EPR study of the effect of formate on cytochrome C oxidase

The addition of formate to oxidized cytochrome $\text{c}$ oxidase (ferrocytochrome $\text{c}$: oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high spin heme signal at g = 6 and a splitting of g = 3 signal to g = 2.98 and 3.07. When formate-cytochrome $\text{c}$ oxidase is reduced, the g = 2.98 signal decreases significantly. The spectrophotometric studies showed that formate is a specific ligand to cytochrome $\text{a}$₃. Data suggest that binding of formate to oxidized cytochrome $\text{c}$ oxidase produces a ligand-$\text{a}$₃ interaction leading to the splitting of g = 3 signal hitherto considered as due to cytochrome $\text{a}$. Thus both cytochrome $\text{a}$ and $\text{a}$₃ contribute to the resonance of g = 3 signal of cytochrome $\text{c}$ oxidase.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

A cytochrome $\text{c}$ - cytochrome $\text{c}$ oxidase complex containing 0.8–1.0 moles of cytochrome $\text{c}$ per mole of cytochrome $\text{c}$ oxidase (heme a + a₃) was isolated as described by Ferguson-Miller, S., Brautigan, D.L., and Margoliash E., J. Biol. Chem. $\text{251}$, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 Å bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome $\text{c}$ was cross-linked to subunit II of cytochrome $\text{c}$ oxidase. Other cross-linked products were formed involving different subunits of cytochrome $\text{c}$ oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. Experiments are also described using N,N′-bis(3-succinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 Å bridging bifunctional reagent was a pair containing II+VI.     

A new photoaffinity-labeled derivative of mitochondrial cytochrome c

Three lysine residues of horse heart cytochrome $\text{c}$ were modified by reaction with methyl-4-mercaptobutyrimidate hydrochloride and the free SH group of the latter was covalently linked to p-azidophenacyl bromide yielding a photoaffinity-labeled cytochrome $\text{c}$. The photoaffinity-labeled cytochrome $\text{c}$ was bound by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase.     

Effects of anti-NADPH-cytochrome c reductase and anti-cytochrome b5 antibodies on the hepatic and pulmonary microsomal metabolism and covalent binding of the pulmonary toxin 4-ipomeanol.

An antibody prepared against purified rat liver NADPH-cytochrome $\text{c}$ reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome $\text{c}$ reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b₅, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome $\text{c}$ reductases, and was inactive against the respective NADPH-dependent cytochrome $\text{c}$ reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b₅ is rate-limiting in lung microsomes.     

Crude oil effects on microsomal mixed-function oxidase system components in the striped mullet (Mugil cephalus)

The effects of exposure to two types of crude oil on microsomal mixed-function oxidase system components in livers of juvenile striped mullet ($\text{Mugil cephalus}$) were investigated. Mullet were exposed for 4 days to emulsified Empire Mix or Saudi Arabian crude oils at an initial concentration of 75 ppm and an average of 1 ppm in the water column. Liver size was increased by about 50% following exposure to both oils. Since neither total hepatic protein nor microsomal protein increased as rapidly as did liver size, the concentrations of both were reduced following oil exposures. The proportion of microsomal protein to total hepatic protein or wet weight was not altered following crude oil exposure. Both cytochromes P-450 and $\text{b}$₅ were induced following oil treatment. NADPH-dependent enzymes assayed with cytochrome $\text{c}$ and dichlorophenolindophenol as substrates showed increases in activity after exposure to Empire Mix crude oil but only the latter enzyme activity was increased on a microsomal protein basis following Saudi Arabian crude oil treatment. Activities of NADH cytochrome $\text{c}$ and NADH cytochrome $\text{b}$₅ reductases appeared to vary with the protein level. However, since liver size was increased, oil-treated mullet had more of all parameters measured than did control mullet. Although the acute toxicity of Saudi Arabian crude oil to mullet is greater than that of Empire Mix crude oil, Empire Mix crude oil had greater inductive effects on microsomal oxidase components.     

