Functional Cytochrome Research Articles

Characterization of two Drosophila melanogaster cytochrome c genes and their transcripts.

Analysis of total Drosophila melanogaster DNA by genomic blot hybridization indicates that two cytochrome c-like sequences exist in the Drosophila genome. These two sequences, DC3 and DC4, have been isolated from a Charon 4A-D. melanogaster genomic library. DC3 and DC4 are located within a 4 kb region of DNA, at position 36A 10-11, on the left arm of chromosome 2. The nucleotide sequence of these two clones has been determined. Both DC3 and DC4 can encode functional cytochrome c proteins. The polypeptide sequences predicted by these two genes, however, differ at 32 amino acid residues. DC4 is expressed at varying, but relatively high levels throughout Drosophila development. In contrast, DC3 is expressed at constant, but relatively low levels throughout development.

Simultaneous presence of two terminal oxidases in the respiratory system of dark aerobically grown Rhodospirillum rubrum

Rhodospirillum rubrum CAF10, a spontaneous cytochrome oxidase defective mutant, was isolated from strain S1 and used to analyze the aerobic respiratory system of this bacterium. In spite of its lack of cytochrome oxidase activity, strain CAF10 grew aerobically in the dark although at a decreased rate and with a reduced final yield. Furthermore, aerobically grown mutant cells took up O2 at high rates and membranes isolated from those cells exhibited levels of NADH and succinate oxidase activities which were similar to those of wild type membranes. It was observed also that whereas in both strains O2 uptake (intact cells) and NADH and succinate oxidase activities (isolated membranes) were not affected by 0.2 mM KCN, the cytochrome oxidase activity of the wild type strain was inhibited about 90% by 0.2 mM KCN. These data indicate the simultaneous presence of two terminal oxidases in the respiratory system of R. rubrum, a cytochrome oxidase and an alternate oxidase, and suggest that the rate of respiratory electron transfer is not limited at the level of the terminal oxidases. It was also found that the aerobic oxidation of cellular cytochrome c 2 required the presence of a functional cytochrome oxidase activity. Therefore it seems that this electron carrier, which only had been shown to participate in photosynthetic electron transfer, is also a constituent of the respiratory cytochrome oxidase pathway.

Energetic aspects of growth of Paracoccus denitrificans: oxygen-limitation and shift from anaerobic nitrate-limination to aerobic succinate-limitation

1. Growth yields and efficiency of energy conservation were the same for aerobic succinate-limited and oxygen-limited cells of Paracoccus denitrificans. 2. A shift from anaerobic nitrate-limitation to aerobic succinatelimitation showed that before and after the shift cells grew with the same capacity of energy conservation. 3. Respiration-driven proton translocation showed the presence of H+-translocating sites 1 and 2, which translocate respectively 2–3 and 4 protons per 2 electrons in oxygen-, anaerobic nitrate-and aerobic succinate-limited cells. 4. Cytochrome spectra and flash-photolysis spectra of oxygen- and nitrate-limited cells gave evidence for the presence of an alternative oxidase, cytochrome a1, never before recognized in Paracoccus denitrificans. 5. Only a-type cytochromes liganded with CO could be flash-photolysed. No evidence for a functional cytochrome o was found in photolysis experiments. 6. Fast oxidation, before photolysis, of the bc-pool after introduction of oxygen in a CO-liganded sample at-20° to-30° C, indicated the presence of a cytochrome oxidase other than cytochrome a1 with a very high affinity for oxygen and a low affinity for CO. 7. In photochemical action spectra, light released CO-inhibition of respiration, but the release was independent of the wavelength used (560–610 nm).

Membrane-bound respiratory chain of Pseudomonas aeruginosa grown aerobically. A KCN-insensitive alternate oxidase chain and its energetics.

There was found to be a KCN-insensitive, alternate oxidase chain branching from the ordinary oxidase chain in the respiratory chain of Pseudomonas aeruginosa grown aerobically. The alternate oxidase activity was highly resistant to KCN, and had a lower affinity for oxygen than ordinary cytochrome oxidase did. The branching point of the alternate oxidase chain from the ordinary oxidase chain was shown to be localized behind cytochrome b. The KCN-insensitive alternate oxidase chain was inhibited slightly with antimycin A and intensively with 2-thenoyltrifluoroacetone. The former inhibited the respiration behind cytochrome b and the latter before cytochrome b. N,N,N',N'-Tetramethyl-p-phenylenediamine oxidase-negative mutant (T105) was prepared from P. aeruginosa. The mutant clearly lacked a functional ordinary cytochrome oxidase, but had the KCN-insensitive alternate oxidase chain and could grow aerobically. The KCN-insensitive alternate oxidase chain had a H+/O ratio of 4, suggesting the existence of two energy-coupling sites in the chain. Under the conditions where both ordinary oxidase and alternate oxidase chains were functioning, the H+/O ratio of the parent strain was 5.6. From these data, we also discuss the energetics of the ordinary oxidase chain in the respiratory chain of aerobic P. aeruginosa.

