Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C₇ having a relatively weak carbon–hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen (¹ΔgO₂) and ozone (O₃) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ_5,6 –double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.     

Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin

The efficacy of the diphenol curcumin as a cancer chemopreventive agent is limited by its chemical and metabolic instability. Non-enzymatic degradation has been described to yield vanillin, ferulic acid, and feruloylmethane through cleavage of the heptadienone chain connecting the phenolic rings. Here we provide evidence for an alternative mechanism, resulting in autoxidative cyclization of the heptadienone moiety as a major pathway of degradation. Autoxidative transformation of curcumin was pH-dependent with the highest rate at pH 8 (2.2 μM/min) and associated with stoichiometric uptake of O(2). Oxidation was also catalyzed by recombinant cyclooxygenase-2 (COX-2) (50 nm; 7.5 μM/min), and the rate was increased ≈10-fold by the addition of 300 μM H(2)O(2). The COX-2 catalyzed transformation was inhibited by acetaminophen but not indomethacin, suggesting catalysis occurred by the peroxidase activity. We propose a mechanism of enzymatic or autoxidative hydrogen abstraction from a phenolic hydroxyl to give a quinone methide and a delocalized radical in the heptadienone chain that undergoes 5-exo cyclization and oxygenation. Hydration of the quinone methide (measured by the incorporation of O-18 from H(2)(18)O) and rearrangement under loss of water gives the final dioxygenated bicyclopentadione product. When curcumin was added to RAW264.7 cells, the bicyclopentadione was increased 1.8-fold in cells activated by LPS; vanillin and other putative cleavage products were negligible. Oxidation to a reactive quinone methide is the mechanistic basis of many phenolic anti-cancer drugs. It is possible, therefore, that oxidative transformation of curcumin, a prominent but previously unrecognized reaction, contributes to its cancer chemopreventive activity.     

Theoretical mechanism of the formation of cholesteryl chloride from cholesterol and thionyl chloride

This work describes a theoretical approach to the substitution reaction mechanism involving the conversion of cholesterol to cholesteryl chloride. Two chlorosulfite ester molecules were formed as intermediates. An iso-steroid was found as the transition state. The final product was cholesteryl chloride and the side products were HCl and SO₂. Calculations were carried out at high level Hartree–Fock theory, using the 6–31G* basis set. From the electronic structure of the reactants, the most important physicochemical properties involved in the reaction pathway were used. Thus, to determine the participation of each molecule and to explain the mechanism of reaction; the total energy, HOMO and LUMO, atomic orbital contribution to frontier orbitals formation, electrostatic potentials, atomic charges, hardness and dipole moment were used. Characterization of intermediates and transition state was supported by their respective energy minima, fundamental frequencies and equilibrium geometry.Figure Synopsis of the reaction pathway. The reaction starts when the lone pair of the Ch oxygen interacts with the sulfur atom, releasing a chloride ion. As a result, the first intermediate is formed. Next, in the first intermediate the nucleophilic chloride ion bonds the electrophilic hydrogen atom, releasing HCl and yielding the second intermediate. In the second intermediate, the electrophilic H-atom from HCl bonds with the lone pair of the Cl atom adjacent to the sulfur atom, restoring HCl. Concurrently, SO₂ is liberated and causes the formation of the C3-C5 partial bond and breaking of the C5-C6 -bond leading to the transition state. In the transition state, the electrophilic H from HCl bonds with the Cl lone pair at C6-Cl, forming HCl again and leaving the C6 atom electron-deficient, which restores the C5-C6 -bond and breaks the C3-C5 partial bond. Finally, the electrophilic C3 atom and the nucleophilic Cl atom form a bond, yielding cholesteryl chloride. HCl and SO₂ are also formed as side products. The arrows show the rearrangement of electrons.     

Efficient nitrosation of glutathione by nitric oxide

Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO₂ or N₂O₃) formed in the course of NO autoxidation. Because the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg²⁺. These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation.     

