A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis

Abstract

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.

Figure 1

Schematic alignment of heterodisulfide reductase from Methanothermobacter marburgensis (Mt Hdr), heterodisulfide reductase from Methanosarcina barkeri (Mb Hdr), and thiol: fumarate reductase from Methanothermobacter marburgensis (Mt Tfr). Homologous subunits are indicated by the same filling patterns. HdrB, the C-terminal part of HdrD, and the C-terminal part of TfrB contain 10 conserved cysteine residues (2 × 5C) in two CX_31–39CCX_35–36CXXC sequence motifs designated as CCG domains. Subunits HdrE, HdrA, and TfrA have no sequence similarity and different functions in the different enzymes.

Figure 2

UV–visible absorption spectra of HdrB produced in E. coli. (A) Spectrum of purified HdrB (0.7 mg of protein/mL) obtained from cell extracts without reconstitution. The inset to A shows the spectrum of nonreconstituted HdrB as purified (solid line) and after addition of 0.1 mM sodium dithionite and an incubation of 60 min (dashed line). (B) Spectrum of HdrB (1 mg of protein/mL) purified from cell extracts after in vitro reconstitution. The inset to B shows spectra of reconstituted HdrB (2 mg of protein/mL): in the “as-purified” state (solid line), reduced by 0.1 mM sodium dithionite (dotted line), and the sodium dithionite reduced protein after reoxidation by 0.2 mM duroquinone (dashed line). Protein samples were in 50 mM Tris/HCl pH 7.6. The spectra were recorded with a Zeiss Specord UV VIS S10 diode array spectrophotometer.

Figure 3

EPR spectra of HdrB. (A) HdrB (350 μM) before reconstitution. (B) HdrB (100 μM) after reconstitution. HdrB was oxidized by 2 mM duroquinone. (C) EPR spectrum of HdrB incubated with CoM-³³SH. HdrB (100 μM) was reduced with 5 mM sodium dithionite; excess sodium dithionite was removed by ultrafiltration (10 kDa cutoff) and subsequently oxidized by 2 mM duroquinone in the presence of 2 mM CoM-³³SH. Dotted lines represent simulations of spectra B and C. Simulation parameters: g_zyx = 2.0147, 1.9949, 1.9503; W_zyx = 3.00, 1.60, 1.50 mT; (³³S, I = 3/2) A_zyx = 4.00, 3.60, 0.00 mT. (D) Spectrum of CoM-HDR, shown for comparison. HdrABC (40 μM) was oxidized by 2 mM duroquinone in the presence of 1 mM CoM-SH. EPR conditions: temperature, 20 K; microwave power, 2.007 mW; microwave frequency, 9.458 GHz; modulation amplitude, 0.6 mT.

Figure 4

EPR-monitored redox titration of HdrB and spectra obtained at different potentials. Titrations were performed as described in Experimental Procedures. HdrB (60 μM) was in 50 mM Tris/HCl pH 7.6. For EPR conditions, see Figure 3A. Titration in the absence of CoM-SH. (B) Titration in the presence of 1 mM CoM-SH. Data points correspond to the amplitude of the trough centered at g = 1.950 because in the low potential range the radical signals of the dyes overlap in the g = 2.0 region. The maximal spin concentration was 0.25 per enzyme molecule. The solid lines are n = 1 Nernst plots with E_m = −175 mV (in the presence of CoM-SH) or −120 mV (in the absence of CoM-SH). (C) EPR spectrum obtained at −70 mV. (D) EPR spectrum obtained at −313 mV. The spectra in C and D are from the titration in the presence of CoM-SH. The arrow in Figure 3D indicates a radical signal resulting from reduced redox dyes. The dotted line indicates the respective simulation. Simulation parameters for D: g_zyx = 2.0550, 1.9770, 1.8416; W_zyx = 5.70, 4.00, 6.50 mT. At higher potentials (>0 mV) and prolonged incubation partial cluster breakdown was observed. Therefore, the data points indicated by open circles were not used for the Nernst plots.

Figure 5

⁵⁷Fe Davies ENDOR spectra. Solid lines represent spectra of ⁵⁷Fe-enriched HdrB recorded at different positions of the EPR line according to (a) B||g₃, (b) B||g₂, (c) B||g₁, and (d) outside the spectrum of HdrB. The inset displays the selected fields in the EPR line. The ⁵⁷Fe spectra of CoM-HDR are displayed as dotted lines for reference (8). Visible ⁵⁷Fe doublets that were previously assigned to the resonances of CoM-HDR are indicated (15). HdrB (1 mM) was in 50 mM Tris/HCl pH 7.6 containing 10% (v/v) glycerol.

Figure 6

EPR spectra of oxidized HdrB: wild type and mutants. (A) Representation of the cysteine residues in the two CCG domains and the two nonconserved cysteine residues of HdrB. The Cys to Ser mutants generated are shown above the sequence. (B) EPR spectra. Samples were prepared as described in Figure 3B. For EPR conditions see Figure 3. The spectrum labeled as DS1 is the difference spectrum which was obtained after subtraction of the spectrum of the C234S mutant at 60% of its intensity from the spectrum of the C231S mutant. The spectrum labeled as DS2 is the difference spectrum which was obtained after subtraction of the spectrum of the C153S mutant at one-third of its intensity from the spectrum of the C283S/C287S mutant. (C) EPR spectra of the C153S mutant at different temperatures.

Figure 7

Zn XAS spectra. (A) Zn K-edge spectra. (B) Fourier transforms (k³ weighted, k = 2.0–13.0 Å⁻¹) of HdrB (solid line), duroquinone-oxidized HdrABC (long-dashed line), and duro-quinone-oxidized HdrABC in the presence of CoM-SH (short-dashed line). (B, inset) k³-weighted EXAFS.