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.     

Membrane-bound cytochromes in a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F

Cytoplasmic membranes were isolated from the cells of a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F and membrane-bound cytochromes were characterized. Redox difference spectra at 77 K revealed the presence of cytochromes with the alpha peaks at 552 and 556 nm while CO-binding difference spectra showed the presence of o-type cytochrome(s). Partial purification of the cytochromes demonstrated that the membranes contain cytochromes c550, c551, c556 and possibly d1 besides high molecular mass cytochrome c and cytochrome c3. It turned out that two kinds of novel CO-binding c-type cytochromes are present in the membrane. The membranes and a partially purified fraction showed weak ubiquinol-1 oxidase activity but no cytochrome c oxidase activity. Results suggest that D. vulgaris does not express the heme-copper terminal oxidase under our growth conditions in spite of the presence of the col gene, which is homologous to the gene of subunit I of the aa3-type oxidase.     

Pathways of terminal respiration in marine invertebrates. II. The cytochrome system of Aplysia

The classic spectrophotometric method for identification and characterization of respiratory enzymes has been used for the study of the cytochrome system of Aplysia. Particles have been prepared from the buccal mass and the gizzard muscles. Difference spectra taken on isolated particle suspensions show the presence of a complete cytochrome system composed of five components: cytochrome a, b, c, c(1), and a(3). As indicated by the peaks of the sharp absorption bands of their reduced forms, they are very similar to the cytochromes of mammals and yeast. Cytochrome a(3) has been identified as the terminal oxidase of Aplysia muscle by means of the spectrophotometric study of its carbon monoxide compound. Further evidence for the presence of a cytochrome system in Aplysia was obtained by assays of the catalytic activities of the isolated particles: succinic dehydrogenase, cytochrome oxidase, DPNH cytochrome c reductase. The cytochrome oxidase activity was strongly inhibited by carbon monoxide in the dark; the inhibition was totally relieved by light. Cytochrome c has been extracted and purified from muscle tissue. Its spectrum is almost identical with that of the mammalian pigment both in the oxidized and reduced forms. From the hepatopancreas a new respiratory enzyme has been extracted which has many physical and chemical properties in common with cytochrome h from terrestrial snails.     

Effects of azide on gastric mucose

Sodium azide, a classical inhibitor of cytochrome oxidase, is an effective inhibitor of gastric acid secretion in bullfrog and skate gastric mucosae at low concentrations. While a portion of the oxygen uptake in these tissues is sensitive to azide (KI less than 2 mM), there remains a large fraction (25-60%) with a KI more than 10 times this value, suggesting the presence of a second oxidase. The spectra of cytochromes c and b change with oxygen-nitrogen alternation in the presence of high azide concentrations which essentially eliminate the reactivity of cytochrome oxidase. In both species two additional components are observed in the spectra. The first has a peak at 590 nm, is not the cytochrome oxidase-CO complex, is fully reactive in the presence of azide and accounts for the asymmetry of the oxidase peak. The second is a component at 557 nm which can only be separated from cytochromes c and b by spectral deconvolution, and seems to react in a manner similar to cytochrome c. It is suggested that the 590 compound may be the alternate cytochrome oxidase.     

Participation of cytochromes in some oxidation-reduction systems in Campylobacter fetus.

Campylobacter species are rich in c-type cytochromes, including forms which bind carbon monoxide. The role of the various forms of cytochromes in Campylobacter fetus has been examined in cell-free preparations by using physiological electron donor and acceptor systems. Under anaerobic conditions, NADPH reduced essentially all of the cytochrome c in crude cell extracts, whereas the reduction level with succinate was 50 to 60%. The carbon monoxide spectrum with NADPH was predominated by the cytochrome c complex; evidence of a cytochrome o type was seen in the succinate-reduced extracts and in membrane fractions. Succinate-reduced cytochrome c was oxidized by oxygen via a cyanide-sensitive, membrane-associated system. NADPH-reduced cytochrome c was oxidized by a cyanide-insensitive system. Partially purified carbon monoxide-binding cytochrome c, isolated from the cytoplasm, could serve as electron acceptor for NADPH-cytochrome c oxidoreductase; the reduced cytochrome was oxidized by oxygen by a cyanide-insensitive system present in the cytoplasmic fraction. Horse heart cytochrome c was also reducible by NADPH and by succinate; the reduced cytochrome was oxidized by a cyanide-sensitive system in the membrane fraction. NADPH and NADH oxidase activities were observed aerobically and under anaerobic conditions with fumarate. NADPH was more active than NADH. NADP was also more effective than NAD as an electron acceptor for the coenzyme A-dependent pyruvate and alpha-ketoglutarate dehydrogenase activities found in crude extracts. These dehydrogenases used methyl viologen and metronidazole as electron acceptors; they could be loci for oxygen inhibition of growth. It is proposed that energy provision via the high-potential cytochrome c oxidase system in the cytoplasmic membrane is limited by oxygen-sensitive primary dehydrogenases and that the carbon monoxide-binding cytochrome c may have a role as an oxygen scavenger.     

