Liviu Clejan

Synergistic interactions between NADPH-cytochrome P-450 reductase, paraquat, and iron in the generation of active oxygen radicals

The toxicity associated with paraquat is believed to involve the generation of active oxygen radicals and the production of oxidative stress. Paraquat can be reduced by NADPH-cytochrome P-450 reductase to the paraquat radical; this results in consumption of NADPH. A variety of ferric complexes, including ferric-ATP, -citrate, -EDTA, ferric diethylenetriamine pentaacetic acid and ferric ammonium sulfate, produced a synergistic increase in the paraquat-mediated oxidation of NADPH. This synergism could be observed with very low concentrations of iron, e.g. 0.25 microM ferric-ATP. Very low rates of hydroxyl radical were generated by the reductase with paraquat alone, or with ferric-citrate or -ATP or ferric ammonium sulfate in the absence of paraquat; however, synergistic increases in the rate of hydroxyl radical generation occurred when these ferric complexes were added together with paraquat. Ferric-EDTA and -DTPA catalyzed some production of hydroxyl radicals, which was also synergistically elevated in the presence of paraquat. Ferric desferrioxamine was essentially inert in the absence or presence of paraquat. This enhancement of hydroxyl radical generation was sensitive to catalase and competitive scavengers but not to superoxide dismutase. The interaction of paraquat with NADPH-cytochrome P-450 reductase and ferric complexes resulted in an increase in oxygen radical generation, and various ferric complexes increased the catalytic effectiveness and potentiated significantly the toxicity of paraquat via this synergistic increase in oxygen radical generation by the reductase.

Oxidation of glycerol to formaldehyde by rat liver microsomes

Rat liver microsomes catalyzed the oxidation of glycerol to a Nash-reactive material in a time- and protein-dependent manner. Omission of the glycerol or the microsomes or any of the components of the NADPH-generating system resulted in almost a complete loss of product formation. Apparent Km and Vmax values for glycerol oxidation were about 18 mM and 2.5 nmol formaldehyde per min per mg microsomal protein. Carbon monoxide inhibited glycerol oxidation indicating a requirement for cytochrome P-450. That the Nash-reactive material was formaldehyde was validated by a glutathione-dependent formaldehyde dehydrogenase positive reaction. These studies indicate that glycerol is not inert when utilized with microsomes or reconstituted mixed function oxidase systems, and that the production of formaldehyde from glycerol may interfere with assays of other substrates which generate formaldehyde as product.

The binding of dicyclohexylcarbodiimide to cytochrome b of complex III isolated from yeast mitochondria

The cytochrome b-cl complex (complex III) of the mitochondrial respiratory chain catalyzes electron transport, coupled to ATP synthesis and ion transport, from coenzyme Q to cytochrome c. This span of the respiratory chain has been shown to eject protons with an observed stoichiometry of H+/2 eapproaching 4 when studied in mitochondria in which other proton-conducting pathways of the inner membrane are selectively inhibited [l-3]. Moreover, similar stoichiometries have been observed when purified cytochrome b-c, complexes from either heart [4,5] or yeast [6] are reconstituted into proteoliposomes. Two different mechanistic models have been proposed to explain proton ejection in mitochondria. Mitchell’s Q cycle [7,8] postulates that proton ejection occurs by a ligand conduction mechanism in which coenzyme Q is the only proton carrier. Papa, however, originally suggested that some unidentified polypeptide of the cytochrome b-c1 complex might undergo a protonation-deprotonation cycle coupled to the oxidation-reduction reaction, such that protons were translocated across the membrane [9]. Subsequently, the cytochrome b dimer was proposed as the proton pump based on the observation that its redox potential displayed a pH dependence in the physiological range [lo]. in the yeast cytochrome b-cl complex reconstituted into liposomes [6]. The electrogenic ejection of protons was blocked in the DCCDtreated b-cl complex as well as reversed electron flow from cytochrome b to coenzyme Q, driven by a K+-diffusion potential. Similar inhibitions of proton translocation by DCCD at site 2 of the respiratory chain have also been reported in intact mitochondria isolated from both beef heart [12] and rat liver [ 131. It was thus of some interest to investigate the possibility that the inhibition of proton translocation by DCCD in the b-cl complex involves a specific covalent linking of DCCD to the enzyme as has been shown for H+-translocation ATPases [14-171, cytochrome c oxidase [18,19] and other proton-translocating proteins [20]. The results obtained indicate that DCCD binds selectively to cytochrome b in complex III, suggesting that this protein is involved in proton translocation at site 2 of the respiratory chain.

Role of iron, hydrogen peroxide and reactive oxygen species in microsomal oxidation of glycerol to formaldehyde

