Presence of cytochromed in respiratory particles ofStreptomyces griseus and its function in the terminal oxidation system

The cytochromed inStreptomyces griseus was studied by means of difference spectra measured under dithionite- or substrate-reduction. Results indicated that the pigment 625 ofStreptomyces griseus is ad-type cytochrome. Its pyridine hemochrome showed the difference maximum at 613 nm. The difference maximum of the reduced cytochromed was shifted to the red by the addition of carbon monoxide or nitric oxide. Cytochromed apparently acts as one of the respiratory pigments under aerobic conditions. The respiratory particle showed nitrite reductase activity at the acidic pH, and the substrate-reduced cytochromesb andd were oxidized in the presence of nitrite under anaerobic conditions.     

Electron redistribution in mixed valence cytochrome oxidase following photolysis of carboxy-oxidase

Absorbance changes at 446 nm in purified cytochrome oxidase following flash photolysis of carboxy-oxidase poised in the mixed valence state at +220 mV show biphasic kinetics. One phase corresponds to CO recombination to ferrous cytochromea ₃ with an energy of activation of 9 kcal/mol; the second phase is 3–5 times faster with an energy of activation of 9.15 kcal/mol. Following flash photolysis at approximately –60°C, cytochromesa andc and the 840-nm CuA species are observed to undergo reduction as electrons from ferrous unliganded cytochromea ₃ equilibrate with the equipotential redox centers of the oxidase; as CO recombines with ferrous cyochromea ₃, these centers are oxidized and the mixed valence carboxy-oxidase is regenerated. Electron redistribution between centers of the oxidase in the forward and reverse directions occurs faster than does the binding of CO.     

A durohydroquinone oxidation site in the mitochondrial transport chain

Although duroquinone had little effect upon NADH oxidation in neutral lipid depleted mitochondria, durohydroquinone was oxidized by ETP at a rate sensitive to antimycin A. Fractionation of mitochondria into purified enzyme systems showed durohydroquinone: cytochromec reductase to be concentrated in NADH: cytochromec reductase, absent in succinate:cytochromec reductase, and decreased in reduced coenzyme Q:cytochromec reductase. Durohydroquinone oxidation could be restored by recombining reduced coenzyme Q:cytochromec reductase with NADH:coenzyme Q reductase. Pentane extraction had no effect upon either durohydroquinone or reduced coenzyme Q₁₀ oxidation, indicating lack of a quinone requirement between cytochromesb andc. Both chloroquine diphosphate and acetone (96%) treatment irreversibly inhibited NADH but not succinate oxidation. Neither reagents had any effect upon durohydroquinone oxidation but both inhibited reduced coenzyme Q₁₀ oxidation 50%, indicating a site of action between Q₁₀ and duroquinone sites. Loss of chloroquine sensitive reduced coenzyme Q₁₀ oxidation after acetone extraction suggests two sites for Q₁₀ before cytochromeb.     

Effect of membrane potential on equilibrium poise between cytochromea and cytochromec in rat liver mitochondria

The object of this work was to test the suggestion that the equilibrium poise between cytochromea and cytochromec in mitochondria might be influenced by the membrane potential.

1. The midpoint potentials of cytochromes (c+c ₁) and cytochromea (CO present) were found to be 250 mV and 245 mV, respectively, by equilibrating rat liver mitochondria with mixtures of ferrocyanide and ferricyanide anaerobically in presence of antimycin A and measuring the redox state of the cytochromes spectrophotometrically. In absence of CO, cytochrome oxidase gave an anomalous redox titration curve with a “midpoint” at about 275 mV.
2. When the mitochondria were equilibrated with ferricyanide/ferrocyanide, the redox poise of cytochromea (CO present) and of cytochromes (a+a ₃) but not of cytochromes (c+c ₁) was dependent on the sign and magnitude of the membrane potential developed by treating the mitochondria as follows: by adding ATP, by chaging the composition of the suspension medium so as to vary the Donnan or Nernst potential, by adding valinomycin in a medium of low K⁺ ion content, or by adding a pulse of acid or alkali when the membrane was made permeable to protons with FCCP.
3. The findings agree with the suggestion that the respiratory chain is arranged across the cristae membrane with cytochromesc ₁ andc in contact with the outer phase and cytochromesa anda ₃ plugged through, so that the equilibrium distribution of electrons between thec anda cytochromes is influenced by the electric field across the membrane.

