Cytochrome System Research Articles

Adaptation in oxidative metabolism of Trypanosoma rhodesiense during transformation in culture

1. 1. Significant increases in rates of oxygen uptake supported by l-glycerol-3-phosphate, α-oxoglutarate, NADH, succinate and l-proline were observed and were maximal within 7 days of transfer of trypomastigote forms into culture. 2. 2. Cyanide inhibited these oxidations within 3 days of transfer into culture. Inhibitions by antimycin A, rotenone and amytal were only partial. 3. 3. The culture (insect mid-gut) form was characterized by a high rate of oxidation of proline. 4. 4. The results indicate that the mitochondrial systems of the citric acid cycle and of an atypical cytochrome system are synthesized within 72 hr of transfer into culture.

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.

Mechanisms of Active Transport in Isolated Membrane Vesicles: I. THE SITE OF ENERGY COUPLING BETWEEN d-LACTIC DEHYDROGENASE AND β-GALACTOSIDE TRANSPORT IN ESCHERICHIA COLI MEMBRANE VESICLES

Abstract Transport of a wide variety of amino acids and sugars by membrane vesicles isolated from Escherichia coli ML 308-225 is coupled primarily to d-lactic dehydrogenase. This membrane-bound, flavin-linked primary dehydrogenase is coupled to oxygen via a cytochrome system also present in the vesicle membrane. Spectrophotometric evidence shows that d-lactic dehydrogenase, succinic dehydrogenase, l-lactic dehydrogenase, and NADH dehydrogenase all utilize the same cytochrome system. There is no relationship between rates of oxidation of electron donors by the respiratory chain (succinate g NADH = d-lactate g l-lactate) and the ability of these compounds to stimulate lactose transport (d-lactate >> succinate g l-lactate g NADH). Furthermore, d-lactate in combination with other electron donors is no more effective than d-lactate alone for support of lactose transport. These findings indicate that the site of energy coupling of d-lactic dehydrogenase to active transport lies between the primary dehydrogenase and cytochrome b1. Supportive evidence for this conclusion is obtained from experiments showing that N-ethylmaleimide and p-chloromercuribenzoate inhibit transport and d-lactate-induced respiration. However, these sulfhydryl reagents do not inhibit d-lactic dehydrogenase with dichlorophenolindophenol as an artificial acceptor, nor do they significantly block NADH-induced respiration.

Energy conversion in autotrophically grown Pseudomonas saccharophila.

1 Studies were undertaken to investigate the energy coupling mechanism in autotrophically grown Pseudomonas saccharophila. The cell-free extracts from Pseudomonas saccharophila contained flavoproteins and cytochromes of b, c, a, and o type. 2 Added NADH was oxidized rapidly but it was not coupled to phosphorylation. The oxidation of generated NADH, was mediated, however, by the flavoprotein and cytochrome systems and yielded P/O ratios ranging from 0.86 to 1.23. 3 The oxidation of hydrogen was mediated by the cytochrome systems but NAD+ was not involved; H2 oxidation yielded P/O ratios between 0.45 and 0.73. 4 Succinate oxidation resulted in a P/O ratio of 0.15 while ascorbate oxidation was not coupled to phosphorylation. 5 The phosphorylation coupled to the oxidation of hydrogen, NADH or succinate was completely uncoupled by various uncouplers of oxidative phosphorylation and phosphate esterification associated with the oxidation of NADH was quite sensitive to the flavoprotein inhibitors such as rotenone, amytal, and atabrine. These inhibitors had no effect, however, on the phosphorylation coupled to hydrogen or succinate oxidation. 6 The ATP formation associated with the oxidation of hydrogen, NADH or succinate was inhibited by antimycin A or 2-n-nonyl-4-hydroxyquinoline-N-oxide as well as by cyanide or azide.

An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase.

The distribution of catalase and superoxide dismutase has been examined in various micro-organisms. Strict anaerobes exhibited no superoxide dismutase and, generally, no catalase activity. All aerobic organisms containing cytochrome systems were found to contain both superoxide dismutase and catalase. Aerotolerant anaerobes, which survive exposure to air and metabolize oxygen to a limited extent but do not contain cytochrome systems, were found to be devoid of catalase activity but did exhibit superoxide dismutase activity. This distribution is consistent with the proposal that the prime physiological function of superoxide dismutase is protection of oxygen-metabolizing organisms against the potentially detrimental effects of the superoxide free radical, a biologically produced intermediate resulting from the univalent reduction of molecular oxygen.

Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria. II. Duroquinone reductase activity

1. Enzymatic reduction of 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) by NADH can be used in an assay procedure for the NADH dehydrogenase. The reduction of this quinone occurs in the region of the electron transport system between the primary dehydrogenase and the cytochrome system as defined by the almost complete loss of reductase activity following piericidin A treatment. 2. Duroquinone reduction can be distinguished from ubiquinone 2 reduction by the marked inhibition of the former following phospholipase C, poly- l-lysine, or chloroquine diphosphate treatment. In addition, duroquinone reduction requires the presence of endogenous ubiquinone 10 specifically whereas ubiquinone 2 reduction does not require the presence of endogenous quinone. These observations are consistent with the nonequivalency of the reduction sites of duroquinone and ubiquinone 2. 3. Duroquinol can be utilized as an electron donor for the energy-linked reduction, of NAD +. Duroquinol reduction of NAD + is dependent upon the presence of ATP, is inhibited by oligomycin, carbonyl cyanide p-trifluoro methoxyphenylhydrazone and piericidin A, and is not inhibited by antimycin A at levels which inhibit electron transport. 4. Duroquinone reduction as well as ubiquinone 2 reduction are inhibited almost completely by phospholipase A, p-chloromercuribenzoate, o-phenanthroline, and Triton X100 treatments.

Anaerobiosis and the activity of enzymes of pyrimidine biosynthesis in Staphylococcus aureus.

SUMMARY: Staphylococcus aureus synthesizes pyrimidines using a pathway similar to that established in Escherichia coli. Soluble and bound forms of dihydro-orotate dehydrogenase are present. Both forms are active in 2,6-dichloro-phenolindophenol reduction assays; that the particulate enzyme normally links to oxygen via a cytochrome system was shown in studies of a haem-requiring mutant. Activities of the enzymes of pyrimidine synthesis are six- to 20-fold greater in anaerobically or semi-anaerobically grown bacteria than in those grown in air, unless uracil is present. Aerobic cultures rapidly accumulate dihydroorotate in the medium after transfer to anaerobic conditions; derepression of the dehydrogenase also ensues. These findings show that the requirement for uracil displayed by Staphylococcus aureus when growing anaerobically is due to its inability to dehydrogenate dihydroorotate in the absence of oxygen: they cannot explain the ability of Escherichia coli to grow anaerobically without uracil.

Nematode biochemistry: XII. Mitochondria from the free-living nematode Turbatrix aceti

1. 1. Biochemically active mitochondria have been isolated from the free-living nematode, Turbatrix aceti. Electron photomicrographs indicate that the preparation consists mostly of intact organelles. 2. 2. The mitochondria oxidized all of the tricarboxylic acid metabolites tested, as well as NADH. α-Phosphoglycerol, dihydroxyacetone, and palmityl-cocnzvme A were inactive. 3. 3. The mitochondria contain a functioning cytochrome system. Cytochrome a- a 2, appears to be the terminal oxidase. 4. 4. Spectra of oxidized and reduced cytochromes are reported. 5. 5. An additional pigment is present in the mitochondria in 6-fold greater quantity than the other cytochromes. The material binds CO, and probably CN −. It is reduced by dithionite and, in the presence of CO, by succinatc or NADH. The function of this pigment is unknown. It does not appear to be a terminal oxidase.

