A mechanism accounting for the robust catalase activity in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. In Mycobacterium tuberculosis, KatG is the sole catalase and is also responsible for peroxidative activation of isoniazid, an anti-tuberculosis pro-drug. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy both at the X-band and at the D-band, and mutagenesis are used to identify catalase reaction intermediates in M. tuberculosis KatG. In the presence of millimolar H 2 O 2 at neutral pH, oxyferrous heme is formed within milliseconds from ferric (resting) KatG, whereas at pH 8.5, low spin ferric heme is formed. Using rapid freeze-quench EPR at X-band under both of these conditions, a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical and the unique heme intermediates persist in wild-type KatG only during the time course of turnover of excess H 2 O 2 (1000-fold or more). Mutation of Met

Unlike direct ESR, spin trap methodology depends on the absolute fidelity of the spin trap reaction. Two alternative reactions of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) leading to radical adduct artifacts have been discovered and investigated: inverted spin trapping and the Forrester-Hepburn nucleophilic mechanisms. These two alternate pathways to radical adducts are a combination of one-electron oxidation and nucleophilic addition, in either order. In biological systems, serious artifacts have been reported due to the Forrester-Hepburn mechanism, which is initiated by the addition of a nucleophile to DMPO. It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) can react with DMPO via a nonradical, nucleophilic reaction, and it has been further proposed that DMPO/•SO3− formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical (•SO3−). The one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the absence of DMPO, the initial rate of (bi)sulfite-dependent oxygen and H2O2 consumption was determined to be half of the initial rate of DMPO/•SO3− radical adduct formation as determined by ESR, demonstrating that, under our experimental conditions, DMPO exclusively forms the radical adduct by trapping the •SO3−.

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the alpha/beta hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k(cat)/K(m,A) and k(cat)/K(m,O)()2, were 1.4 x 10(8) and 3.0 x 10(5) M(-1) s(-1) with I and 1.2 x 10(5) and 0.41 x 10(5) M(-1) s(-1) with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the alpha/beta hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D(2)O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.