Adaptation of terminal respiration pathways of Tetrahymena pyriformis ST to growth with added iron or carbonyl cyanide m-chlorophenylhydrazone

1. 1. Tetrahymena pyriformis grown in medium which contained 0.6 μg/ml iron possesses at least three pathways of terminal respiration which are inhibited by cyanide, azide and salicythydroxamic acid respectively. 2. 2. Mid-exponential phase cultures grown in the presence of 5.6 μg/ml iron and glucose showed the same respiratory properties in terms of respiration rate and inhibitor sensitivity as cultures grown in the absence of added iron and glucose. 3. 3. The respiration of mid-exponential phase cultures adapted to growth in the presence of 2.5 μ m-carbonyl cyanide m-chlorophenylhydrazone was incompletely inhibited by a combination of cyanide, azide and salicylhydroxamic acid. 4. 4. The residual respiration accounted for between 25 and 35% of the total, indicating synthesis of a novel terminal oxidase or gross modification of existing terminal respiratory pathways in the presence of the uncoupler of energy conservation. 5. 5. The sole functional cytochrome oxidase present in organisms grown in the absence or presence of added iron and glucose or after adaptation to uncoupler was cytochrome a 620; no other CO-reacting haemoprotein was detected in photochemical action spectra.

Biochemical characterization of the OXI mutants of the yeast Saccharomyces cerevisiae

OXI mutants in Saccharomyces cerevisiae lack a functional cytochrome c oxidase. Wild type and OXI mutants were grown in the presence of radioactive δ-amino[ 14C]levulinic acid, a precursor of porphyrin and heme, and [ 3H]mevalonic acid, a precursor of the alkyl side-chain of heme a. SDS polyacrylamide gel electrophoresis of the delipidated mitochondria showed that δ-amino[ 14C]levulinic acid was distributed into three bands migrating in the regions of M r 28 000, 13 500, and 10 000, while [ 3H]mevalonic acid was found in a single band with apparent M r of 10 000. The immunoprecipitates obtained by incubating the solubilized mitochondria of any OXI mutant with antibodies against cytochrome c oxidase, showed, after delipidation, a high specific radioactivity due to δ-amino[ 14C]levulinic acid and [ 3H]mevalonic acid. This suggested that a prophyrin a was present in all these OXI mutants. HCl fractionation confirmed the presence of porphyrin a in the apooxidase of these mutants. Atomic absorption spectra of the immunoprecipitate of cytochrome c oxidase showed that copper was not detectable in the mutant OXI IIIa which lacked subunit 1, but was present in the mutant OXI IIIb, which exhibited a minor alteration in the electrophoretic mobility of subunit 1. In OXI I and II mutants there was a 50% reduction in the amount of copper in the immunoprecipitated cytochrome c oxidase. These observations may be interpretable as follows: (1) alterations in polypeptide biosynthesis due to the OXI mutations lead to an improper configuration of cytochrome c oxidase, so that ferrochelatase cannot transfer iron into porphyrin a; (2) subunit I is the binding site for copper, but the mutations in subunits II and III alter the binding site of one of the two copper atoms in subunit I.

Barley nitrate reductase contains a functional cytochrome b557

NADH-nitrate reductase (NR) from barley ( Hordeum vulgare L. cv. Steptoe) leaves was purified to specific activity of 19 μmol NO 2 − produced per min per mg protein with 27% yield by blue A sepharose affinity chromatography. Reduction of NR by NADH resulted in spectral absorption maxima at 422 (γ), 528 (β) and 556 (α) nm while oxidation by nitrate caused disappearance of α and β peaks and a shift of the γ peak to 413 nm. These results demonstrate the presence of a functional cytochrome b 557 in barley nitrate reductase.

Structure of the Schizosaccharomyces pombe cytochrome c gene.