Interaction of androgens with the adrenal mitochondrial cytochrome system: The influence of androgen treatment on the levels of cytochrome in rat adrenal cortical mitochondria and on substrate-induced spectral changes

The treatment of female rats with high doses of the synthetic androgens, 17α-methylandrostenediol (MAD) and 17α-methyltestosterone (MT), leads to inhibition of corticosterone formation from progesterone in adrenal homogenates. Mitochondria were isolated from the adrenals of control and treated animals and several changes were noted. 11β-Hydroxylation rates were about 40 and 70% of control rates in adrenal mitochondria from MT- and MAD-treated rats, respectively. Associated with the androgen treatment there was a doubling in the levels of cytochromes a and a 3 in the adrenal mitochondria with no change in the levels of cytochromes b or c and c 1. The cytochrome P-450 levels were about 60% of control levels in adrenal mitochondria isolated from MT- and MAD-treated rats. In addition to these alterations in adrenal mitochondria cytochrome levels, there was evidence that the MT was bound to the adrenal mitochondria. This binding resulted in lower DOC-induced spectral changes in adrenal mitochondria isolated from MT-treated rats. Such binding of androgen and the lower levels of cytochrome P-450 would appear to explain the decreased 11β-hydroxylation rates.

Mitochondrial responses to carbon monoxide toxicity.

CO not only bonds hematin iron in blood hemoglobin but also ligands hematin iron atoms of cytochrome a/sub 3/. Nonuniform distribution of CO means that rapidly metabolizing tissue are more susceptible to CO toxicity. Mitochondria are protected by branching and cushioning mechanism so CO toxicity response is not linear as in COHb formation. O/sub 2/ flux in respiratory change is not altered by CO. In these in vitro experiments, as little as 100 ppM CO transiently disrupted cytochrome system in transition from anoxia to nonmoxia, slowing the oxidaton of the chain. Pigeon mitochondria were extremely sensitive to CO when in metabolically-active (uncoupled) state. Increased binding of CO to cytochrome a/sub 3/ during anoxia was observed.

The hemoprotein content of Chromatium sp. strain D

1. The hemoprotein content of autotrophic Chromatium strain D has been measured from difference spectra, using solvent-extracted and intact cells. 2. The quantities of the individual cytochromes were estimated by absorbance differences at single wavelengths and by peak-trough differences in spectra obtained from reduced versus oxidized, and CO-treated reduced versus reduced preparations. The concentrations found in intact cells weer 1.15, 0.49 and 0.56 nmoles heme per mg protein for cytochromes c 553, cc′ and c 555 ∗∗ ∗∗ The components of the cytochrome system in Chromatium were named according to the recommendations of the International Union of Biochemistry on Nomenclature and Classification of Enzymes 1. , respectively. In intact cells, the corresponding values were 1.33, 0.47 and 0.67 nmoles heme per mg protein. 3. The mesoheme content of the cells, estimated as pyridine hemochromogen, was 2.08 nmoles heme per mg protein, while the amount of protoheme was negligible.

A Cytochrome-Related Inherited Disorder of the Nervous System and Muscle

INVESTIGATION of an unusual hereditary disease of the nervous system and muscle revealed a marked reduction in the demonstrable cytochrome b content of the muscle mitochondria and other abnormalities of the cytochrome system. The purpose of this report is to present the clinical, morphological, and biochemical studies of the two patients in whom these unique defects were found, in the hope that these studies will contribute to the further understanding of the pathogenesis of similar neurological disorders. Report of Cases CASE 1.— This 46-year-old man was in excellent health until age 33, when his gait became unsteady and his speech became slurred. Shortly thereafter, movements of his hands became clumsy and his handwriting deteriorated, and, within two years, he experienced weakness of the muscles of his legs and shoulders. All of these symptoms were insidiously progressive, but cramplike muscle pains, which persisted for approximately six years after the onset of

The Electron Transport System of Kappa Particles from Paramecium aurelia Stock 51

Surmmary NADH oxidase activity demonstrated spectrophotometrically in kappa of Paramecium aurelia stock 51, was inhibited by KCN, antimycin A and HOQNO. Using a split beam spectrophotometer we obtained low temperature reduced minus oxidized spectra of whole paramecia and of the mitochondrial fraction of paramecia. Two absorption maxima at 588 and 556 nm. were revealed in addition to the peaks of the known a and c cytochromes. From the reduced minus oxidized difference spectrum, the carbon monoxide difference spectrum and other studies, we concluded that kappa has a cytochrome system very different from that of its host but very much like that of bacteria in the families of Brucellaceae and Enterobacteriaceae. The slight contamination in the preparations could not be responsible for the spectra obtained. These findings strongly support the recent belief that kappas and other symbionts in P. aurelia are procaryotic in nature and origin.