     

The electron transport chain ofStreptomyces erythreus: Possible functions of cytochromeaa 3 and a novel green pigment

The cytochromes of the bacteriumStreptomyces erythreus have been investigated. Membrane-bounda-, b-, andc-type cytochromes were found together with a green pigment, which was found in both a soluble and membrane-bound form. Cells containing the green pigment exhibited cyanide-insensitive oxygen uptake. The CO-binding pigments included cytochromea ₃, ab-type cytochrome, cytochrome P450, and the green pigment. Photodissociation spectra at various low temperatures, in the presence or absence of oxygen, revealed cytochromeaa ₃ to be the predominant cytochrome terminal oxidase. The green pigment was capable of electron transport; the relationship of the pigment to the remainder of the electron transport chain remains to be ascertained.     

Studies on the cytochromef ofBryopsis maxima in vivo andin vitro

Cytochromec (553.7Bryopsis maxima) isolated fromB. maxima had absorption maxima at 553.7, 523.0, 417.1 and 317.5 nm in its reduced form. Isosbestic points in the reduced minus oxidized difference spectrum were located at 561, 543, 528, 511, 436, 411 and 334 nm. The purified protein exhibited a molecular weight of 10,700. The midpoint potential for the cytochromec was estimated to be 372±5 mVin vitro at pH 7.0 and 365±5 mVin vivo.In vivo 80% of the cytochromec was in the reduced form. This cytochrome was located only in chloroplasts indicating that it functions in the photosynthetic electron transport as cytochromef. Chloroplasts contained one molecule of this cytochrome per 360 molecules of chlorophyll. The magnitude of the chemically induced absorbance changes for the cytochromoesin vivo were much smaller than the light-induced absorbance change at 561 nm. It is concluded that the light-induced 561 nm absorbance change characteristic of this alga is not mainly attributable to the redox reaction of cytochromesb andf in the chloroplasts.     

EPR studies on cytochrome components in phosphorylating particles ofAzotobacter vinelandii

Oxidized particles ofA. vinelandii show high-spin ferric signals with an axial and a rhombically distorted component with g-values at 5.94 and 6.24, 5.51, respectively. The signals behave similarly on variation of temperature and/or power and are, assigned to cytochromed. The addition of ligands such as cyanide and carbon monoxide to oxidized particles mainly affects the rhombic component of the signal in the g=6 region. Prolonged, incubation of cyanide with oxidized particles results in the appearance of two new low-spin ferric heme signals at g=2.99 and at g=3.23 which are tentatively assigned to low-spin forms of cyanide-liganded cytochromed. With computer signal-averaging of the EPR spectrum of oxidized particles, the presence of resonances in the g=3–4 region could be demonstrated. These resonances are assigned to cytochromeb ₁ (g-values at 3.68, 3.43),c-type cytochromes (g-values at 3.43, 3.25) and cytochromea ₁, and possibly a low-spin form of ac-type cytochrome (g-value at 3.03). These EPR results represent, to our knowledge, the only such studies reported on the membrane-boundb ₁ andc-type cytochromes of a bacterial respiratory-linked phosphorylating electron-transport chain.     

Amino acid identities in the three redox center-carrying polypeptides of cytochromebc 1/b 6 f complexes

The comparison of primary structures is extended to 22 cytochromesb orb ₆, 12 cytochromesc ₁ orf, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochromeb each.     