The cytochrome c gene of the fission yeast Schizosaccharomyces pombe has been cloned by using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. The DNA sequence and the 5' termini of the mRNA transcripts of the gene have been determined. The DNA sequence has confirmed, with two exceptions, the previously determined protein sequence. The nonrandom distribution of silent third base differences which was observed between the two cytochrome c genes of S. cerevisiae does not extend to the S. pombe cytochrome c gene, suggesting that there are no constraints other than protein function and codon usage which have acted to conserve the cytochrome DNA sequences of the two yeasts. Introduction of the S. pombe cytochrome c gene on a yeast plasmid into a S. cerevisiae mutant which lacked functional cytochrome c transformed that recipient strain for the ability to grow on a nonfermentable carbon source. This implies that the S. pombe cytochrome c gene has all the regulatory signals which are required for its expression in S. cerevisiae, and that none of the amino acid differences between the cytochrome c proteins of the two yeasts has a drastic effect on the function of the protein in vivo.

Structure of the Schizosaccharomyces pombe cytochrome c gene.

The cytochrome c gene of the fission yeast Schizosaccharomyces pombe has been cloned by using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. The DNA sequence and the 5' termini of the mRNA transcripts of the gene have been determined. The DNA sequence has confirmed, with two exceptions, the previously determined protein sequence. The nonrandom distribution of silent third base differences which was observed between the two cytochrome c genes of S. cerevisiae does not extend to the S. pombe cytochrome c gene, suggesting that there are no constraints other than protein function and codon usage which have acted to conserve the cytochrome DNA sequences of the two yeasts. Introduction of the S. pombe cytochrome c gene on a yeast plasmid into a S. cerevisiae mutant which lacked functional cytochrome c transformed that recipient strain for the ability to grow on a nonfermentable carbon source. This implies that the S. pombe cytochrome c gene has all the regulatory signals which are required for its expression in S. cerevisiae, and that none of the amino acid differences between the cytochrome c proteins of the two yeasts has a drastic effect on the function of the protein in vivo.

Mitochondrial biogenesis during fungal spore germination: respiration and cytochrome c oxidase in Neurospora crassa.

The germination of conidiospores of wild-type Neurospora crassa was found to be dependent upon the function of the cytochrome-mediated electron transport pathway. The cyanide-insensitive alternate oxidase did not contribute significantly to the respiration of these germinating spores. The dormant spores contained all of the cytochrome components and a catalytically active cytochrome c oxidase required for the activity of the standard respiratory pathway, and these preserved components were responsible for the accelerating rates of oxygen uptake which began immediately upon suspension of the spores in an incubation medium. Mitochondria of the dormant spores contained all of the subunit peptides of the functional cytochrome c oxidase; nevertheless, de novo synthesis of these subunits began at low rates in the first stages of germination. Reactivation of the respiratory system of germinating N. crassa spores seems not to be dependent initially upon the function of either the mitochondrial or cytoplasmic protein-synthesizing systems. The respiratory activity of spores of three mutant cytochrome c oxidase-deficient strains of N. crassa also was found to depend upon the function of the cytochrome electron transport pathway; the dormant and germinating spores of these strains contained a catalytically active cytochrome c oxidase. Cytochrome c oxidase may be present in the dormant and germinating spores of these strains as the result of a developmental-phase-specific synthesis of and requirement for the enzyme.

Mechanism of the inhibitory action of isoniazid on microsomal drug metabolism

Addition of isoniazid (isonicotinic acid hydrazide, INH) to rat liver microsomes produced an immediate decrease in the binding of carbon monoxide to reduced cytochrome P-450. Preincubation of the microsomes with INH in the presence of NADPH produced a further decrease of carbon monoxide binding to cytochrome P-450. The latter decrease of functional cytochrome P-450 was dependent upon NADPH and oxygen and was transitory. Examination of compounds structurally related to INH indicated that both the hydrazine moiety and the aromatic ring were needed to produce both effects. Incubation of microsomes with INH also resulted in gradual increases in absorbance at 449 nm and at 493 nm which also were transitory. Thus, the decreased binding of carbon monoxide to cytochrome P-450 may have occurred concurrently with formation of these spectral intermediates. Microsomal N-demethylation and aniline p-hydroxylation were inhibited by isoniazid. Preincubation of the microsomes with INH and NADPH increased the inhibition. Thus, the decreased availability of cytochrome P-450 as observed may account for the inhibition of the mixed function oxidases by isoniazid.

Effect of methylation on the stability of cytochrome c of Saccharomyces cerevisiae in vivo.