Carbon monoxide-binding cytochromes in the respiratory chain of the thermophilic bacterium PS3 grown with sufficient or limited aeration

The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and aa3, while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome a (a3). The CO-difference spectra indicated that the terminal oxidase was cytochrome aa3 for high aeration conditions and the cytochrome o for low aeration conditions. Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions. The carbon monoxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25 degrees C. At liquid nitrogen temperature, CO-rebinding did not occur. The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al. (1979) FEBS Lett. 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome aa3. Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome aa3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o.     

Oxidation of reduced nicotinamide adenine dinucleotide by particles from Mycobacterium lepraemurium.

Particles from Mycobacterium lepraemurium catalysed the oxidation of NADH with oxygen as the terminal electron acceptor. The preparations contained cytochromes of the a + a3'b and c types, as well as CO-binding pigments. The NADH oxidase activity was sensitive to inhibitors of the flavoprotein system as well as to HQNO and antimycin A. In addition, a cytochrome oxidase sensitive to cyanide was also present. The system was inhibited by the thiol-binding agent, PCMB, and thus indicated the involvement of sulphydryl group in the enzymatic oxidation of NADH. The sensitivity of the NADH oxidase system to all the inhibitors of the respiratory chain and the effect of these inhibitors on the absorption spectra suggested that cytochromes of the b, c, a + a3 types are involved in the transfer of electrons in NADH oxidation.     

Cardiolipin is associated with the terminal oxidase of an extremely halophilic archaeon

Membranes having an a high content of cardiolipin were isolated from an extremely halophilic archaeon Halorubrum sp. Absorbance difference spectra of detergent-solubilized plasma membranes reduced by dithionite suggested the presence of b-type cytochromes. Non-denaturing gel electrophoresis revealed only one fraction having TMPD-oxidase activity in which cardiolipin was the major lipid component. The electroeluted fraction showed a cytochrome c oxidase activity characterized by the reduced minus oxidized difference spectra as a terminal heme-copper oxidase. The cytochrome c oxidase activity of the archaeal cardiolipin-rich membranes was inhibited by the cardiolipin-specific fluorescent marker 10-N-nonyl acridine orange (NAO) in a dose-dependent manner. The results indicate that an archaeal analogue of cardiolipin is tightly associated to archaeal terminal oxidases and is required for its optimal functioning.     

Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation.

Cultured cells of a Rhizobium phaseoli wild-type strain (CE2) possess b-type and c-type cytochromes and two terminal oxidases: cytochromes o and aa3. Cytochrome aa3 was partially expressed when CE2 cells were grown on minimal medium, during symbiosis, and in well-aerated liquid cultures in a complex medium (PY2). Two cytochrome mutants of R. phaseoli were obtained and characterized. A Tn5-mob-induced mutant, CFN4201, expressed diminished amounts of b-type and c-type cytochromes, showed an enhanced expression of cytochrome oxidases, and had reduced levels of N,N,N',N'-tetramethyl-p-phenylenediamine, succinate, and NADH oxidase activities. Nodules formed by this strain had no N2 fixation activity. The other mutant, CFN4205, which was isolated by nitrosoguanidine mutagenesis, had reduced levels of cytochrome o and higher succinate oxidase activity but similar NADH and N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activities when compared with the wild-type strain. Strain CFN4205 expressed a fourfold-higher cytochrome aa3 content when cultured on minimal and complex media and had twofold-higher cytochrome aa3 levels during symbiosis when compared with the wild-type strain. Nodules formed by strain CFN4205 fixed 33% more N2 than did nodules formed by the wild-type strain, as judged by the total nitrogen content found in plants nodulated by these strains. Finally, low-temperature photodissociation spectra of whole cells from strains CE2 and CFN4205 reveal cytochromes o and aa3. Both cytochromes react with O2 at -180 degrees C to give a light-insensitive compound. These experiments identify cytochromes o and aa3 as functional terminal oxidases in R. phaseoli.     

