A catalytic di-heme bis-Fe(IV) intermediate, alternative to an Fe(IV)=O porphyrin radical

Abstract

High-valent iron species are powerful oxidizing agents in chemical and biological catalysis. The best characterized form of an Fe(V) equivalent described in biological systems is the combination of a b-type heme with Fe(IV)=O and a porphyrin or amino acid cation radical (termed Compound I). This work describes an alternative natural mechanism to store two oxidizing equivalents above the ferric state for biological oxidation reactions. MauG is an enzyme that utilizes two covalently bound c-type hemes to catalyze the biosynthesis of the protein-derived cofactor tryptophan tryptophylquinone. Its natural substrate is a monohydroxylated tryptophan residue present in a 119-kDa precursor protein. An EPR-silent di-heme reaction intermediate of MauG was trapped. Mössbauer spectroscopy revealed the presence of two distinct Fe(IV) species. One is consistent with an Fe(IV)=O (ferryl) species (delta = 0.06 mm/s, DeltaE(Q) = 1.70 mm/s). The other is assigned to an Fe(IV) heme species with two axial ligands from protein (delta = 0.17 mm/s, DeltaE(Q) = 2.54 mm/s), which has never before been described in nature. This bis-Fe(IV) intermediate is remarkably stable but readily reacts with its native substrate. These findings broaden our views of how proteins can stabilize a highly reactive oxidizing species and the scope of enzyme-catalyzed posttranslational modifications.

Scheme. 1.

MauG-dependent TTQ biosynthesis.

Fig. 1.

EPR analysis of the formation and decay of the intermediate formed by reaction of di-ferric MauG with a stoichiometric amount of H₂O₂. After mixing EPR spectra were recorded at time intervals of 0 (a), 0.03 (b), 2 (c), 8 (d), and 20 (e) minutes. Each sample contained 200 μM MauG. EPR parameters were temperature 10 K, microwave power 1 mW, modulation amplitude 5 G, time constant 40.96 ms, and sweep time 83.89 s. Each spectrum is the average of five scans.

Fig. 2.

Proposed mechanism for the formation of the bis-Fe(IV) intermediate formed by reaction of di-ferric MauG with a stoichiometric amount of H₂O₂.

Fig. 3.

Mössbauer spectra of MauG recorded at 4.2 K in a 53-mT magnetic field. (a) The spectrum of ferric MauG (hashed marks) is overlaid with spin Hamiltonian simulations of a high-spin Fe(III) heme (25% of total Fe, dashed line) and of a low-spin Fe(III) heme (75% of total Fe, solid line) with the following parameters: S = 5/2, g_5/2 = 2, D_5/2 = 10 cm⁻¹ (E/D)_5/2 = 0, δ = 0.50 mm/s, ΔE_Q = 2.0 mm/s, η = 0, A/g_Nβ_N = (−18.0, −18.0, −18.0) T and S = 1/2, g_1/2 = (1.87, 2.19, 2.54), δ = 0.25 mm/s, ΔE_Q = 1.97 mm/s, η = −3, A/g_Nβ_N = (−36.2, +9.3, +30.0) T. (b) Spectrum of di-ferric MauG, which was reacted with H₂O₂ for 45 s (hashed marks). The solid line indicates the contribution of ferric MauG (≈34% of total Fe). Removal of these features yields the reference spectrum of the intermediates (c, in which the x axis scale has been expanded), which can be simulated with two quadrupole doublets: δ₁ = 0.06 mm/s and ΔE_Q1 = 1.70 mm/s (15%, dashed line) and δ₂ = 0.17 mm/s and ΔE_Q2 = 2.54 mm/s (35%, solid line).

Fig. 4.

EPR analysis of the reaction of the high-valent MauG intermediate with its natural substrate. (a) High-valent MauG intermediate spectrum generated from reaction of di-ferric MauG with a stoichiometric amount of H₂O₂. (b) Spectrum of a further reacted with a stoichiometric amount of the biosynthetic precursor of MADH for 15 s before freezing in liquid nitrogen. (Inset) Spectrum of the g = 2 region. (c) Parallel sample of b further reacted with 2 mM hydroxyurea for 2 min. EPR parameters were temperature 10 K, microwave power 0.5 mW and modulation amplitude 5 G.