Orientation of complex III in the yeast mitochondrial membrane: Labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [¹²⁵I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochromeb revealed that the two core proteins and cytochromeb were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochromec ₁ and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromesb andc ₁ was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15–30% of the total dithionite-reducibleb and 100% of the cytochromec ₁: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochromeb but insignificant amounts of cytochromec ₁. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.Abbreviations used: DABS, diazobenzene sulfonate; DBH₂, reduced form of decyl analogue of coenzyme Q (2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.     

The reversibility of the ethidium bromide-induced alterations of mitochondrial structure and function in the cellular slime mold,Dictyostelium discoideum

Addition of ethidium bromide to ameboid cultures of the slime mold,Dictyostelium discoideum, caused a cessation of cell division after 1 or 2 generations. The replication of mitochondrial DNA was immediately blocked as indicated by the 50% decrease in the DNA content of purified mitochondria from ethidium-bromide-treated cultures. The activity of the respiratory chain was also inhibited, resulting in a 75% decrease in cyanide-sensitive whole cell respiration. Spectral analysis at low temperature indicated that the amount of cytochromec ₁ was decreased 80% and that of cytochromec increased 100% in mitochondria from treated cells. Two cytochromesb absorbing at 556 and 561 nm were observed in mitochondria from both control and ethidium-bromide-treated cultures. The content of cytochromeb ₅₆₁ appeared to decline more than didb ₅₅₆, but it is hard to quantitate the decrease. The effects of ethidium bromide were fully reversible. When the drug was removed, the cells resumed a normal growth rate without any discernible lag. The activity of oligomycin-sensitive ATPase, cytochrome oxidase, and succinate-cytochrome-c reductase as well as the cytochrome content began to increase after 1 day returning to control levels within 5 days. Electron micrographs of whole cells treated with ethidium bromide revealed that mitochondrial profiles were elongated and had greatly reduced cristae. Numerous membrane whorls were apparent, as was a profound loss of rough endoplasmic reticulum. Three days after removal of ethidium bromide, mitochondria were again ovoid in shape and contained well-developed cristae. In all of the cells during recovery, there was a single large vacuole that appeared to enclose a large portion of the cell volume, forming a new cellular compartment that may simplify the breakdown of previously damaged organelles.This work is in partial fulfillment of the requirements for the Doctor of Philosophy degree at the City University of New York.     

The sequence of electron carriers in the reaction of cytochromec oxidase with oxygen

Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochromec by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochromec, to Cu_A, to cytochromea, and then to the binuclear (i.e., cytochromea ₃-Cu_B) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochromec to Cu_A 5–7 Å, cytochromec to cytochromea 20–25 Å, Cu_A to cytochromea 14–16 Å and cytochromea to cytochrome a₃-Cu_B 8–10 Å. It is proposed that the step from cytochromea to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.     

Effect of Brestan on Saccharomyces cerevisiae during continuous cultivation

An increase in Brestan concentration in nutrient media decreased the content of protein, phosphorus, total ribonucleic acid, activity of pyruvate carboxylase and isocitrate lyase in cells ofSaccharomyces cerevisiae parent strain and respiratory deficient (RD) mutant while the trehalose content increased. The respiration quotient value for the RD mutant was higher than for the parent strain. The RD mutant lacked cytochromeaa ₃; cytochromec andb contents were lower than those of the parent strain.     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Cytochromec and evolution of the energy acquiring system