Dechlorination of DDT by membranes isolated from Escherichia coli.

The capacity of cellular components of Escherichia coli to metabolize DDT-14C was investigated. Washed membrane fractions were obtained by lysozyme treatment followed by osomotic shock. Gas chromatography with electron capture detection and thin-layer chromatography were used to measure the effect of exogenous Kreb’s cycle cofactors and intermediates on the metabolism of DDT by particulate components of the bacteria. Reductive dechlorination of DDT to TDE occurred in the membranous fraction of the bacterial cell and was stimulated by a factor (s) present in the cytoplasmic fraction. Isolated membranes produced substantial amounts of TDE only in the presence of exogenous flavin adenine dinucleotide (FAD), and the prior reduction of FAD is essential to the production of TDE. The presence of Kreb’s cycle energy inhibited TDE production, indicating that DDT does not accept electrons produced by the oxidation of Kreb’s cycle intermediates directly from the cytochrome system.

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

The biochemistry of cestode mitochondria, i. aerobic metabolism of mitochoindria from Taenia taeniaeformis

1. 1. The oxidative capacity of mitochondrial fractions isolated from larval and adult Taenia taeniaeformis was assayed polarographically in the presence of various substrates and inhibitors. α-Glycerophosphate was the substrate most readily oxidized. Malate and NADH were oxidized at substantially lower rates; other tricarboxylic acid cycle intermediates, amino-acids, and fatty acids were either less effective or inert as substrates. 2. 2. Dihydroxyacetone phosphate was the product of α-glycerophosphate oxidation. Hydrogen peroxide was not detected, but the presence of an active catalasc may have prevented its identification. 3. 3. α-Glycerophosphate oxidation in the mitochondrial fractions was not mediated by pyridine nucleotide, but was stimulated by vitamin Kj. The cytoplasm of the parasite contained an extramitochondrial α-glycerophosphate dehydrogenase that catalysed the oxidation of NADH in the presence of dihydroxyacetone phosphate. This pair of enzymes provides the essential components of the a-glycerophosphate cycle. 4. 4. The activity of the mitochondrial a-glycerophosphate oxidase was inhibited by dicoumarol. Antimycin and cyanide were less effective, and there was little definitive evidence of a classic cytochrome system participating in the terminal electron transport. 5. 5. Oxidation of a-glycerophosphate was inhibited by several metal-chelating agents. These observations, together with the finding of substantial amounts of non-haem relative to haem iron in the mitochondrial preparations, suggest that non-haem iron has a functional role in the tapeworm respiratory chain. 6. 6. Both oxidized and reduced pyridine nucleotide were found in these preparations, and inhibition of malate oxidation was demonstrated with rotenone or Amytal (amylobarbitone). 7. 7. The respiratory chain of T. taeniaeformis appears to contain the following components: flavoprotein dehydrogenases, non-haem iron, pyridine nucleotide, and a branched cytochrome system, one portion sensitive to cyanide and the other insensitive. 8. 8. The presence of high endogenous ATPase activity in the mitochondrial fractions may indicate that the organelles were functionally altered during isolation with respect to the coupled phosphorylation reactions, which were not demonstrable.

On the role of quinones in bacterial electron transport. The respiratory system of Bacillus megaterium.

