Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters

Abstract

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]¹⁺ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Figure 1.. The proposed catalytic mechanisms for plant-type FTR, FDR and AFTR.

Residue numbering left of the slash mark is for FTR, middle is for FDR and to the right is for AFTR. Structures in brackets are proposed one-electron-reduced intermediates.

Figure 2.. Comparison of FDR and AFTR active sites.

The model of AFTR was obtained using AlphaFold (29). The active sites of FDR (A) and AFTR (B) containing the [4Fe-4S] cluster and active-site disulfides. Atoms are color coded for iron (brown), sulfur (yellow), carbon (gray), nitrogen (blue), and oxygen (red).

Figure 3.. Spectral changes accompanying reduction of as-purified wild type AFTR and wild type NEM-AFTR.

(A) wild type (34 μM), and (B) wild type NEM-AFTR (43 μM). Key (—) air-oxidized, (---) recorded 1.0 min after addition of 0.3 mM (final concentration) sodium dithionite.

Figure 4.. Ferredoxin-dependent reduction of wild type AFTR.

The complete reaction mixture (700 μl) contained CODH/ACD (26 μg or 0.12 μM considering the molecular weight of CODH complex is 297 kDa based on as reported for CODH complex from M.thermophila (32)), AFTR (45 μM), Fdx (28 μg or 6.4 μM), and NaCl (150 mM) in 50 mM phosphate buffer (pH 7.4). The anaerobic reactions were contained in a serum-stoppered cuvette (2 ml) with 1 atm CO and initiated by the addition of CODH/ACD. Panel A, UV-Vis spectra. Key: (—) complete mixture minus CODH/ACD and Fdx, (- - -) complete mixture at time zero, and (….) complete mixture after 5 min incubation. Panel B, time course for reduction monitored at 412 nm. Key: (—) complete mixture minus CODH, (- - - ) complete mixture minus Fdx, and (….) complete mixture containing AFTR/CODH/Fdx.

Figure 5.. Ferredoxin dependent disulfide reductase activity of wild type AFTR.

The complete reaction mixture (400 μl) contained 20 μg of CODH/ACD, 8.6 μM Fdx, 7 μM wild type AFTR, 12.5 μM Trx2, and 400 μg insulin in 50 mM phosphate buffer (pH 7.4). The reaction mixture was contained in a serum-stoppered vial (2.0 ml) with 1.0 atm CO. The reactions were started by adding CODH/ACD. Reduction of insulin and Trx2 was monitored with Ellman’s reagent detecting the formation of thiols. Reaction mixtures: (■) complete, (•) minus Fdx, (Δ) minus insulin, (▴) minus Trx2. Thiol levels detected for reactions minus AFTR or minus AFTR and insulin (not shown) were not significantly different from the reaction mixture minus Fdx. Data points and bars are the mean and standard deviation of three experiments.

Figure 6.. Insulin disulfide reductase activity of Trx2 reduced with wild type AFTR or H85Y/A variants.

The reaction mixture contains 6μM of AFTR (solid line), H85Y-AFTR (dashed line), H85A-AFTR (dotted line) 50μg Trx2, and 1 mg Insulin. The reaction was started by adding dithionite (1.8mM). No increase in absorbance at 650 nm was observed in the following control reactions: (i)DT, Insulin & Trx2 (ii) DT, Insulin, AFTR (iii)/(iv), DT, Insulin, H85Y/H85A.

Figure 7.. Spectroscopy of the [4Fe4S] cluster in as-purified, one-electron reduced, and two-electron reduced wild type AFTR.

Panels A and B. Mössbauer spectra of 0.778mM ⁵⁷Fe AFTR proteins as purified (A) and after incubation with 20 equivalent of dithionite for 30 minutes (B) showing [4Fe4S]²⁺ cluster signals. Black lines represent experimental spectra and the associated statistical error; red lines show total spectral simulations; simulations of the subcomponents are color coded: green, mixed-valent Fe²⁺Fe³⁺ pair; blue, valence localized Fe²⁺ site; yellow, valence localized Fe³⁺ site. Panels C and D. X-band cw-EPR spectra of reduced wild type AFTR in the presence of methyl viologen, showing the S = 7/2 species (C) and the S = ½ species (D). The EPR sample was prepared by mixing 0.2 mM of WT-AFTR with 0.1 eq of MV and 10 eq of DT, and incubating the solution for 80 seconds before freezing it for EPR analysis. Black and red lines show the experimental data and the spectral simulations, respectively. The insert in panel C contains the scaled (10x) spectral signal and the simulation showing the weak resonance at g = 12.49. Sample information and measurement conditions are indicated in the Figure. For Mössbauer spectra, the external field is parallel to the γ-radiation. Mössbauer simulation parameters are listed in Table 1. EPR simulation parameters: for the S = 7/2 species, g = [2.03, 2.01, 2.09], D = −1.45 cm⁻¹, E/D = 0.116, σ(E/D) = 0.012, concentration = 0.017 mM; for S = ½ cluster, g = [2.06, 1.95, 1.91], σ_g = [0.010, 0.0051, 0.007], concentration = 0.011 mM; for viologen radical: g = 2.002. Protein concentration = 0.2 mM.