The reactivity of cytochromesc derived from various organisms withPseudomonas aeruginosa nitrite reductase and cow cytochrome oxidase has been studied.Generally, cytochromesc isolated from primitive organisms react very rapidly with the bacterial nitrite reductase but do not react with cow cytochrome oxidase while those from higher organisms react poorly with the nitrite reductase but react very rapidly with the animal oxidase. The reactivity of cytochromec with the bacterial nitrite reductase reflects very well the evolutionary position of the organism from which it is isolated, while that with cow cytochrome oxidase seems to be related to the extent of adaptation of the parent organism to molecular oxygen. The results obtained in the present investigation suggests that cytochromec molecule which reacts very rapidly with the bacterial nitrite reductase but does not react with cow cytochrome oxidase has evolved to that which reacts very poorly with the nitrite reductuase but reacts very rapidly with the animal oxidase. It is also inferred that the evolution of cytochromec molecule may be caused by the evolution of cytochrome oxidase, and that the latter may be intimately related to genesis of molecular oxygen in the biosphere.Special Symposium on Photochemistry and the Origins of Life, Sixth International Congress on Photobiology, Bochum, Germany.     

Changes in cytochrome levels during growth of the yeast-like fungusAureobasidium pullulans

Cytochromes ofAureobasidium pullulans have been identified and partially characterized using low-temperature and carbon-monoxide-difference spectroscopy. The presence ofa-,b-, andc-type cytochromes is demonstrated, as are other unidentified redox components. During exponential growth in batch culture, cytochrome levels showed complex changes. Changes in respiration rates and in the levels of cytochromea+a ₃ closely paralleled cellular growth: both increased exponentially until stationary phase, when no further increase occurred. Theb- andc-type cytochromes showed biphasic increases, initially doubling every, generation time and then increasing more slowly during the stationary phase. Sensitivity of respiration to 100M potassium cyanide gradually decreased during exponential growth, falling from virtually 100% inhibition after about 20 h growth to 30% inhibition in the stationary phase. The results suggest that in stationary-phase cultures, an alternative cyanide-insensitive but salicylhydroxamic-acid-sensitive terminal oxidase also operates.     

l-Aspartate fermentation by a free-livingCampylobacter species

In the fermentation ofl-aspartate by a free-livingCampylobacter spec., the products formed were acetate, succinate, carbon dioxide and ammonia. The oxidative part of the fermentation pathway yielded acetate, succinate, carbon dioxide and ammonia, and the reductive part gave rise to the formation of succinate and ammonia. When grown anaerobically with aspartate, cells contained cytochromesb andc as well as menaquinone. Reduced cytochromeb, but not reduced cytochromec could be reoxidized by fumarate. In the presence of nitrate, 90% of the available electrons were transferred to nitrate, which was reduced to nitrite; the remainder was transported via the fumarate reductase system. Cells grown with aspartate and excess of formate converted aspartate quantitatively to succinate.Abbreviation Used TLC thin layer chromatography     

The metabolism ofStreptomyces griseus

The electron transfer pathway in the respiratory particles ofStreptomyces griseus was studied. Vitamins K₃ and K₅,α- andβ-naphthoquinones, served as the hydrogen acceptors in succinate oxidation, and succinate- and reduced nicotinamide adenine dinucleotide (NADH)-cytochromec reductase activities, but were ineffective for NADH oxidase activity. Vitamin K seemed to mediate the hydrogen from NADH-diaphorase to cytochromec. Chlorpromazine inhibited electron transfer in the respiratory particles. Cyanide completely inhibited the electron transfer system initially, however, oxygen consumption increased gradually with time. AlthoughS. griseus possesses cytochromesa, b, c and pigment 625 (probablyd), the electron transfer chain was complicated. Two terminal oxidase activities (cytochromec oxidase and cytochromec peroxidase activities) were detected in the respiratory particles ofS. griseus. Dedicated to Prof. Shoichiro Usami celebrating his sexagenary birthday.     