Bound cytochrome C as proton donor and acceptor during enzymic oxidoreduction

When ferrocytochrome $\text{c}$ reacts with delipidated cytochrome oxidase under conditions which prevent oxidation, one proton is taken up per molecule of ferrocytochrome $\text{c}$ bound to cytochrome oxidase. When ferricytochrome $\text{c}$ reacts with delipidated Complex III, one proton is released per molecule of ferricytochrome $\text{c}$ bound to Complex III. From these data it can be concluded that the oxidation of ferrocytochrome $\text{c}$ by cytochrome oxidase leads to the release of a proton and an electron, whereas the reduction of ferricytochrome $\text{c}$ by Complex III leads to the uptake of a proton and an electron. Thus ferrocytochrome $\text{c}$ like QH₂ and NADH is both an electron and proton donor, and ferricytochrome $\text{c}$ like Q and O₂ is both an electron and proton acceptor. The pattern for the three mitochondrial electron transfer sequences NADH → Q, QH₂ → ferricytochrome $\text{c}$ and ferrocytochrome $\text{c}$ → O₂ involves separation of an electron and proton on the side of the membrane where electron transfer is initiated and recombination of an electron and a proton in the terminal acceptor on the side of the membrane where electron transfer terminates.     

Properties of rat liver mitochondria with intermembrane Cytochrome c.

When rat liver mitochondria were suspended in 0.15 m KCl, the cytochrome c appeared to be solubilized from the binding site on the outside of the inner membrane and trapped in the intermembrane space. When the outer membrane of these mitochondria was disrupted with digitonin at a digitonin concentration of 0.15 mg/mg of protein, the solubilized cytochrome c could be released from mitochondria along with adenylate kinase. When mitochondria were suspended in 0.15 m KCl instead of 0.33 m sucrose, the $\text{ADP}\text{O}$ ratio observed with succinate, β-hydroxybutyrate, malate + pyruvate or glutamate as substrates was little affected. A number of cycles of State 4-State 3-State 4 with ADP was observed. The respiratory control ratios, however, were decreased, particularly when glutamate was used as the substrate. Cytochrome c oxidase activity was also decreased to 55% when assayed using ascorbate + N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD) as substrates. Suspension of mitochondria in 0.15 m KCl resulted in an enhancement of the very low NADH oxidation by intact mitochondria and a twofold enhancement of sulfite oxidation. Trapped cytochrome c in outer membrane vesicles prepared from untreated and trypsin-treated intact mitochondria was found to be readily reduced by NADH and suggests that some cytochrome b₅ is located on the inner surface of the outer membrane. The enhanced NADH oxidase could therefore reflect the ability of cytochrome c to mediate intermembrane electron transport. The enhanced sulfite oxidase activity was sensitive to cyanide inhibition and coupled to oxidative phosphorylation ($\text{ADP}\text{O}\text{< 1}$) unlike the activity of mitochondria in sucrose medium. These results suggest that free cytochrome c in the intermembrane space can mediate electron transfer between the sulfite oxidase and the inner membrane.     

Cobalt cytochrome c: enzymic assays.

An improved synthesis for cobalt-cytochrome $\text{c}$ has been developed; its half reduction potential is ?140 ± 20mV. Reduced ^Cocyt-$\text{c}$³ is oxidized by bovine heart cytochrome $\text{c}$ oxidase at a rate ～45% that of the native cytochrome $\text{c}$. It is not reduced by mitochondrial NADH or succinate cytochrome $\text{c}$ reductase nor by microsomal NADH or NADPH cytochrome $\text{c}$ reductase.     

The cytochrome c binding site on cytochrome c oxidase.

Cytochrome $\text{c}$ was chemically coupled to cytochrome $\text{c}$ oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome $\text{c}$ binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome $\text{c}$ reaction with oxidase, binds to the same oxidase subunit as does cytochrome $\text{c}$, subunit IV in the gel system used.     

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome $\text{c}$ oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome $\text{c}$, the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome $\text{c}$ oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome $\text{c}$ oxidase. There are, however, definite differences between spectra of solubilized cytochrome $\text{c}$ oxidase and electron transport particles in the oxidized states.     

The effect of enzyme induction on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes

The effect of pretreatment with phenobarbitone, rifampicin, β-naphthoflavone, antipyrine and spironolactone on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes has been examined and the corresponding changes in microsomal P-450 content and cytochrome $\text{c}$ reductase activity measured. Rifampicin produced the greatest increase (220%) in irreversible binding while phenobarbitone produced the greatest increase in both microsomal P-450 content (172%) and cytochrome $\text{c}$ reductase activity (210%). There was no correlation of irreversible binding with either microsomal P-450 content or with cytochrome $\text{c}$ reductase activity.     

Binding of cytochrome c to cytochrome c-oxidase in intact mitochondria. A study with radioactive photoaffinity-labeled cytochrome c

[³H]-p-Azidophenacylbromide-(methyl-4-mercaptobutyrimidate)-cytochrome $\text{c}$ from $\text{Saccharomyces cerevisiae}$ was prepared and its properties determined. The radioactive photoaffinity-labeled cytochrome $\text{c}$ was linked by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase. Analysis of the complex on SDS-polyacrylamide gels showed that cytochrome $\text{c}$ bound to one of the smaller subunits of cytochrome $\text{c}$ oxidase with an apparent molecular weight of 15,000.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Effect of thyroid hormone administration on synthesis and degradation of cytochrome c in rat liver.