The menaquinone of Bacillus megaterium (14581 ATCC) is localized exclusively in the cytoplasmic membrane as are the cytochromes. Menaquinone is contained in a 5 to 10‐fold molar excess over cytochrome b1. The constituent menaquinone is almost completely reduced to menaquinole in the anaerobic state with the substrates respired by the membrane fraction. On the addition of oxygen the menaquinole is reoxidized to menaquinone. Menaquinone and menaquinole are the only forms of MK detected in the steady states of the electron transport. The redox reactions of menaquinone indicate its participation in the electron transport from the substrates α‐glycerophosphate, malate and NADH, to the acceptors oxygen and fumarate. Menaquinone is in a steady state in all the flow systems established with each substrate and both acceptors, and exhibits the steady state response expected for a respiratory component. The cytochrome system of this strain of B. megaterium consists of a b‐type cytochrome with a redox potential of + 8 mV and absorption bands close to those of pure cytochrome b1, two cytochrome oxidases, and α‐type cytochrome and cytochrome o and probably a small amount of c‐type cytochrome. Cytochrome b1 is completely reducible by each of the substrates. 2‐n‐Heptyl‐4‐hydroxyquinoline‐N‐oxide inhibits the electron flow at about the concentration of the constituent menaquinone between menaquinone and the cytochromes. The electron flow from each of the substrates to both acceptors depends completely on the reaction sensitive to the inhibitor. The electron transport from α‐glycerophosphate, malate and NADH to oxygen and fumarate as well as the reduction of cytochrome b1 is dependent on the presence of MK. This is demonstrated by pentane extraction and reincorporation of the MK. The reincorporation leads to the reconstitution of the original pathway, since MK and cytochrome b1 participate in the electron transport and the same sensitivity to 2‐n‐heptyl‐4‐hydroxyquinoline‐N‐oxide is found. The position of menaquinone in the respiratory system of B. megaterium is between the dehydrogenases for α‐glycerophosphate, malate and NADH and cytochrome b1 to which the fumarate reductase and the cytochrome oxidases are linked.

RESPIRATORY METABOLISM IN ACHLYA AMBISEXUALIS

Manometric and spectrophotometric techniques were utilized in studying respiratory metabolism in intact mycelia and cell‐free extracts of the heterothallic water mold, Achlya ambisexualis Raper. Enzymes representing the hexose monophosphate pathway, the Embden‐Meyerhof‐Parnas scheme, the tricarboxylic acid (TCA) cycle and a terminal cytochrome system were demonstrated. In addition, glycerol phosphate dehydrogenase and lactic dehydrogenase activities were observed. Activities of selected enzymes were observed to decline with culture‐age, with the exceptions of glucose‐6‐phosphate dehydrogenase and glycerol‐phosphate dehydrogenase. Oxygen uptake in the male strain more than doubled in the presence of sexual hormone A.

Enzymic sulphide oxidation by Thiobacillus concretivorus

1. 1. The oxidation of sulphide by Thiobacillus concretivorus, Thiobacillus thiooxidans and Thiobacillus thioparus is an enzymic process associated with cell membranes. 2. 2. The first reaction is an oxidative one involving electron transfer through the cytochrome system to oxygen. It is inhibited by sodium diethyldithiocarbamate and Tris-HCl, but not by CO or sodium azide. 3. 3. The K m of sulphide oxidations is 2·10 −6 M. 4. 4. Conjugates sulphur compounds with distinctive spectral properties are subsequently fromed. These may be polysulphides. This step is inhibited by CO. 5. 5. A native cytochrome c which has been purified 120-fold from T. concretivorus is involved in the formation of the polysulphur intermediates. CO binds to this cytochrome, and inhibits the production of the polysulphur compounds which absorb around 320 mμ. 6. 6. A role for copper and the formation of a bound intermediate is proposed. 7. 7. A heat-stable factor, essential for sulphide oxidation, has been extracted in acetone from the membrane fraction.

Energy and Electron Transfer Systems of Chlamydomonas reinhardi. I. Photosynthetic and Respiratory Cytochrome Systems of the Pale Green Mutant

The role of cytochromes in photosynthetic electron transfer system has been studied using the pale green mutant of Chlamydomonas reinhardi (ATCC 18302). The existence of cytochromes b(563) and f is confirmed, while no significant amount of ascorbate-reducible cytochrome b(559) is detected in this mutant. The presence of cytochrome c and a small amount of a-type cytochrome is determined in these cells.Light-induced oxidation of cytochrome b(563) is eliminated by oxygen-induced oxidation, and oxygen-induced oxidation is greatly diminished under illumination. Antimycin A diminishes the oxygen-induced oxidation of cytochrome b(563), but does not affect light-induced oxidation. In the aerobic state in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide, cytochrome b(563) is reduced by illumination with far red light; this reduction is not inhibited by 3-(4'-chlorophenyl)-1,1-dimethylurea.A tentative scheme for the electron transfer system in chloroplasts, which involves a cyclic pathway via cytochrome b(563) and its interaction with oxygen, is proposed.