Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC

Abstract

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by ¹⁵N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using ¹H and ²H ENDOR. In addition, ²H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary ¹H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Figure 1.

Reaction catalyzed by MbnBC and crystallographic model for the diiron active site. (a) The MbnA from M. trichosporium OB3b consists of a leader sequence (shown as amino acid one-letter codes) followed by the core sequence. The cysteine residues that are modified to oxazolone/thioamide groups are colored orange and labeled. The ²H atoms present in the ^DMbnA substrate are shown in blue. (b) The coordination of the two Fe ions in the M. trichosporium OB3B MbnBC active site (PDB accession code 7TCX). The third Fe ion, which is not required for activity, is not shown.

Figure 2.. ¹⁵N Q-band Davies ENDOR of ¹⁵N labeled MV MbnBC.

The brackets represent twice the Larmor frequency (2ν_15N), but some ν₋ extend into frequencies unmeasurable. The ▼ represents |A/2|. (A) ¹⁵N Davies ENDOR at g₁ (g = 1.93). (B) ¹⁵N Davies ENDOR at g₂ (g = ~1.85). As the field increases, the presence of an addition Fe(III) ¹⁵N coupling is resolved. (C) ¹⁵N Davies ENDOR at the high field end of the EPR envelope near g₃ (g =1.76) and (D) (g=1.75). Davies ENDOR conditions: 2 K, 34.63 GHz microwave frequency, 100 ms repetition time, π = 60 ns, τ = 600 ns, T_RF = 35 μs, RF tail = 10 μs, ~150 scans each spectrum.

Figure 3.

¹H frequency-swept (high rf frequency to low) Q-band CW ENDOR spectra of MV MbnBC in H₂O buffer (black), exchanged into buffer prepared in D₂O (gray) at canonical g-values A, g₁=1.95; B, g₂=1.88; and C, g₃=1.77, and relevant portions of simulations from Fe(III)-OH_x ligands (blue) and μ-OH_x ligand (red). Couplings corresponding to Fe(II)OH_x are labeled (green). The ν₊ features are better resolved than the ν₋ features, so the ν₊ portion of the spectrum is magnified to show these features more clearly; those features are labeled by the associated hyperfine couplings (signs not determined). CW ENDOR conditions: 2 K, 34.88 GHz (H₂O) and 34.83 GHz (D₂O) MW frequency, 4 G modulation, 1.8 μW MW power, 0.5 MHz/s scan, 32 ms time constant, 200 KHz RF bandwidth broadening, ~200 scans each. Complete simulations are shown in Figure S4, and simulation parameters are given in the legend to Figure S4.

Figure 4.

The MV MbnBC active site chemical composition as determined through a combination of X-ray crystallography (black), ¹⁵N Davies ENDOR (blue and red), and ¹H CW ENDOR (gold).

Figure 5.. ²H Q-band pulsed Mims ENDOR of MbnBC with ^DMbnA added anaerobically.

The ²H ENDOR across the EPR envelope at A, g = 1.93; B, g = 1.89; C, g = 1.85; and D, g = 1.82 reveals relatively anisotropic coupling. The * signifies signals from the ¹H harmonic, and not ²H ENDOR responses. Red brackets represent |A|,▼ represents the ²H Larmor frequency, and the black bracket represents ²H quadrupole splitting (3P). Mims ENDOR conditions: 2 K, 34.70 GHz, 100 ms repetition time, π/2 = 50 ns, τ = 500 ns, T_RF = 35 μs, RF tail = 5 μs.

Figure 6.

Displacement of solvent ligand from MbnBC by MbnA binding. (A) ¹H Q-band CW ENDOR of MV MbnBC (black) and MV MbnABC complex (red) at g₁. The bracket represents ¹H |A| and is centered at the ¹H Larmor frequency. The loss of the 24 MHz coupling when MbnA is added is associated with MbnA displacing a terminal H_xO to coordinate the Fe(III) center. CW ENDOR conditions: 2 K, 34.81 GHz, 4 G Modulation, 1.8 μW MW power, 0.5 MHz/s scan, 32 ms time constant, reverse sweep, ~200 scans each. (B) Model of the substrate-bound active site of MbnBC as determined above.

Figure 7.

Mechanistic scheme for MbnBC as determined by this study and suggested in previous work.^,