Carbon monoxide-cytochrome interactions in the brain of the fluorocarbon-perfused rat

Reflectance spectrophotometry through the skull was used to investigate carbon monoxide (CO) binding by tissue hemoproteins in the brains of barbiturate-anesthetized Sprague-Dawley rats. After splenectomy and extensive perfluorotributylamine exchange transfusion, steady-state spectral scans were obtained in Soret and visible wave-length regions during O2 ventilation, during subsequent exposure to O2-enriched gases containing 1, 3, or 5% CO, and finally after N2 anoxia. These CO exposures were well-tolerated and electroencephalograph (EEG) activity continued to be present. Initial difference spectra were influenced by CO binding to residual hemoglobin, but spectral evidence of CO-mediated b-type cytochrome reduction was obtained in the visible region as CO concentration was increased to 3 or 5%. This was associated with Soret spectra compatible with formation of the reduced cytochrome a3-CO complex. Reduction of cytochrome a at 605 nm and cytochrome c + c1 at 550 nm was absent. These findings may indicate respiratory chain branching through b cytochromes, either to a separate a3-like oxidase independent of the classical cytochrome aa3 or to an unidentified alternative CO-sensitive oxidase.     

Cyanide- and carbon monoxide-resistant mutants of Vitreoscilla: altered cytochromes and respiratory properties

Two respiratory mutants of the aerobic bacterium, Vitreoscilla, have been studied: a CO-resistant mutant that can grow in 50% CO-50% oxygen, and a cyanide-resistant mutant that can grow in 1 mM KCN. Wild-type cells are unable to grow under either condition. This report presents evidence that the resistance of the CO mutant is due to an altered membrane-bound cytochrome o [cytochrome o(m)], and that of the cyanide mutant is due to the presence of an increased amount of cytochrome d, which has a lower affinity for cyanide than cytochrome o(m). The evidence was obtained from spectral studies on the three types of intact cells as well as enzymatic and ligand-binding techniques on the cytoplasmic cytochromes o[cytochrome o(s)] and the respiring membrane vesicles isolated from these cells. Carbon monoxide difference spectra of intact cells revealed a 5-nm shift in an absorption maximum of a CO-binding pigment in the CO mutant relative to that of the wild type. The formation of oxygenated cytochrome o(s) and its conversion to the reduced form when the cells became anaerobic due to cellular respiration were inhibited when 1 mM KCN was added to a cell suspension of wild-type cells; the cyanide mutant cells showed resistance to cyanide in this experiment. Cytochrome o(s) purified from all three cell types had identical physical, electron transferring, and ligand binding properties within experimental error. Respiring membrane vesicles isolated from the two mutants showed more resistance to inhibition by cyanide and carbon monoxide than those from the wild type. Carbon monoxide difference spectra of these membrane vesicles revealed that there was a fivefold increase in the amount of cytochrome d in the cyanide mutant relative to the wild type. A CO absorption band of the membrane-bound cytochrome o in the CO mutant membrane vesicles showed a 5-nm shift relative to that of the wild type.     

Comparative studies on redox proteins from ammonia-oxidizing bacteria

Abstract Soluble fractions prepared from representatives of 3 genera of ammonia-oxidising bacteria all contained CO-binding c -type cytochromes and the cytochrome P-460 chromophore. Hydroxylamine oxidase from terrestrial strains of Nitrosomonas europaea was immunologically related to the analogous protein from marine strains of the same genus. Soluble cytochrome oxidase nitrite reductase activity was detected only in extracts prepared from terrestrial strains of N. europaea among the organisms examined.     