Heme-bound nitroxyl, hydroxylamine, and ammonia ligands as intermediates in the reaction cycle of cytochrome c nitrite reductase: a theoretical study

In this article, we consider, in detail, the second half-cycle of the six-electron nitrite reduction mechanism catalyzed by cytochrome c nitrite reductase. In total, three electrons and four protons must be provided to reach the final product, ammonia, starting from the HNO intermediate. According to our results, the first event in this half-cycle is the reduction of the HNO intermediate, which is accomplished by two PCET reactions. Two isomeric radical intermediates, HNOH^? and H₂NO^?, are formed. Both intermediates are readily transformed into hydroxylamine, most likely through intramolecular proton transfer from either Arg₁₁₄ or His₂₇₇. An extra proton must enter the active site of the enzyme to initiate heterolytic cleavage of the N–O bond. As a result of N–O bond cleavage, the H₂N⁺ intermediate is formed. The latter readily picks up an electron, forming H₂N^+?, which in turn reacts with Tyr₂₁₈. Interestingly, evidence for Tyr₂₁₈ activity was provided by the mutational studies of Lukat (Biochemistry 47:2080, 2008), but this has never been observed in the initial stages of the overall reduction process. According to our results, an intramolecular reaction with Tyr₂₁₈ in the final step of the nitrite reduction process leads directly to the final product, ammonia. Dissociation of the final product proceeds concomitantly with a change in spin state, which was also observed in the resonance Raman investigations of Martins et al. (J Phys Chem B 114:5563, 2010).     

Glyoxysomal β-oxidation of long-chain fatty acids: completeness of degradation

The ability of glyoxysomes from sunflower (Helianthusannuus L.) cotyledons to completely degrade long-chain fatty acids into their constituent acetyl units and the time courses of the appearance of acyl-CoA intermediates during β-oxidation have been studied using ¹⁴C-labelled substrates at non-saturating concentrations (1.3 to 1.8 μmol · l⁻¹). [¹⁴C]Acetyl-CoA was formed from [18-¹⁴C]oleate metabolized at a yield of up to 80%, and from [U-¹⁴C]palmitate and [U-¹⁴C]linoleate to an extent indicating that a maximum of 80% and 30%, respectively, of the substrate β-oxidized had been degraded beyond the C₄-CoA intermediate level. To obtain the latter values, an acetyl-CoA-removing system was required during β-oxidation. Constant re-oxidation of the NADH formed during the β-oxidation did not replace the effect of acetyl-CoA removal. Neither the completeness of the linoleate β-oxidation nor the rate of reaction were influenced by NADPH. Medium- and short-chain acyl-CoA intermediates were predominantly detected during β-oxidation of the long-chain substrates employed. The degradation of these intermediates appeared to be stimulated mainly in the presence of an acetyl-CoA-removing system. The time courses of the appearance of intermediates corresponded to a precursor-product relationship between intermediates of decreasing chain lengths. Received: 12 December 1997 / Accepted: 26 January 1998     

Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions

3-Hydroxykynurenine (3-OHKyn) is a tryptophan metabolite that is readily autoxidised to products that may be involved in protein modification and cytotoxicity. The oxidation of 3-OHKyn has been studied here with a view to characterising the major products as well as determining their relative rates of formation and the role that H₂O₂ and hydroxyl radical (HO^·) may play in modifying the autoxidation process. Oxidation of 3-OHKyn generated several compounds. Xanthommatin (Xan), formed by the oxidative dimerisation of 3-OHKyn, was the major product formed initially. It was, however, found to be unstable, particularly in the presence of H₂O₂, and degraded to other products including the p-quinone, 4,6-dihydroxyquinolinequinonecarboxylic acid (DHQCA). A compound that has a structure consistent with that of hydroxy-xanthommatin (OHXan) was also formed in addition to at least two minor species that we were unable to identify. Hydrogen peroxide was formed rapidly upon oxidation of 3-OHKyn, and significantly influenced the relative abundance of the different autoxidation species. Increasing either pH (from pH 6 to 8) or temperature (from 25°C to 35°C) accelerated the rate of autoxidation but had little impact on the relative abundance of the autoxidation species. Using electron paramagnetic resonance (EPR) spectroscopy, a clear phenoxyl radical signal was observed during 3-OHKyn autoxidation and this was attributed to xanthommatin radical (Xan^·). Hydroxyl radicals were also produced during 3-OHKyn autoxidation. The HO^· EPR signal disappeared and the Xan^· EPR signal increased when catalase was added to the autoxidation mixture. The HO^· did not appear to play a role in the formation of the autoxidation products as evidenced using HO^· traps/scavengers. We propose that the cytotoxicity of 3-OHKyn may be explained by both the generation of H₂O₂ and by the formation of reactive 3-OHKyn autoxidation products such as the Xan^· and DHQCA.     

