Probing bis-Fe(IV) MauG: experimental evidence for the long-range charge-resonance model

Abstract

The biosynthesis of tryptophan tryptophylquinone, a protein-derived cofactor, involves a long-range reaction mediated by a bis-Fe(IV) intermediate of a diheme enzyme, MauG. Recently, a unique charge-resonance (CR) phenomenon was discovered in this intermediate, and a biological, long-distance CR model was proposed. This model suggests that the chemical nature of the bis-Fe(IV) species is not as simple as it appears; rather, it is composed of a collection of resonance structures in a dynamic equilibrium. Here, we experimentally evaluated the proposed CR model by introducing small molecules to, and measuring the temperature dependence of, bis-Fe(IV) MauG. Spectroscopic evidence was presented to demonstrate that the selected compounds increase the decay rate of the bis-Fe(IV) species by disrupting the equilibrium of the resonance structures that constitutes the proposed CR model. The results support this new CR model and bring a fresh concept to the classical CR theory.

Keywords: charge resonance; electronic structure; heme proteins; high-valence iron; near-infrared spectroscopy.

Figure 1

Structural orientation of the hemes and the intervening tryptophan residue (PDB entry: 3L4M). The distance between the two iron ions and the edge-to-edge distances between the aromatic moieties are labelled.

Figure 2

Type III CR in bis-Fe(IV) MauG. (a) Proposed resonance structures in the Type III CR model. “H” represents a third aromatic moiety (i.e., the Trp93 residue in this case), which functions as a hopping relay to facilitate ET between the two primary aromatic moieties. The two resonance structures (Cpd ES* and Cpd I*) that can be potentially targeted by small molecule ligands are highlighted with a grey background. (b) Specific targeting of Cpd ES* and Cpd I* by CN⁻ to disrupt the Type III CR in the bis-Fe(IV) species.

Figure 3

Disruption of bis-Fe(IV) MauG by small molecule ligands. (a) Addition of CN⁻ accelerated the decay of the NIR band of bis-Fe(IV) MauG. CN⁻ was added immediately after bis-Fe(IV) formation. The solid lines are fits of the data to single-exponential decay. (b) Effect of different small molecule ligands on the decay of the NIR band. Each small molecule ligand (25 mM) was added immediately after bis-Fe(IV) formation. (c) Decay rates of the NIR band in the absence and presence of small molecule ligands.

Figure 4

EPR spectra of MauG. Gray trace: as-isolated di-ferric MauG; black trace: MauG + 25 mM CN⁻; blue trace: MauG + 1× H₂O₂; red trace: MauG + 1× H₂O₂ (frozen 75 s after reaction); green trace: MauG + 1× H₂O₂ + 25 mM CN⁻ (CN⁻ was added immediately after addition of H₂O₂ and the sample was frozen 75 s after addition of CN⁻). The arrows indicate the CN⁻ adduct of Heme_5C at g = 3.37.

Figure 5

Temperature effect on the decay rate of the NIR band of bis-Fe(IV) MauG. The bis-Fe(IV) species was generated by addition of a stoichiometric amount of H₂O₂ to di-ferric MauG (15 μM). The data were fit to the Arrhenius equation (solid trace) to calculate the activation energy (E_a) and to the Marcus equation (dashed line) to calculate the reorganization energy (λ) of the ET reaction, respectively.