Thyrotoxicosis can induce increases in the concentrations of the cytochromes of the inner mitochondrial membrane in rat liver. The purpose of this study was to determine whether the increase in hepatic cytochrome c concentration in thyrotoxic rats is maintained by an increase in the rate of synthesis, a decrease in the rate of degradation, or a combination of the two. The turnover of cytochrome c labeled with δ-amino [¹⁴C]levulinate was measured in the livers of thyrotoxic rats that were in steady state with respect to liver cytochrome c concentration, liver weight, and body weight. Cytochrome c concentration was increased 3.4-fold in the livers of the thyrotoxic animals. The $\text{t}\text{1}\text{2}$ of liver cytochrome c was 3.7 days in the thyrotoxic and 5.7 days in euthyroid animals. It was calculated that the 3.4-fold increase in cytochrome c concentration was maintained, in the face of a 63% increase in k_d, by a 5.5-fold increase in synthesis rate.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

The distribution of copper in neonatal mottled mutant mice after exposure to copper and penicillamine

Tissue copper levels of brindled ($\text{Mo}$^br) mice and normal litter-mates after single and repeated dosing with CuCl₂ and/or D-penicillamine are examined, together with a study of the cytosol distribution of copper after CuCl₂ treatment. The results confirm that the mutant mouse kidney is capable of extensive copper accumulation in association with the low MW copper-binding protein. Deficient tissues such as brain, heart and spleen are able to sequester sufficient of the exogenous copper to raise their levels to the normal control level, whereas mutant liver levels, even after copper treatment, remain below normal, indicating that the $\text{Mo}$ gene affects the ability of the liver to retain copper.     

Cold lability and dissociation of the F1-ATPase from Bacillus stearothermophilus

Effects of inadequate vitamin E (E) and/or selenium (Se) nutrition on the activities of cytochrome P-450 mixed function oxidase system (heme hydroperoxidase, p-nitroanisole O-demethylase), and epoxide hydrolase have been investigated. Heme hydroperoxidase activity of liver and lung microsomes was significantly decreased in E deficiency. In the liver, Se deficiency resulted in a significant increase in hydroperoxidase activity. In contrast to the peroxidase activity, liver demethylase activity was only marginally affected in $\text{E}\text{Se}$ deficiency states. However, kidney demethylase activity was increased two fold in Se deficient states. Liver microsomal epoxide hydrolase activity was significantly increased in both E and Se deficiency states.     

Selectivity of oxidase and reductase activity of horse heart cytochrome c

The ascorbate-TMPD-cytochrome $\text{c}$ oxidase and succinate cytochrome $\text{c}$ reductase activities and the redox potentials of native and chemically modified cytochromes $\text{c}\text{—NBS-cytochrome}\text{c}$ with modification of Trp-59 and Met-65, nitro-cytochrome $\text{c}$ with modification of Tyr-67, and a new preparation, Chloramine-T-cytochrome m$\text{c}$ with modification of Met-80 and -65 to methionine sulfoxide—have been compared at pH 7.8 in 25 mM cacodylate-Tris buffer. These modifications exhibit (i) a slight lowering of redox potential, from 260 mV to 180, 215 and 170 mV, respectively, (ii) destabilization of the cytochrome $\text{c}$-reductase complex, 6 to 12 fold, but without alteration of the cytochrome $\text{c}$-oxidase complex, and (iii) a slight lowering of the maximum velocity for both the oxidase and reductase reactions. The selective destabilization of the cytochrome $\text{c}$-reductase complex is interpreted as an indication of a two-path, two-function model for the oxido-reduction function of cytochrome $\text{c}$.     

Charge distribution in electron transport components: cytochrome c and cytochrome c oxidase mixtures

Mixtures of cytochrome $\text{c}$ oxidase and cytochrome $\text{c}$ have been titrated by coulometrically generated reductant, methyl viologen radical cation, and physiological oxidant, O₂. Charge distribution among the heme components in mixtures of these two redox enzymes has been evaluated by monitoring the absorbance changes at 605 and 550 nm. Differences in the pathway of the electron transfer process during a reduction cycle as compared to an oxidation cycle are indicated by variations found in the absorbance behavior of the heme components during successive reductive and oxidative titrations. It is apparent that the potential of the cytochrome $\text{a}$ heme is dependent upon whether oxidation or reduction is occurring.