Microbial transformation of curcumin by Rhizopus chinensis

Abstract

Curcumin (1) is a potent antioxidant and antitumor natural product. In spite of its efficacy and safety, its clinical use is hindered mainly by poor water solubility and bioavailability. Structural modification to introduce hydrophilic functions is a promising approach to resolve this problem. In the present study we first found that curcumin could be efficiently converted into glucosides by filamentous fungi including Rhizopus chinensis IFFI 03043, Absidia coerulea AS 3.3389 and Cunninghamella elegans AS 3.1207. Curcumin 4′-O-β-d-glucoside (2), together with hexahydrocurcumin (3), was isolated from a preparative-scale biotransformation with R. chinensis IFFI 03043 and characterized fully by NMR and MS. A time-course study revealed that curcumin could be efficiently converted into curcumin 4′-O-β-d-glucoside within 8 h when administered at 0.05 mmol L^?1 and the productivity was 57%. Additionally, the biotransformation products of curcumin by different fungal strains were analyzed by LC/MS. At least 15 metabolites were detected, and the predominant biotransformation reaction was glucosylation. This study provides a simple, efficient and less expensive approach for the preparation of curcumin glucosides. The introduction of the glucosyl function might be able to enhance the bioavailability of curcumin.     

Time-Resolved Resonance Raman Investigation of Oxygen Reduction Mechanism of Bovine Cytochrome c Oxidase

Six oxygen-associated resonance Raman bands were identified for intermediates in the reaction of bovine cytochrome c oxidase with O₂ at room temperature. The primary intermediate, corresponding to Compound A of cryogenic measurements, is an O₂ adduct of heme a ₃ and its isotope frequency shifts for ¹⁶O¹⁸O have established that the binding is of an end-on type. This is followed by two oxoheme intermediates, and the final intermediate appearing around 3 ms is the Fe–OH heme. The reaction rate between the two oxoheme intermediates is significantly slower in D₂O than in H₂O, suggesting that the electron transfer is regulated by proton translocations at this step. It is noted that the reaction intermediates of oxidized enzyme with hydrogen peroxide yield the same three sets of oxygen isotope-sensitive bands as those of oxoheme intermediates seen for O₂ reduction and that the O–O bond has already been cleaved in the so-called peroxy form (or 607 nm form).     

Intracellular ROS protection efficiency and free radical-scavenging activity of curcumin

Curcumin has many pharmaceutical applications, many of which arise from its potent antioxidant properties. The present research examined the antioxidant activities of curcumin in polar solvents by a comparative study using ESR, reduction of ferric iron in aqueous medium and intracellular ROS/toxicity assays. ESR data indicated that the steric hindrance among adjacent big size groups within a galvinoxyl molecule limited the curcumin to scavenge galvinoxyl radicals effectively, while curcumin showed a powerful capacity for scavenging intracellular smaller oxidative molecules such as H₂O₂, HO^•, ROO^•. Cell viability and ROS assays demonstrated that curcumin was able to penetrate into the polar medium inside the cells and to protect them against the highly toxic and lethal effects of cumene hydroperoxide. Curcumin also showed good electron-transfer capability, with greater activity than trolox in aqueous solution. Curcumin can readily transfer electron or easily donate H-atom from two phenolic sites to scavenge free radicals. The excellent electron transfer capability of curcumin is because of its unique structure and different functional groups, including a β-diketone and several π electrons that have the capacity to conjugate between two phenyl rings. Therfore, since curcumin is inherently a lipophilic compound, because of its superb intracellular ROS scavenging activity, it can be used as an effective antioxidant for ROS protection within the polar cytoplasm.     