The in vivo stability of methylated and unmethylated cytochrome c in Saccharomyces cerevisiae was studied by pulse-labeling the hemoproteins with [methyl-3H]-methionine and/or [2-14C]methionine and following the fate of these proteins under anaerobiosis and in the presence of cycloleucine. These two conditions will respectively block further cytochrome c synthesis and inhibit methylation by lowering the cellular S-adenosyl-L-methionine pool and, thus, permit an unambiguous interpretation of the data. The results showed that the rate of degradation of unmethylated cytochrome c was constant throughout the chase period, while methylated cytochrome c degradation was seen only in the later part of cold chase. At the end of the chase period (40 h), the extent of degradation of the unmethylated species was three times higher than the methylated species. This indicated that the methylation of cytochrome c has a protective effects against the intracellular proteolytic enzyme attack on itself. Furthermore, this protective effect was considerably reduced in the petite mutant, which lacks high affinity cytochrome c binding sites, functional cytochrome c reductase, and oxidase, and possesses a less integrated and organized mitochondrial membrane. These results led us to the conclusion that the mechanism of methylated cytochrome c stabilization is best explained by a higher efficacy of binding to the mitochondria.

Hydrogen peroxide-supported oxidation of benzo [ a]pyrene by rat liver microsomal fractions

In the presence of liver microsomes from phenobarbital-pretreated rats, hydrogen peroxide oxidized benzo [ a]pyrene to a number of biologically significant products at a rate that was approximately 20 per cent as fast as that seen by us and others with NADPH and oxygen. As with NADPH-dependent reactions [J. Capdevila, R. W. Estabrook, and R. A. Prough, Archs. Biochem. Biophys. 200, 186 (1980)], the hydrogen peroxide-dependent reactions resulted in the production of relatively large quantities of dihydrodiols as metabolites. This was in marked contrast to the product distribution observed when cumene hydroperoxide was utilized as a cosubstrate (foregoing reference). The formation of the various organic-soluble metabolites was dependent on the presence of functional liver microsomal cytochrome P-450 in the reaction mixture. Approximately 48 per cent of the benzo[ a]pyrene metabolites, however, was observed to be bound to microsomal protein, and inhibition of cytochrome P-450 function, by metyrapone or N-octylamine did not affect the extent of covalent binding of the hydrocarbon to the microsomal protein. The differences noted during benzo[ a]pyrene metabolism using hydrogen peroxide strongly suggest that at least two distinct mechanisms exist to account for the oxidation of the hydrocarbon, i.e. epoxidation and one-electron oxidation reactions.

A comparison of the structural and functional properties of cytochrome c oxidase isolated from beef ( Bos tauros), camel ( Camelus dromedarius), chicken ( Gallus domesticus) and rat ( Rattus norvegicus)

1. 1. Cytochrome c oxidases from a number of eukaryotic organisms have been isolated and show spectral properties similar to those of the bovine enzyme. All possess both Cu and Fe atoms. 2. 2. Ultracentrifuge studies reveal that the camel oxidase is monomeric yet exhibits similar properties to the bovine enzyme. 3. 3. Investigations of the subunit composition have shown 7 subunits for all the species examined and similarities are suggested on the basis of the 203Hg labelling of the subunits. 4. 4. Steady state kinetic measurements show two kinetically functional cytochrome c binding sites on all the oxidases examined. The pH optima of the monomeric camel enzyme (pH 7.2) is significantly different to the dimeric bovine enzyme (pH 6.2). 5. 5. Pre-steady state measurements show a fast initial electron entry from ferrocytochrome c to ferricytochrome a followed by a slower second phase. These results are similar for all the oxidases studied and comparable to those already established for the bovine enzyme. 6. 6. A complete cycle of full reduction followed by oxidation by molecular oxygen leads to an activated (“pulsed”) form of cytochrome oxidase. This property was exhibited by the enzymes from all the species examined.

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3.

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.

Structural studies on reconstituted reaction center-phosphatidylcholine membranes

Reaction center protein, isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 mutant, was incorporated into phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. Cytochrome c, added to preformed reaction center-phosphatidylcholine vesicles, rapidly reduced up to 90% of the laser-generated (BChl) ⨥ 2 of the reaction center (with kinetics of electron transfer similar to those in the chromatophore membrane) which suggests that the portion of the reaction center which accommodates functional cytochrome c binding sites is exposed predominantly on the exterior of the vesicles. Unit cell electron density profiles were derived from lamellar X-ray diffraction from oriented reaction center-phosphatidylcholine membrane multilayers at varying lipid/protein ratios. The analysis of these profiles showed that the reaction center protein incorporates into the phosphatidylcholine membrane with unique sidedness and that the profile of the reaction center protein itself is asymmetric and spans the membrane.