Rat liver microsomes can oxidize glycerol to formaldehyde. This oxidation is sensitive to catalase and glutathione plus glutathione peroxidase, suggesting a requirement for H2O2 in the overall pathway of glycerol oxidation. Hydrogen peroxide can not replace NADPH in supporting glycerol oxidation; however, added H2O2 increased the NADPH-dependent rate. Ferric chloride or ferric-ATP had no effect on glycerol oxidation, whereas ferric-EDTA was inhibitory. Certain iron chelators such as desferrioxamine, EDTA or diethylenetriaminepentaacetic acid, but not others such as ADP or citrate, inhibited glycerol oxidation. The inhibition by desferrioxamine could be overcome by added iron. Neither superoxide dismutase nor hydroxyl radical scavengers had any effect on glycerol oxidation. With the exception of propyl gallate, several antioxidants which inhibit lipid peroxidation had no effect on formaldehyde production from glycerol. The inhibition by propyl gallate could be overcome by added iron. In contrast to glycerol, formaldehyde production from dimethylnitrosamine was not sensitive to catalase or iron chelators, thus disassociating the overall pathway of glycerol oxidation from typical mixed-function oxidase activity of cytochrome P450. These studies indicate that H2O2 and nonheme iron are required for glycerol oxidation to formaldehyde. The responsible oxidant is not superoxide, H2O2, or hydroxyl radical. Cytochrome P450 may function to generate the H2O2 and reduce the nonheme iron. There may be additional roles for P450 since rates of formaldehyde production by microsomes exceed rates found with model chemical systems. Elevated rates of H2O2 production by certain P450 isozymes, e.g., P450 IIE1, may contribute to enhanced rates of glycerol oxidation.

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1.

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH‐cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti‐pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non‐heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole‐treated, but not phenobarbital‐treated rats, glycerol oxidation was inhibited by anti‐pyrazole P450 IgG, anti‐hamster ethanol‐induced P450 IgG, and monoclonal antibody to ethanol‐induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti‐rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.—Clejan, L. A.; Cederbaum, A. I. Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes. FASEB J. 6: 765‐770; 1992.

Structural determinants for alcohol substrates to be oxidized to formaldehyde by rat liver microsomes

Glycerol can be oxidized to formaldehyde by rat liver microsomes and by cytochrome P450. The ability of other alcohols to be oxidized to formaldehyde was determined to evaluate the structural determinants of the alcohol which eventually lead to this production of formaldehyde. Monohydroxylated alcohols such as 1- or 2-propanol did not produce formaldehyde when incubated with NADPH and microsomes. Geminal diols such as 1,3-propanediol, 1,3-butanediol, or 1,4-butanediol also did not yield formaldehyde. However, vicinal diols such as 1,2-propanediol or 1,2-butanediol produced formaldehyde. With 1,2-propanediol, the residual two-carbon fragment was found to be acetaldehyde, while with 1,2-butanediol, the residual three-carbon fragment was propionaldehyde. Oxidation of 1,2-propanediol to formaldehyde plus acetaldehyde involved interaction with an oxidant derived from H2O2 plus nonheme iron, since production of the two aldehydic products was completely prevented by catalase or glutathione plus glutathione peroxidase and by chelators such as desferrioxamine or EDTA. The oxidant was not superoxide or hydroxyl radical. Product formation was fivefold lower when NADH replaced NADPH, and was inhibited by substrates, ligands, and inhibitors of cytochrome P450. A charged glycol such as alpha-glycerophosphate (but not the geminal beta-glycerophosphate) was readily oxidized to formaldehyde, suggesting that interaction of the glycol with the oxidant was occurring in solution and not in a hydrophobic environment. These results indicate that the carbon-carbon bond between 1,2-glycols can be cleaved by an oxidant derived from microsomal generated H2O2 and reduction of non-heme iron, with the subsequent production of formaldehyde plus an aldehyde with one less carbon than the initial glycol substrate.

[17] Preparation of complex III from yeast mitochondria and related methodology

Publisher Summary This chapter outlines the procedures for purification of complex III from yeast mitochondria, for biochemical characterization of the soluble complex, and for the study of the bioenergetic properties of the complex reconstituted into proteoliposomes. Complex III, also called the cytochrome bc 1 complex, is a multiprotein enzyme complex that catalyzes ubiquinol-cytochrome- c oxidoreductase activity with a concomitant unidirectional and electrogenic movement of protons across the inner mitochondrial membrane. The procedures for isolation and purification of complex III from beef heart mitochondria are based on the extraction of the complex from the inner membrane in the presence of bile salts and subsequent fractionation with ammonium sulfate. These procedures for the isolation and purification of complex III from various species procedures include the use of nonionic detergents such as Triton X-100 for the extraction of the complex from the membrane and chromatography on hydroxyapatite columns, immobilized cytochrome c -Sepharose 4B columns, or cytochrome c -thiol-activated Sepharose columns for fractionation of the complex.

Effect of Temperature on Protein Synthesis and Leucine Transport by Yeast Mitochondria

Protein synthesis in yeast mitochondria shows biphasic Arrhenius plots both in vivo and in vitro, with a twofold increase in the activation energy below the transition temperature suggesting a functional association between mitochondrial protein synthesis and the inner membrane. Analysis by gel electrophoresis of mitochondrial translation products labeled in vivo showed that the same proteins are synthesized and then inserted into the membrane above and below the transition temperature of the membrane. The rate of leucine uptake into mitochondria was decreased at least fivefold in the presence of chloramphenicol, suggesting that leucine is used mainly for protein synthesis. In the absence of chloramphenicol, the rate of leucine uptake was always slightly higher but comparable to the incorporation rate of leucine into protein at all temperatures studied, suggesting that the transport of leucine into mitochondria is not rate-limiting for protein synthesis. The ionophore valinomycin or the uncoupler carbonyl phenylhydrazone (CCCP) inhibited 75-80% of the leucine uptake in the presence of chloramphenicol. In addition, the omission of respiratory chain substrates and the ATP-regenerating system led to a 93% inhibition of uptake, suggesting that leucine uptake may occur by an active transport mechanism.