Roles of multiple-proton transfer pathways and proton-coupled electron transfer in the reactivity of the bis-FeIV state of MauG

The high-valent state of the diheme enzyme MauG exhibits charge–resonance (CR) stabilization in which the major species is a bis-Fe^IV state with one heme present as Fe^IV=O and the other as Fe^IV with axial heme ligands provided by His and Tyr side chains. In the absence of its substrate, the high-valent state is relatively stable and returns to the diferric state over several minutes. It is shown that this process occurs in two phases. The first phase is redistribution of the resonance species that support the CR. The second phase is the loss of CR and reduction to the diferric state. Thermodynamic analysis revealed that the rates of the two phases exhibited different temperature dependencies and activation energies of 8.9 and 19.6 kcal/mol. The two phases exhibited kinetic solvent isotope effects of 2.5 and 2.3. Proton inventory plots of each reaction phase exhibited extreme curvature that could not be fit to models for one- or multiple-proton transfers in the transition state. Each did fit well to a model for two alternative pathways for proton transfer, each involving multiple protons. In each case the experimentally determined fractionation factors were consistent with one of the pathways involving tunneling. The percent of the reaction that involved the tunneling pathway differed for the two reaction phases. Using the crystal structure of MauG it was possible to propose proton–transfer pathways consistent with the experimental data using water molecules and amino acid side chains in the distal pocket of the high-spin heme.MauG (1) is a diheme enzyme that catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) (2) to complete the biosynthesis of its protein-derived cofactor (3) tryptophan tryptophylquinone (TTQ) (4). The hemes of MauG are unusual in several respects. One is a high-spin five-coordinate heme that is ligated by His35. The other is a low-spin six-coordinate heme with two ligands provided by His205 and Tyr294 (1, 5). The latter is, to our knowledge, the first example of natural His–Tyr ligation of a protein-bound heme cofactor, and the first example of Tyr ligation of a c-type heme. An intervening residue, Trp93, “connects” the two hemes (Fig. 1) via rapid electron transfer (ET) (6–9). A unique feature of MauG is that the oxidation of diferric MauG by H₂O₂, or of diferrous MauG by O₂, generates a high-valent bis-Fe^IV state (8) in which the high-spin heme is present as Fe^IV=O with the His35 ligand, and the other heme is present as Fe^IV with the His–Tyr axial ligation retained (5, 10, 11). Formation of the bis-Fe^IV state is accompanied by changes in the visible absorbance spectrum. One observes a decrease in intensity and shift of the Soret peak from 406 to 408 nm and appearance of minor peaks at 526 and 559 nm (Fig. 2) (9, 12).Open in a separate windowFig. 1.Diheme site of MauG. A portion of the crystal structure of the MauG-preMADH complex Protein Data Bank (PDB) ID code 3L4M] is shown with MauG in pink, the MADH β-subunit in green, and the α subunit in blue. Shown in sticks are the hemes of MauG, the intervening Trp93, the three Met residues that are susceptible to autooxidation, the residues on preMADH that are modified by MauG, and Trp-199 which mediates ET from preMADH to bis-Fe^IV MauG. This figure was produced using PyMOL (www.pymol.org).Open in a separate windowFig. 2.Changes in the absorption spectrum of MauG caused by addition of H₂O₂ to diferric MauG. Spectra of MauG were recorded before (solid line) and after (dashed line) the addition of a stoichiometric amount of H₂O₂.The entire absorbance spectrum (A) is presented and the changes in the Soret region (B) and NIR region (C) are magnified.Despite being a highly potent oxidant, the bis-Fe^IV species displays extraordinary stability with a half-life of several minutes in the absence of its substrate (8). A basis for this stability was inferred from the observation of a near-infrared (NIR) electronic absorption feature centered at 950 nm that was observed in bis-Fe^IV MauG (Fig. 2C). This spectral feature is characteristic of a charge–resonance (CR) transition phenomenon (6, 9). A model was presented in which the CR occurs in the absence of direct heme–heme contact by ultrafast and reversible ET between the two high-valent hemes, via hopping through the intervening Trp93 residue (9). In this model the high-valent form of MauG comprises an ensemble of resonance structures including compound ES-like and compound I-like forms of the hemes, with the bis-Fe^IV as the dominant species.The catalytic mechanism of MauG is unusual in that the preMADH substrate does not make direct contact with either heme but instead binds to the surface of MauG several angstroms away (5). Catalysis requires long-range ET to bis-Fe^IV MauG from the residues on preMADH that are modified via a hole-hopping mechanism through Trp199 (13, 14), which resides at the MauG–preMADH interface (Fig. 1). Concomitant with this ET is the formation of free-radical intermediates on preMADH that go on to form the TTQ product (15). In the absence of preMADH, the autoreduction of the bis-Fe^IV redox state to the diferric state leads to inactivation of MauG (16). Analysis of the damaged MauG revealed that this process involves the oxidation of three Met residues (108, 114, and 116) which are located 7.5–15.2 Å from the high-spin heme iron (Fig. 1) (17).To further investigate the dynamic nature of the ensemble of resonance forms of MauG that comprise the high-valent state and the basis for its stability, temperature-dependence and kinetic solvent isotope effect (KSIE) studies were performed. These studies provide evidence for a redistribution within the ensemble of resonance structures before loss of CR stabilization of the high-valent redox state which is linked to the reduction to the diferric state. Thermodynamic analysis of the rates of reaction of these processes reveals that the rates of the initial redistribution of the ensemble of resonance structures and the subsequent loss of CR stabilization exhibit different dependencies on temperature. This accounts for the fact that the early phase is only observable at lower temperatures. Proton inventories of the KSIE indicate that the rates of both the initial redistribution of the ensemble of high-valent species and the loss of CR stabilization are rate-limited by multiple proton-transfer (PT) steps involving two alternative pathways. The likely pathways are identified from the crystal structure of MauG.