Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum--mitochondrion complex in rat liver

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum--mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum--mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.

Biosynthesis of cytoplasmic subunits of cytochrome C oxidase in rat liver

The biosynthesis of the cytoplasmic subunits of cytochrome oxidase from rat liver has been studied in vitro by translating liver poly (A)-containing RNA in the wheat germ cell-free system and immunoprecipitating the products with anti-cytochrome oxidase antibody. Analysis of the labelled immunoprecipitate on SDS-gels does not reveal the presence of a polyprotein precursor. On the other hand discrete products which are either slightly bigger or closely similar to the mature subunits present in purified cytochrome oxidase have been detected.

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.

Construction of a cDNA clone for a nuclear-coded subunit of cytochrome c oxidase from rat liver

A cDNA library for 6S–9S poly(A)-containing RNA from rat liver was constructed in Image . Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3′-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.

Reversible electrochemistry of cytochrome c on recompressed, binderless exfoliated graphite electrodes

The direct electrochemistry of cytochrome c (cyt-c) has been investigated on exfoliated graphite (EG) electrodes. The as-polished and roughened (using SiC emery sheet) EG surfaces are inactive for the direct electron transfer. However, when the EG electrode was sonicated before the experiment, a pair of redox waves were obtained for freely diffusing cyt-c in the solution phase. The formal potential was found to be 0.01 V (vs. SCE) in 0.1 M phosphate buffer at a pH of 7.1. The electrochemical response for the adsorbed cyt-c on sonicated EG electrodes, which is shown to have carbonyl functional groups on its surface, shows nearly reversible voltammograms in the same electrolyte. However, the formal potential in the adsorbed state is more negative than that observed for the solution phase cyt-c. A structure based on an open heme conformation proposed by Hildebrandt and Stockburger is probably present on the EG surface. It is suggested that the electrochemistry at the EG electrode is essentially governed by favourable electrostatic interactions.

Brown adipose tissue mitochondria are cytochrome c — subsaturated

The oxidative activity of mitochondria freshly isolated from brown adipose tissue of rats was stimulated two-fold on the addition of small concentrations of exogenous cytochrome c to the reaction medium. Loss of membrane-bound cytochrome c did not occur during isolation of mitochondria. Estimation of the high-affinity binding sites on the organelle membrane indicated that less than a third of these sites remained saturated with cytochrome c. The pigment is thus shown to be a functionally limiting electron transport component in brown adipose tissue.

Functions Of Cytochrome-C In Regulation Of Electron-Transfer And Protein Folding

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.

Effect of beta-lactamase-catalyzed hydrolysis of cephalosporins on peroxynitrite-mediated nitration of serum albumin and cytochrome c

The hydrolysis of beta-lactam antibiotics by beta-lactamases is one of the major bacterial defense systems. These enzymes generally hydrolyze a variety of antibiotics including the latest generation of cephalosporins, cephamycins and imipenem. In this paper, the effect of cephalosporins-based antibiotics on the peroxynitrite-mediated nitration of protein tyrosine is described. Although some of the antibiotics have weak inhibitory effect on the nitration reactions in the absence of beta-lactamase, they exhibit very strong inhibition in the presence of beta-lactamase. This is due to the elimination of heterocyclic thiol/thione moieties from cephalosporins by beta-lactamase-mediated hydrolysis. After the elimination, the thiols/thiones effectively scavenge peroxynitrite, leading to the inhibition of the nitration reactions.

Inhibition of peroxidase-catalyzed protein tyrosine nitration by antithyroid drugs and their analogues

In this paper, we describe the effect of some commonly used thiourea-based antithyroid drugs and their analogues on the peroxidase-catalyzed nitration reactions. The nitration of bovine serum albumin (BSA) and cytochrome c was studied using the antibody against 3-nitro-L-tyrosine. This study reveals that the thione-based antithyroid drugs effectively inhibit lactoperoxidase (LPO)-catalyzed nitration of BSA. These compounds show very weak inhibition towards the nitration of cytochrome c. Some of these compounds also inhibit myeloperoxidase (MPO)-catalyzed nitration of L-tyrosine. A structure-activity correlation study on the peroxidase-catalyzed nitration of L-tyrosine reveals that the presence of thione/selone moiety is important for the inhibition. Although the presence of a free N-H group adjacent to C=S moiety is necessary for most of the thiones to inhibit the LPO-catalyzed nitration, the corresponding selones do not require the presence of any free N-H group for their activity. Furthermore, experiments with different concentrations of H2O2 suggest that the antithyroid drugs and related thiones inhibit the nitration reaction mainly by coordinating to the Fe(III)-center of the enzyme active site as previously proposed for the inhibition of peroxidase-catalyzed iodination. On the other hand, the selenium compounds inhibit the nitration by scavenging H2O2 without interacting with the enzyme active site. This assumption is based on the observations that catalase effectively inhibits tyrosine nitration by scavenging H2O2, which is one of the substrates for the nitration. In contrast, superoxide dismutase (SOD) does not alter the nitration reactions, indicating the absence of superoxide radical anion (O-2 center dot(-)) during the peroxidase-catalyzed nitration reactions. (C) 2010 Elsevier B.V. All rights reserved.

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish peroxidase and H2O2 and decreased the ratio of scopoletin (consumed):H2O2 (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH: O-2 of 2:1. On step-wise addition of a small concentration of H2O2 a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H2O2 ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound;d accumulated during oxidation of NADH. The effectiveness of a small concentration of H2O2 in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H2O2 during scopolerin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b 5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Electron transfer amongst flavo- and hemo-proteins: diffusible species effect the relay processes, not protein-protein binding

Hitherto, electron transfer (ET) between redox proteins has been deemed to occur via donor-acceptor binding, and diffusible reactive species are considered as deleterious side-products in such systems. Herein, ET from cytochrome P450 reductase (CPR, an animal membrane flavoprotein) and horseradish peroxidase (HRP, a plant hemoprotein) to cytochrome c (Cyt c, a soluble animal hemoprotein) was probed under diverse conditions, using standard assays. ET in the CPR-Cyt c system was critically inhibited by cyanide and sub-equivalent levels of polar one-electron cyclers like copper ions, vitamin C/Trolox and superoxide dismutase. In the presence of lipids, inhibition was also afforded by amphipathic molecules vitamin E, palmitoyl-vitamin C and the membrane hemoprotein, cytochrome b(5). Such nonspecific inhibition (by diverse agents in both aqueous and lipid phases) indicated that electron transfer/relay was effected by small diffusible agents, whose lifetimes are shortened by the diverse radical scavengers. When CPR was retained in a dialysis membrane and Cyt c presented outside in free solution, ET was still observed. Further, HRP (taken at nM levels) catalyzed oxidation of a phenolic substrate was significantly inhibited upon the incorporation of sub-nM levels of Cyt c. The findings imply that CPR-Cyt c or HRP-Cyt c binding is not crucial for ET. Further, fundamental quantitative arguments (based on diffusion/collision) challenge the erstwhile protein-protein binding-assisted ET hypothesis. It is proven beyond reasonable doubt that mobile and diffusible electron carriers (ions and radicals) serve as ``redox-relay agents'' in the biological ET models/setup studied.