Multistep electron tunneling through tryptophans in the KatG bifunctional peroxidase monitored by a nonperturbing spin probe

The bifunctional heme peroxidase from B. pseudomallei (BpKatG) utilizes heme and three tryptophans (Trp139, Trp153, and Trp330) as unique redox cofactors in the peroxidase-like catalytic cycle, as was shown previously by multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with isotope labeling and site-directed mutagenesis. In this work, we exploited the redox properties of a strategically attached nitroxide as a direct probe of the long-range multistep electron-tunneling pathway between Trp153 • and the high-valent heme intermediate, thereby showing it is mediated by Trp94 and Trp95. We also demonstrated that the equilibrium between the [Fe IV = O Trp153 • ] and [Fe IV = O Trp139 • ] intermediates, which is observed in the absence of substrate, is preferentially shifted toward catalytic oxidation of isoniazid substrate by Trp139 • . Our EPR experimental data confirm that Trp139 • oxidatively activates the isoniazid prodrug, in sharp contrast to the current view that [Fe IV = O Por •+ ] is the oxidant, as in canonical peroxidases.