Conversion of a Mitochondrial Precursor Polypeptide into Subunit 1 of Cytochrome Oxidase in the mi‐3 Mutant of Neurospora crassa

1. The cytochrome-alpha alpha 3-deficient mi-3 cytoplasmic mutant of Neurospora crassa synthesizes a mitochondrial translation product which crossreacts with antibodies specific to subunit 1 of cytochrome oxidase. The immunoprecipitated polypeptide migrates more slowly during gel electrophoresis than the authentic 41 000-Mr subunit 1 of the wild-type enzyme. An apparent molecular weight of about 45 000 was estimated for the mutant product. 2. Radioactive labelling experiments in vivo show that the crossreacting material found in the mutant is relatively stable and does not form complexes with other subunits of the oxidase. 3. After induction of a functional cytochrome oxidase in the mutant cells with antimycin A, the 45 000-Mr polypeptide is converted to a 41 000-Mr component, which exhibits the same electrophoretic mobility as subunit 1 of the oxidase. Pulse-chase labelling kinetics reveal a typical precursor product relationship. 4. The converted polypeptide becomes assembled with other enzyme subunits to form a protein complex which has the immunological characteristics of cytochrome oxidase. A possible physiological role of the post-translational processing of the mitochondrially synthesized component is discussed.

The role of oxygen in the biosynthesis of cytochrome c oxidase of yeast mitochondria.

The formation of cytochrome c oxidase in yeast is dependent on oxygen. In order to examine the oxygen-dependent formation of the active enzyme, the effect of oxygen on the synthesis and the assembly of cytochrome c oxidase subunits was studied. Pulse-labeling experiments revealed that oxygen has no significant immediate effect on the synthesis of the three mitochondrially made subunits I to III; however, its presence causes subunits I and II to form a complex with the cytoplasmically made subunits VI and VII. This "assembly-inducing" effect can be demonstrated with intact yeast cells as well as with isolated mitochondria. It is independent of cytoplasmic or mitochondrial protein synthesis. After anaerobic growth for 10 or more generations, the intracellular concentrations of individual cytochrome c oxidase subunits drop 10- to 100-fold. Most of these residual subunits are not assembled within a functional cytochrome c oxidase molecule.

Incorporation of exogenous heme into hepatic cytochrome P-450 in vivo.

Parenteral administration of [3H]heme to allylisopropylacetamide-treated rats resulted in the formation of a radiolabeled allylisopropylacetamide-porphyrin adduct in the liver, which was isolated and purified. Since formation of this adduct requires the heme moiety of a functional cytochrome P-450 species, our findings indicate that exogenously supplied heme had been incorporated into the hepatic microsomal hemoprotein. Moreover, they suggest that exogenously supplied heme readily gains access to an "unassigned" heme pool, which provides heme for cytochrome P-450 formation.

Cytochrome Formation, Oxygen-induced Proton Extrusion and Respiratory Activity in Streptococcus faecalis var. zymogenes Grown in the Presence of Haematin

Streptococcus faecalis var. zyrnogenes was grown in oxygen-limited continuous culture in a lactate/tryptone/yeast extract medium containing 7·5 μg haematin ml-l. Low temperature difference spectra of whole organisms and washed membranes showed two peaks in the cytochrome b region, at 558 nm and 562 nm, and a peak at 627 nm indicative of a d-type cytochrome. A CO-binding cytochrome was also present in the haematin-grown bacteria. These cytochromes were not detected in bacteria grown without haematin. The haematin- grown bacteria produced proton pulses in a weakly buffered medium when pulsed with oxygen-saturated buffer. The average →H+/O ratio of such pulses was 1·4 and they were abolished by carbonyl cyanide m-chlorophenylhydrazone (CCCP). The haematin-grown bacteria had a high activity of particulate NADH oxidase, at least 10 times that of bacteria grown without haematin. They also had a high lactate Q 02 which was strongly inhibited by CCCP and gramicidin whereas bacteria grown without haematin had a very low lactate Q 02 largely insensitive to the uncoupling agents. The glucose Q 02 was similar in bacteria grown in the presence or absence of haematin but was stimulated by uncoupling agents in haematin- grown organisms and slightly inhibited by these agents in organisms grown without haematin. These results confirm earlier findings of the ability of S. faecalis to form a functional cytochrome system in the cell membrane when supplied with haematin and show that electron transport to oxygen by such a system is coupled to proton translocation.