Characterization of the Arene-Oxidizing Intermediate in ToMOH as a Diiron(III) Species

Abstract

We report the generation and characterization of a diiron(III) intermediate formed during reaction with dioxygen of the reduced hydroxylase component of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1. The decay rate of this species is accelerated upon mixing with phenol, a substrate for this system. Under steady state conditions, hydrogen peroxide was generated in the absence of substrate. The oxidized hydroxylase also decomposed hydrogen peroxide to liberate dioxygen in the absence of reducing equivalents. This activity suggests that dioxygen activation may be reversible. The linear free energy relationship determined from hydroxylation of para substituted phenols under steady state turnover has a negative slope. A value of ρ < 0 is consistent with electrophilic attack by the oxidizing intermediate on the aromatic substrates. The results from these steady and pre-steady state experiments provide compelling evidence that the diiron(III) intermediate is the active oxidant in ToMO and a peroxodiiron(III) transient, despite differences between its optical and Mössbauer spectroscopic parameters and those of other peroxodiiron(III) centers.

Structural consequences of effector protein complex formation in a diiron hydroxylase

“…Other studies have shown that ligand binding can induce spectral changes and shifts in hydroxylase helices (7,23,24), but these previous efforts showed neither the extent nor the detail of the interaction now revealed by the stoichiometric T4moHD complex. We propose that the extensive changes in ␣A, ␣E, ␣F, and ␣H in the complex, which provide Ͼ20 new hydrogen bonds along the helices of TmoA, may allow the rearranged T4moH configuration to persist after T4moD has dissociated, thus providing a structural basis for a long-lived conformational state.…”

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

“…His62, in particular, seems important for proper orientation of the substrate, analogous to Lys127 in MIOX, which is essential for activity (6). With the exception of MIOX, all dinuclear nonheme-iron oxygenases and oxidases studied to date use the fully reduced forms of their cofactors to activate O 2 (32)(33)(34)(35)(36)(37)(38), and the mixedvalent Fe II /Fe III forms are usually only marginally stable in these enzymes (28,39,40). By contrast, MIOX employs the mixedvalent form of its diiron cluster to promote substrate and O 2 activation and accordingly stabilizes this state, allowing its accumulation in 60-70% yield (17 (Fig.…”

The catalase activity of diiron adenine deaminase

“…These samples exhibited a spectral pattern typical of high-spin Fe III ions with rhombic symmetry (E/D $ 1/3). 5,6 The corresponding electron paramagnetic resonance (EPR) spectrum [ Fig. 3(a) Figure 4.…”

“…5,6 The latter are found in monooxygenase enzymes and model compounds. 5,6,19 Perhaps the best studied of these is toluene/o-xylene monooxygenase from Pseudomonas sp. OX1.…”

“…OX1. 6 For this enzyme, the two irons are anti-ferromagnetically coupled and the diferric form is stable in air. This is typical for diiron centers that have no catalase activity.…”