Reactions of Low Valent Transition Metal Complexes with Hydrogen Peroxide. Are they “Fenton-Like” or not? 4. The Case of Fe(II)L,L = Edta; Hedta and Tcma

The question whether hydroxyl free radicals are formed in the reactions of divalent iron complexes Fe(II)L; L = edta; hedta; tcma (tcma = l-acetato-l,4,7-triazacyclononane) with hydrogen peroxide in neutral and slightly acidic solutions was studied by using the β elimination reaction as an assay for the formation of hydroxyl free radicals, OH. The results show that at pH<5.5 the iron(II)peroxide intermediate complex decomposes rapidly to yield free hydroxyl radicals for L=edta and hedta. This is in contrast to the mechanism of the corresponding Fe(II)nta peroxide complex, which probably decomposes to form Fe(IV)nta which then reacts with organic substrates to yield aliphatic free radicals. Thus, the non-participating ligand L has an appreciable effect on the mechanism of reaction of the metal center with hydrogen peroxide. Blank experiments using ionizing radiation as the source of CH₂CR(CH₃)OH, R = H or CH₃ radicals indicate that when L = tcma intermediates of the type LFe^III-CH₂CR(CH₃)OH_aq are formed, but their major mode of decomposition is not the β elimination reaction. Thus, the present assay for the formation of hydroxyl free radicals by the Fenton Reaction does not fit the latter system.     

Curcumin inhibits bTREK-1 K+ channels and stimulates cortisol secretion from adrenocortical cells

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K⁺ channels that set the resting membrane potential. Inhibition of these channels by adrenocorticotropic hormone (ACTH) is coupled to membrane depolarization and cortisol secretion. Curcumin, a phytochemical with medicinal properties extracted from the spice turmeric, was found to modulate both bTREK-1 K⁺ currents and cortisol secretion from AZF cells. In whole-cell patch clamp experiments, curcumin inhibited bTREK-1 with an IC₅₀ of 0.93 μM by a mechanism that was voltage-independent. bTREK-1 inhibition by curcumin occurred through interaction with an external binding site and was independent of ATP hydrolysis. Curcumin produced a concentration-dependent increase in cortisol secretion that persisted for up to 24 h. At a maximally effective concentration of 50 μM, curcumin increased secretion as much as 10-fold. These results demonstrate that curcumin potently inhibits bTREK-1 K⁺ channels and stimulates cortisol secretion from bovine AZF cells. The inhibition of bTREK-1 by curcumin may be linked to cortisol secretion through membrane depolarization. Since TREK-1 is widely expressed in a variety of cells, it is likely that some of the biological actions of curcumin, including its therapeutic effects, may be mediated through inhibition of these K⁺ channels.     

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H₂O or H₂S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5′-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of l-serine and l-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k_obs = 0.96 ± 0.02 and 0.38 ± 0.01 mm⁻¹ s⁻¹, respectively, at 24 °C) with concomitant loss of H₂O and H₂S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k_obs = 40.6 ± 3.8 s⁻¹ and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k_obs = 0.38 ± 0.01 mm⁻¹ s⁻¹. This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.     

The role of superoxide anion in peroxidase-catalyzed chemiluminescence of luminol

The chemiluminescence associated with peroxidation of luminol in buffered aqueous solution is a complex process involving several intermediates. It can be inhibited by removal of oxygen from the incubation medium. Superoxide radical is both an intermediate in this reaction and an essential component in light-producing steps. The importance of O₂^? in propagating this reaction was shown by the inhibition of luminescence by superoxide dismutase. A mechanism was proposed which is consistent with the data. It appears likely that the diverse biological effects of peroxidases are largely due to the reactivities of these intermediates and products.     

