flavoproteins and are labeled as such. DBM, dinucleotide-binding motif.

FIGURE 7.

Redox titration of active site Cys-disulfide-dithiol and bound flavin of Mj-DFTR. Each data point in A, C, and D or each spectrum in B represent an average from three independent measurements. Consequently, each error bar shown is for three independent measurements. In each case the dataset from one biological replicate is shown. The E′₀ values derived from two biological replicates (D) were similar (data not shown). A, redox titration of Cys-disulfide-dithiol: Oxidized Mj-DFTR (30 μm) was incubated in a series of solutions containing 100 mm MOPS buffer, pH 7.0, and set at varying potential (E′) values with mixtures of DTT and oxidized DTT at appropriate ratios. The total concentration of DTT and oxidized DTT was 5 mm. % thiol, portion of the Mj-DFTR molecules with the active site Cys residues at the thiol state was quantified via a fluorimetric assay for mBBr-labeled Cys-thiolate; the fluorescence intensity values for the fully oxidized and fully reduced preparations were set to 0 and 100%. B, UV-visible spectra of Mj-DFTR redox titration mixtures with F₄₂₀H₂ as a reductant. The titration of Mj-DFTR-bound FAD involved the addition of various amounts of F₄₂₀H₂ (at final concentrations of 5–115 μm) to a series of solutions containing oxidized Mj-DFTR (25 μm) and 100 mm potassium phosphate buffer, pH 7.0, inside an anaerobic chamber. The labels on the spectra (0.2, 0.5, 0.7, 1, 1.5, 2, and 3) are the amounts of added F₄₂₀H₂ presented in terms of equivalents with respect to the Mj-DFTR-bound FAD; the label “oxidized Mj-DFTR” is for zero F₄₂₀H₂. The isosbestic points that occurred at 445 and 500 are marked with asterisks. C, changes in extinction coefficient values at 480 (filled circles) and 540 nm (open circles) of the Mj-DFTR mixtures as described in B. D, redox titration of Mj-DFTR-bound FAD with dithionite in the presence of methyl viologen, a redox dye, as a reference. The concentration of Mj-DFTR-bound FAD in a mixture was determined from the respective A_{460 nm} value. % FADH₂, percentage of the bound flavin in the FADH₂ state as calculated from the concentration of Mj-DFTR-bound FAD in a mixture with dithionite and that in a control mixture without dithionite. The E′₀ value shown was obtained by fitting the plotted data to the Nernst equation for a 2-electron reduction reaction (n = 2) (see Equation 2).

FIGURE 8.

Thioredoxins and thioredoxin reductases in representative methanogens. The information is presented on a maximum likelihood inference-based 16S-rRNA phylogenetic tree. Dash, not detectable in homology based searches; black bullet at the branches, a confidence value >70%; scale bar, number of base substitution per site; Trx, thioredoxin; NTR, FTR, and DFTR, NADPH-, ferredoxin-, and deazaflavin-dependent thioredoxin reductase, respectively. The NCBI accession numbers for Trxs and Trx reductases are listed in supplemental Table S1.

FIGURE 9.

Maximum likelihood phylogenetic analysis of DFTR and selected flavin-containing TrxRs. The phylogenetic tree was constructed as described previously (91). Name in boldface, bona fide TrxR validated by direct activity assay; details are in supplemental Table S1. Black and white bullets near branches: bootstrap values >70 (calculated from 100 replicates). Each label appearing on the outer side of the tree; electron donor is utilized by the respective clade. Unknown, TrxR with unknown reductant. Non-NAD(P)H, inability to use NAD(P)H as electron donor as determined via direct activity assay of representative members (supplemental Table S1). Symbols for the departures in the amino acid sequences at select conserved elements of TrxRs as described in Fig. 6: filled black and gray circles, unrecognizable HRR and GGG motifs, respectively; open circle, absence of the GG motif. Ec-DLD, dihydrolipoamide dehydrogenase of E. coli used as an outgroup. Details for the abbreviations for the host organism names and the accession numbers of respective TrxRs are listed in supplemental Table S2.

FIGURE 10.

Catalytic mechanism of Mj-DFTR. The assays were performed anaerobically in a mixture containing 100 mm potassium phosphate, pH 7.0, 2 mm EDTA, 1 mm oxidized glutathione, and varying concentrations of F₄₂₀H₂ (5, 7, 10, 20, 40, and 60 μm) at two fixed concentrations of Mj-Trx1 (2 or 15 μm) at 70 °C. Each initial rate value is an average over three replicates. Error bar, standard deviation of samples.

FIGURE 11.

Diversity of electron donors of low molecular weight flavin-containing thioredoxin reductase (TrxR). e⁻, electron; H^˙̄, hydride. General electron flow scheme: electron donor → bound FAD → disulfide of TrxR → disulfide of Trx. The E′₀ standard mid-point redox potential for a reduced electron carrier/oxidized electron carrier pair.