The cytochromes of luminous bacteria and their coupling to bioluminescence

Vibrio fischeri andV. harveyi possess cytochromes a, b, and c, whereasPhotobacterium leiognathi andP. phosphoreum also contain cytochrome d. In all, cytochrome a as well as some of c binds carbon monoxide. Carbon monoxide does not inhibit bioluminescence (in vivo or in vitro), but carbonyl cyanidem-chlorophenylhydrazone inhibits only in vivo bioluminescence. This inhibition is due to dissipation of the proton motive force which indirectly inhibits bioluminescence by interruption of aldehyde recycling. Bioluminescence is thereby indirectly coupled to the proton motive force.     

Low-temperature studies of electron transfer between different cytochromes c and cytochrome c oxidase.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.     

Immunological and spectral characterization of partly purified cytochrome oxidase from the cyanobacterium Synechocystis 6714

Membranes were isolated by French pressure cell extrusion of lysozyme-preincubated cells of the cyanobacterium Synechocystis 6714 after growth in the presence of 0.4 M NaCl for 4 days. These cells showed up to 6-fold respiratory activity (oxygen uptake) when compared to control cells. Separation of plasma and thylakoid membranes revealed that the major part of cytochrome c oxidase was associated with the latter. Immunoblotting of sodium dodecylsulfate polyacrylamide gel electrophorized membranes with antisera raised against subunit I, subunit II, and the holoenzyme of the aa3-type cytochrome oxidase from Paracoccus denitrificans gave specific and complementary cross-reactions at apparent molecular weights of about 25 and 17-18 kDa, respectively. Crude membranes were solubilized also with n-octyl glucoside, and the cytochrome oxidase was separated from the extract by affinity chromatography using immobilized cytochrome c from Saccharomyces cerevisiae. The enzyme was eluted with KCl/octyl glucoside. Dialysed and concentrated enzyme solution, which was free of b- and c-type cytochromes, gave reduced alpha- and gamma-peaks around 603 and 443 nm, respectively. Upon treatment of the sample with carbon monoxide the peaks were found at 593 and 433 nm, respectively. Photodissociation spectra of the CO-complexed enzyme were in full agreement with cytochrome aa3 being a functional cytochrome oxidase in Synechocystis 6714.     

Do evolutionary changes in cytochrome c structure reflect functional adaptations?

Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.     

A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12

Abstract Five different c -type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media. None of these cytochromes was detectable in aerobically-grown cultures. Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a funtional tor operon located at minute 22 (coordinate 1070 kb) on the E. coli chromosome. The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate. As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes. No other c -type cytochrome was detected under any growth condition tested.     

Structural perturbation of the a3-CuB site in mitochondrial cytochrome c oxidase by alcohol solvents

Ethanol has been observed to cause a perturbation of the catalytic center of the major respiratory protein cytochrome c oxidase. These effects were examined by Fourier transform infrared spectroscopy of carbon monoxide complexes of cytochrome a3Fe and of CuB formed by low-temperature photodissociation of the a3FeCO complex. Carbon monoxide binds to reduced cytochrome oxidase in two major structural forms, alpha and beta, both of which are altered by ethanol. In the absence of ethanol, 15-22% of the total cytochrome oxidase in beef heart mitochondria was observed as beta-forms. Ethanol addition caused a concentration-dependent elimination of the beta-forms with 40% disappearing at 0.05 M (0.23%) ethanol, a concentration that can readily be achieved in the blood of intoxicated individuals. At 0.5 M (2.3%) ethanol and above, almost no beta-forms were detectable. The alpha-CuBCO absorption normally splits into two bands at temperatures below 40 K. This effect was decreased in the presence of ethanol and eliminated by high ethanol concentrations. It appears that ethanol increases the structural fluctuations at the active site of the enzyme, analogous to the effects of increased temperature. There was an 8-10% decrease in the maximum rate of oxygen reduction by mitochondrial cytochrome oxidase in 0.05 M ethanol at 24 degrees C, while higher concentrations of ethanol caused no further inhibition. This is the first demonstration that alpha- and beta-forms of cytochrome c oxidase can be modified by an externally added reagent. Changes in the spectra of alpha-CuBCO in the presence of 50% (v/v) ethylene glycol were quite striking, but variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

Cytochromes c550, c552, and c1 in the Electron Transport Network of Paracoccus denitrificans: Redundant or Subtly Different in Function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.