Differential exposure of components of cytochromeb−c 1 region in beef heart mitochondria and electron transport particles

The reduction of cyctochromesc +c ₁ by durohydroquinone and ferrocyanide in electron transport particles (ETP) and intact cytochromec-depleted beef heart mitochondria has been studied. At least 94% of the ETP are in an inverted orientation. Durohydroquinone reduces 80% ofc +c ₁ in ETP but less than 20% in mitochondria; sonication of mitochondria allows reduction of cytochromesc +c ₁ (80%). Addition of ferrocyanide (effective redox potential +245 mV) to electron transport particles results in 30% reduction of cytochromesc +c ₁. Addition of ferrocyanide to intact cytochromec-depleted mitochondria does not reduce cytochromec ₁; treatment withN,N,N,N-tetramethylphenylenediamine, Triton X-100, or sonic oscillation results in 30% reduction of cytochromesc +c ₁. TheK _m value of ferrocyanide oxidase for K-ferrocyanide is pH-dependent in ETP only, increasing with increasing pH. The extent of reduction of cytochromec ₁ is also pH-dependent in ETP only, the extent of reduction increasing with decreasing pH. On the basis of these data cytochromec ₁ is exposed to the matrix face and cytochromec is exposed to the cytoplasmic face. No redox center other than cytochromec in the segment between the antimycin site and cytochromec is exposed on the C-side.Abbreviations Used: MES, 2(N-morpholino)-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; TMPD,N,N,N,N-tetramethylphenylenediamine; ETP, electron transport particles; NAD-NADH, nicotinamide adenine dinucleotide; PMS, phenazine methosulfate.     

Photo-oxidation of membrane-bound and soluble cytochromec in the green sulfur bacteriumChlorobium tepidum

We studied the photosynthetic electron transfer system of membrane-bound and soluble cytochromec inChlorobium tepidum, a thermophilic green sulfur bacterium, using whole cells and membrane preparations. Sulfide and thiosulfate, physiological electron donors, enhanced flash-induced photo-oxidation ofc-type cytochromes in whole cells. In membranes,c-553 cytochromes with two (or three) heme groups served as immediate electron donors for photo-oxidized bacteriochlorophyll (P840) in the reaction center, and appeared to be closely associated with the reaction center complex. The membrane-bound cytochromec-553 had anE _m-value of 180 mV. When isolated soluble cytochromec-553, which has an apparent molecular weight of 10 kDa and seems to correspond to the cytochromec-555 inChlorobium limicola andChlorobium vibrioforme, was added to a membrane suspension, rapid photo-oxidation of both soluble and membrane-bound cytochromesc-553 was observed. The oxidation of soluble cytochromec-553 was inhibited by high salt concentrations. In whole cells, photo-oxidation was observed in the absence of exogenous electron donors and re-reduction was inhibited by stigmatellin, an inhibitor of the cytochromebc complex. These results suggest that the role of membrane-bound and soluble cytochromec inC. tepidum is similar to the role of cytochromec in the photosynthetic electron transfer system of purple bacteria.     

A soluble CO- and NO-bindingc-type cytochrome inNeisseria meningitidis

Group BNeisseria meningitidis (SDIC) was grown aerobically in Mueller-Hinton medium to stationary phase and then broken either by sonic oscillation or high pressure extrusion from a French pressure cell. The particulate fraction (345,000×g, 3 h) contained cytochromesc, b, o, and evidence of ana-type. The supernatant fraction contained a solublec-type cytochrome (c ₅₄₉). The pyridine hemochrome derivatives of acid-acetone-extracted supernatant fractions were shown to be free of other cytochrome types. The solublec cytochrome increased during the growth cycle of the organism, reaching a maximum after cells entered stationary phase, whereas the cellular level of other cytochromes from the particulate fraction started to decline as cells progressed from log into stationary phase. Osmotic shock, as well as other treatments, failed to selectively release the solublec cytochrome from the cell. When untreated, thec ₅₄₉ in the supernatant fraction remained in a reduced form. While it was not air oxidizable, it was readily oxidizable with K₃Fe(CN)₆. In the reduced form, thec ₅₄₉ bound either CO or NO, but in the oxidized form, only NO was bound. The results obtained from this study indicate that the solublec ₅₄₉ is probably best classified as a cytochromec′.