Iron Autoxidation in Mops and Hepes Buffers

Iron autoxidation in Mops and Hepes buffers is characterized by a lag phase that becomes shorter with increasing FeCl₂ concentration and pH. During iron oxidation in these buffers a yellow colour develops in the solution. When the reaction is conducted in the presence of nitro blue tetrazolium (NBT), blue formazan is formed. Of the many OH' scavengers tested, mannitol and sorbitol are most effective in inhibiting Fe²⁺ oxidation, yellow colour development and NBT reduction. Some inhibition was also noted with catalase. The iron product of the oxidative reaction differs from Fe³⁺ in its absorption spectrum and its low reactivity with thiocyanate. Similar results are obtained when iron autoxidation is studied in unbuffered solutions brought to alkaline pH with NaOH. In phosphate buffer, no lag phase is evident and the absorption spectrum of the final solution is identical to that of Fe³⁺ in this buffer. The iron product reacts immediately with thiocyanate. When iron oxidation is conducted in the presence of NBT the formation of formazan is almost undetectable. Of the many compounds tested only catalase inhibits iron autoxidation in this buffer. The sequence of reactions leading to iron autoxidation in Good-type buffers¹ thus resembles that occurring in unbuffered solutions brought to alkaline pH with NaOH and greatly differs from that occurring in phosphate buffer. These results are in agreement with the observation that these buffers have very low affinity for iron.¹ The data presented define experimental conditions where Fe²⁺ is substantially stable for a considerable length of time in Mops buffer.     

克罗烷二萜的昆虫拒食活性及构效关系研究

以饲料柱称重法测定25个克罗烷二萜新化合物对亚洲玉米螟Ostrinia furnacalis(Guenee)5龄幼虫的拒食活性,并进行构效关系分析。结果表明：立体效应对拒食活性意义 重大;C₉边链、C₁₈位的酯基对活性具有一定影响;C_{4处的螺环氧结构看来并非活性所必需。且在25μg/mL时选择法测定中,活性最高的化合物拒食率为57.9%,而500/μg/mL时非选择法测定中拒食率为43.2%。}     

The role of the reactions of NO with superoxide and oxygen in biological systems: A kinetic approach

In this study we calculate the half-life of ^·NO in its reactions with superoxide and with oxygen under various conditions using the known rate constants for these reactions. The measured half-life of ^·NO in biological systems is 3–5 s, which agrees well with the calculated value for intracellular ^·NO, but not for extracellular ^·NO under normal physiological conditions. The autoxidation of ^·NO to yield NO₂ as a final product cannot be responsible for such a short measured half-life under normal as well as pathologic conditions. Therefore, if there is direct evidence for the occurrence of the reaction of ^·NO with O₂ in the medium, one has to assume that the steady state concentrations of free ^·NO are much lower than those measured. The very low concentrations of free ^·NO in biological systems may result from its reversible strong binding to biological molecules. Simulation of the mechanism of the autoxidation of ^·NO indicates that the binding constants of ^·NO to O₂ or to another ^·NO are too small to account for the very low concentration of free ^·NO in biological systems. Nevertheless, the reaction of ^·NO with oxygen cannot be neglected in biological systems if the intermediate ONOO· reacts rapidly with a biological target. The biological damage caused by ONOO′ is expected to be due to the radical itself and to peroxynitrite, which is most probably formed via the reaction of ONOO· with the biological molecule.     

