Involvement of semiquinone radicals in the in vitro cytotoxicity of cigarette mainstream smoke.

Free radicals in cigarette smoke have attracted a great deal of attention because they are hypothesized to be responsible in part for several of the pathologies related to smoking. Hydroquinone, catechol, and their methyl-substituted derivatives are abundant in the particulate phase of cigarette smoke, and they are known precursors of semiquinone radicals. In this study, the in vitro cytotoxicity of these dihydroxybenzenes was determined using the neutral red uptake (NRU) assay, and their radical-forming capacity was determined by electron paramagnetic resonance (EPR).

Resonance Raman spectroscopy of Compound II and its decay in Mycobacterium tuberculosis catalase-peroxidase KatG and its isoniazid resistant mutant S315T.

The reaction of Mycobacterium tuberculosis KatG and the mutant KatG(S315T) with two different organic peroxides is studied using resonance Raman spectroscopy. For the first time, an intermediate is observed in a catalase-peroxidase with vibrations that are characteristic of Compound II. The observation of this intermediate is consistent with photoreduction of Compound I and is in agreement with the formation of Compound I during the catalytic cycle of KatG.

Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function.

Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm(-)(1) in the ferrous forms of both the wild-type enzyme and KatG(S315T).

Conformational differences in Mycobacterium tuberculosis catalase-peroxidase KatG and its S315T mutant revealed by resonance Raman spectroscopy.

KatG from Mycobacterium tuberculosis is a heme-containing catalase-peroxidase, which belongs to the class I peroxidases and is important for activation of the prodrug isoniazid (INH), a front-line antituberculosis drug. In many clinical isolates, resistance to INH has been linked to mutations on the katG gene, and the most prevalent mutation, S315T, suggests that modification of the heme pocket has occurred. Electronic absorption and resonance Raman spectra of ferric wild-type (WT) KatG and its INH-resistant mutant KatG(S315T) at different pH values and their complexes with INH and benzohydroxamic acid (BHA) are reported.

Analysis of heme structural heterogeneity in Mycobacterium tuberculosis catalase-peroxidase (KatG).

Mycobacterium tuberculosis catalase-peroxidase (KatG) is a heme enzyme considered important for virulence, which is also responsible for activation of the anti-tuberculosis pro-drug isoniazid. Here, we present an analysis of heterogeneity in KatG heme structure using optical, resonance Raman, and EPR spectroscopy. Examination of ferric KatG under a variety of conditions, including enzyme in the presence of fluoride, chloride, or isoniazid, and at different stages during purification in different buffers allowed for assignment of spectral features to both five- and six-coordinate heme.

Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethal concentrations of inorganic arsenic.

Inorganic arsenic (iAs), a known human carcinogen, acts as a tumor promoter in part by inducing a rapid burst of reactive oxygen species (ROS) in mammalian cells. This causes oxidative stress and a subsequent increase in the level of cellular glutathione (GSH). Glutathione, a ubiquitous reducing sulfhydryl tripeptide, is involved in ROS detoxification and its increase may be part of an adaptive response to the oxidative stress.

Identification and characterization of tyrosyl radical formation in Mycobacterium tuberculosis catalase-peroxidase (KatG).

The catalytic function of Mycobacterium tuberculosis catalase-peroxidase (KatG) and its role in activation of the anti-tuberculosis antibiotic isoniazid were investigated using rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiments. The reaction of KatG with peroxyacetic acid was followed as a function of time using x-band EPR at 77 K. A doublet EPR signal appears within 6.

Direct voltammetry and catalysis with Mycobacterium tuberculosis catalase-peroxidase, peroxidases, and catalase in lipid films.

Stable films of dimyristoylphosphatidylcholine and M. tuberculosis catalase-peroxidase (KatG), several peroxidases, myoglobin, and catalase showed reversible FeIII/FeII voltammetry on pyrolytic graphite electrodes and catalytic current for hydrogen peroxide and oxygen. Amperometric responses for these films to H2O2 at 0 V are likely to contain significant contributions from catalytic reduction of oxygen produced during the catalytic cycles.

Characterization of the W321F mutant of Mycobacterium tuberculosis catalase-peroxidase KatG.

A single amino acid mutation (W321F) in Mycobacterium tuberculosis catalase-peroxidase (KatG) was constructed by site-directed mutagenesis. The purified mutant enzyme was characterized using optical and electron paramagnetic resonance spectroscopy, and optical stopped-flow spectrophotometry. Reaction of KatG(W321F) with 3-chloroperoxybenzoic acid, peroxyacetic acid, or t-butylhydroperoxide showed formation of an unstable intermediate assigned as Compound I (oxyferryl iron:porphyrin pi-cation radical) by similarity to wild-type KatG, although second-order rate constants were significantly lower in the mutant for each peroxide tested.