Toward a mechanistic and physiological understanding of a ferredoxin:disulfide reductase from the domains Archaea and Bacteria

Abstract

Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster. UV-visible, EPR, and Mössbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]¹⁺ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR, advancing the physiological understanding of FDR's role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show that FDR homologs are widespread in diverse microbes from the domain Bacteria.

Keywords: archaea; disulfide; enzyme mechanism; stress; thioredoxin.

Figure 1.

Structure comparison of FTR (Protein Data Bank entry IDJ7) and FDR (Protein Data Bank entry 4TPU). A, overall structure of the Synechocystis Fdx-FTR-Trx complex. Reproduced with permission (12). Fdx, the catalytic subunit of FTR, the variable subunit of FTR, and Trx are colored blue, beige, brown, and green, respectively. The [4Fe-4S] cluster is shown in stick representation. Fdx and Trx interact exclusively with the catalytic subunit of FTR in opposite directions. B, active-site structure of FTR. Reproduced with permission (5). The active-site disulfide formed by Cys⁵⁷ and Cys⁸⁷ is contained in the red circle. C, crystal structure of the FDR dimer. Monomers are shown in red and blue. The iron and sulfur atoms of the [4Fe-4S] clusters and [1Fe-0S] centers are shown in a red and yellow ball representation, respectively. D, active-site structure of FDR dimer showing the adjacent C- and N-terminal domains containing the [1Fe-0S] centers and [4Fe4S] clusters, respectively. The active-site disulfide formed by Cys⁵² and Cys⁸⁴ is contained in the red circle. Color codes for active-site structures of FTR (B) and FDR (D) are as follows: iron (green), sulfur (yellow), carbon (gray), nitrogen (blue), and oxygen (red). The Fe–S and S–S interactions are shown in black broken lines with distances in ångstroms. The angles bisecting the S–S interactions in B and D are 128.8 and 140.9° for FTR and FDR, respectively (see “Results”).

Figure 2.

Proposed catalytic mechanism for the plant-type FTR. The square brackets indicate proposed transient intermediates. Residue numbering left of the slash mark is for Synechocystis FTR, and numbering to the right is for FDR. Structures in brackets are one-electron-reduced intermediates hypothesized for FTR.

Figure 3.

UV-visible absorption spectra of resting-state WT and NEM-WT FDR. A, resting-state WT (42 μm). B, oxidized NEM-WT (40 μm). Solid line, air-oxidized; dashed line, recorded 1.0 min after the addition of 0.3 mm (final concentration) dithionite.

Figure 4.

X-band EPR spectra of the [1Fe-0S] center in FDR. A, resting-state WT (75 μm). B, air-oxidized NEM-WT (120 μm). EPR conditions were as follows: temperature, 16 K; microwave power, 1 milliwatt; modulation amplitude, 0.63 mT; microwave frequency, 9.60 GHz.

Figure 5.

Mössbauer spectra of resting-state WT and NEM-WT FDR. A, spectrum of resting-state WT (black vertical bars), collected at 4.2 K with a 53-mT magnetic field applied parallel to the γ-beam; a simulation is overlaid (red solid line). Components of the simulation are shifted upward: a site-differentiated [4Fe-4S]²⁺ cluster constituting ∼95% of the sample iron (yellow solid line) and a ferric [1Fe-0S] center constituting ∼8% of the sample iron (black solid line). B, spectrum of NEM-WT (black vertical bars), collected at 4.2 K with a 53-mT magnetic field applied parallel to the γ-beam; a simulation is overlaid (red solid line). Components of the simulation are shifted upward: a site-differentiated [4Fe-4S]²⁺ cluster constituting ∼55% of the sample iron (orange solid line), a [4Fe-4S]³⁺ cluster constituting ∼38% of the sample iron (blue solid line), and a ferric [1Fe-0S] center constituting ∼8% of the sample iron (black solid line). C, spectrum of NEM-WT (black vertical bars), collected at 4.2 K with an 8-T magnetic field applied parallel to the γ-beam; a simulation is overlaid (red solid line). Components of the simulation are shifted upward: a site-differentiated [4Fe-4S]²⁺ cluster constituting ∼55% of the sample iron (orange solid line), a [4Fe-4S]³⁺ cluster constituting ∼38% of the sample iron (blue solid line), a ferric [1Fe-0S] center constituting ∼8% of the sample iron (black solid line), and a mononuclear ferric component constituting ∼4% of the sample iron (green solid line).

Figure 6.

X-band EPR spectra of the [4Fe-4S] center in FDR. A, resting-state WT (120 μm) reduced with 10 mm (final concentration) dithionite and frozen after 1 min. B, oxidized NEM-WT (105 μm). EPR conditions were as follows: temperature, 16 K; microwave power, 1 milliwatt; modulation amplitude, 0.63 mT; microwave frequency, 9.60 GHz.

Figure 7.

Time course of change in the [4Fe-4S]¹⁺ EPR signal intensity of dithionite-reduced resting-state WT FDR. See Fig. S3 for EPR spectra.

Figure 8.

Mössbauer spectra of dithionite-reduced resting-state WT FDR. A and C, reduced for 1 min. B and D, reduced for 30 min. A and B, collected at 4.2 K with a 53-mT magnetic field applied parallel to the γ-beam. C and D, collected at 4.2 K with an 8-T magnetic field applied parallel to the γ-beam. Experimental spectra are displayed as black vertical bars, with simulations overlaid (red solid lines). Components of the simulation are shifted upward: a site-differentiated [4Fe-4S]²⁺ cluster, (yellow solid lines), a [4Fe-4S]¹⁺ cluster (blue solid lines), and a ferrous rubredoxin site (black solid lines). In the sample reduced for 1 min (A and C), these components constitute ∼57, ∼38, and ∼8% of the sample iron, respectively. In the sample reduced for 30 min (B and D), these components constitute ∼80, ∼14, and ∼8% of the sample iron, respectively.

Figure 9.

Reduction of MRX and Trx5 with two-electron-reduced FDR. The 400-μl reaction mixtures contained 42 μm prereduced FDR, 90 μm MRX (■), or 82 μm Trx5 (▴) in 50 mm Tris-HCl buffer (pH 7.5). ●, reaction mixture minus MRX and Trx5. All solutions were made anaerobic, and the reactions were performed in an anaerobic glove bag containing a 95% N₂ and 4% H₂ atm. FDR was reduced with 2 mm dithionite, and the excess was removed by gel filtration with a PD-10 column. The reactions were monitored by production of thiols determined with Ellman's reagent (36). Reactions were performed in triplicate.