Effect of retinol deficiency and curcumin or turmeric feeding on brain Na+−K+ adenosine triphosphatase activity

The effect of retinol deficiency and curcumin and turmeric feeding on brain microsomal Na⁺-K⁺ ATPase activity was investigated. The brain Na⁺–K⁺ ATPase activity registered an increase of 148.5% as compared to the control group. Upon treating retinol deficient rats with curcumin or turmeric, the abnormally elevated activity showed a decrease of 36.9 and 47.1%, respectively, when compared to the retinol deficient group. An increase in V_max by 67% and K_m by 66% for ATP was observed in the retinol deficient group. Curcumin or turmeric fed retinol-deficient groups reduced the V_max by 25 and 33%, while K_m was reduced by 25 and 31%, respectively, compared to the retinol deficient group. Arrhenius plot of Na⁺–K⁺ ATPase showed a typical bi-phasic pattern in all the groups. Cholesterol: Phospholipid ratio showed a decrease in the retinol-deficient group by 67.8%, which showed a marked increase in curcumin or turmeric treated groups. Detergents could increase the Na⁺–K⁺ ATPase activity more in the control group than in the retinol deficient groups. Curcumin or turmeric improved the detergent action on the enzyme. Subsequent freezing and thawing over a period of 30 min decreased the enzyme activity by 22.8% in the retinol deficient group compared to 15.9% decrease in the control group. Curcumin or turmeric treated groups showed a decrease in the enzyme activity by 22.0 and 19.2%, respectively, when compared to the zero time in each group. In the presence of concanavalin-A (Con-A) there was only 52.4% stimulation in the enzyme activity in retinol deficient groups, compared to 108.0% in the control group. Curcumin or turmeric treated retinol-deficient groups showed a stimulation in the presence of con-A by 70 and 99.5%, respectively.     

Brain Mitochondria Catalyze the Oxidation of 7-(2-Aminoethyl)-3,4-Dihydro-5-Hydroxy-2H-1,4-Benzothiazine-3-Carboxylic Acid (DHBT-1) to Intermediates that Irreversibly Inhibit Complex I and Scavenge Glutathione: Potential Relevance to the Pathogenesis of Parkinson's Disease

Abstract: We have proposed that a very early step in the pathogenesis of idiopathic Parkinson's disease is the elevated translocation of l -cysteine into neuromelanin-pigmented dopaminergic neurons in the substantia nigra. This influx of l -cysteine was proposed to divert the normal neuromelanin pathway by scavenging dopamine-o-quinone, formed by autoxidation of cytoplasmic dopamine, to give initially 5-S-cysteinyldopamine, which is further oxidized to 7 - (2 - aminoethyl) - 3,4 - dihydro - 5 - hydroxy - 2H - 1,4 - benzothiazine-3-carboxylic acid (DHBT-1). In a recent report, it was demonstrated that DHBT-1 evokes inhibition of complex I respiration when incubated with intact rat brain mitochondria and a time-dependent irreversible inhibition of NADH-coenzyme Q₁ (CoQ₁) reductase when incubated with mitochondrial membranes. In this study, it is established that the time dependence of NADH-CoQ₁ reductase inhibition reflects the oxidation of DHBT-1, catalyzed by an unknown constituent of the inner mitochondrial membrane, to an o-quinone imine intermediate that rearranges to 7-(2-aminoethyl) - 5 - hydroxy - 1,4 - benzothiazine - 3 - carboxylic acid (BT-1) and decarboxylates to 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine (BT-2), which are further catalytically oxidized to o-quinone imine intermediates. The electrophilic o-quinone imine intermediates formed in these mitochondria-catalyzed oxidations of DHBT-1, BT-1, and BT-2 are proposed to bind covalently to key sulfhydryl residues at the complex I site, thus evoking irreversible inhibition of NADH-CoQ₁ reductase. Evidence for this mechanism derives from the fact that greater than equimolar concentrations of glutathione completely block inhibition of NADH-CoQ₁ reductase by DHBT-1, BT-1, and BT-2 by scavenging their electrophilic o-quinone imine metabolites to form glutathionyl conjugates. The results of this investigation may provide insights into the irreversible loss of glutathione and decreased mitochondrial complex I activity, which are both anatomically specific to the substantia nigra and exclusive to Parkinson's disease.