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.Abbreviations DCIP 2,6-dichlorophenolindophenol - DMPD N,N-dimethyl-p-phenylenediamine - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]-glycine     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Cytochrome d expression and regulation pattern in free-living Rhizobium phaseoli

The respiratory system of Rhizobium phaseoli CFN42 in free-living cultures was studied. Cytochromes b, c, o and aa ₃ were found in fast growing cells cultured under forced aeration. Stationary aerobic cells, and semianaerobically grown cells showed decreased levels of cytochromes c, aa ₃ and o, concomitant with a significant increase of b type cytochromes and the synthesis of a new cytochrome, tentatively identified as cytochrome d. Cell membranes with the highest content of cytochrome d (semianaerobically grown cells) showed the highest respiratory activities with NADH, succinate, malate or ascorbate-TMPD (N,N,N,N-tetramethyl p-phenylendiamine). In the presence of either of the above electron donors, cytochrome d was clearly reduced. NADH dependent respiration in membranes of fast growing cells (no cytochrome d detected) was abolished by 25 M KCN. This inhibitor concentration caused only 15–20% inhibition in membranes of semianaerobically grown cells (cyt d present). Moreover, in the presence of 1–5 mM KCN, the oxidation of cyt d and a b type cytochromes was spectrally detected. It is suggested that cyt d is a functional cytochrome in the respiratory system of free-living Rhizobia, probably acting as terminal oxidase.     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.     

Cytochrome b type oxidases in the respiratory chains of the phytopathogenic fluorescent bacteria Pseudomonas cichorii and Pseudomonas aptata

Membrane fragments from the phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata have been examined. A branched respiratory chain is operative in P. cichorii whereas a linear electron transport system characterizes the related bacterium P. aptata. Both species contain several b type cytochromes resolved by redox titration analysis, but no a type components may be detected. In contrast, only P. cichorii is endowed with c type cytochromes and hence with cytochrome c oxidase activity. Among the b type cytochromes, two high-potential components, with Em_7.0 at +250 mV and +380 mV, have been kinetically characterized and tentatively associated with cyanideresistant and cytochrome c oxidase activities, respectively. Cytochrome b-250 should correspond to the spectrally detectable cytochrome o whereas cytochrome b-380 is functionally similar to cytochrome b-410 described in Rhodopseudomonas capsulata. This conclusion seems to blur previous reported data on other obligate aerobes in which cytochrome o has been generally associated with cytochrome c oxidase and also suggests that a more accurate reconsideration of the actual physiological role of cyt. o in bacterial respiration is necessary. Furthermore the question arises whether cyt. b-410 like oxidases, i. e. high-potential b's similar to cyt. b-410 of R. capsulata, may be widely distributed among aerobes rather than restricted to facultative photosynthetic prokaryotes.     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt ^– parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Evidence for a dual role of cytochrome c-553 and plastocyanin in photosynthesis and respiration of the cyanobacterium,Anabaena variabilis

The cytochrome oxidase activity (oxygen uptake in the dark) of a membrane preparation from Anabaena variabilis was found to be stimulated by cytochrome c-553 and plastocyanin obtained from this alga. Cytochrome c from horse heart was as active as cytochrome c-553, whereas little or no stimulation of oxygen uptake was obtained with cytochromes c ₂ from two Rhodospirillaceae, the plastidic cytochrome c-552 from Euglena, and plastocyanin from spinach. Cytochrome c-553 (A. variabilis) stimulated photosystem 1 activity in the same preparation much more than cytochrome c (horse heart). The results indicate that cytochrome c-553 and plastocyanin, besides their established function as electron donors of photosystem 1, participate in respiratory electron transport as reductants of a terminal oxidase. Photooxidation and dark oxidation show a different donor specificity.Abbreviations Chl chlorophyll a - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

Do cyanobacteria contain “mammalian-type” cytochrome oxidase?

The cytochrome content of membranes isolated from seven species of cyanobacteria was investigated in terms of conventional difference spectra, carbon monoxide difference spectra, photoaction spectra and photodissociation spectra, and by extraction of acid-labile heme followed by spectral identification. In addition, the effect of various inhibitors and activators on the oxidation of horse heart cytochrome c by the membrane was studied. Both the spectral features and the properties of the cytochrome oxidase reaction catalysed by the membranes suggested the presence of a terminal oxidase strikingly similar to mitochondrial ferrocytochrome c: oxygen oxidoreductase (EC. 1.9.3.1).Abbreviations PMS phenazine methosulfate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Cyt cytochrome     

Effects of copper concentration in continuous culture on the aa 3-type cytochrome oxidase and respiratory chains of Paracoccus denitrificans

The role(s) of copper in a bacterial cytochrome oxidase of the aa ₃-type was investigated by growth of Paracoccus denitrificans NCIB 8944, in batch and steady state continuous culture, in a medium from which the bulk of the copper had been extracted. In a medium containing approximately 0.02 M copper, cellular copper content, cytochromes a+a ₃ and cytochrome a ₃ were reduced to 55%, 58% and 33% respectively of control values and there were also less marked decreases in cytochromes c+c ₁ (to 85%) and a CO-binding b-type cytochrome, possibly cytochrome o (to 71%). Copper deficiency elicited in reduced minus oxidized difference spectra a shift to shorter wavelengths and narrowing of the band width of the -band of the oxidase, and loss of a (negative) band near 830 nm attributable to Cu_A (the copper functionally associated with haem a in the oxidase complex). The oxidase in copper-deficient cells reacted with oxygen to form the oxy Compound A at rates similar to that in control cells but CO recombination to ferrous haem a ₃ was slowed 4-fold in the copper deficient case. The results are interpreted as indicating loss of Cu_A and changes in the proportions of haems a and a ₃ with retention of catalytic activity. Titrations of respiration rates with antimycin suggested that copper deficiency did not result in diversion of electron flux through an antimycin A-insensitive, cytochrome o-terminated branch of the respiratory chain.     

Identification of b,c, and d cytochromes in the membrane of Vitreoscilla

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c ₅₅₆ and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT p-iodonitrotetrazolium violet - RMF respiratory membrane fragments - K _d dissociation constant - CHAPS 3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

The effect of transfer from low to high light intensity on electron transport in Rhodospirillum rubrum membranes

The effects of transfer from low to high ligh intensity on membrane bound electrontransport reactions of Rhodospirillum rubrum were investigated. The experiments were performed with cultures which did not form bacteriochlorophyll (Bchl) for about two cell mass doublings during the initial phase of adaptation to high light intensity. Lack of Bchl synthesis causes a decrease of Bchl contents of cells and membranes. Also, the cellular amounts of photosynthetically active intracytoplasmic membranes decrease.In crude membrane fractions containing both cytoplasmic and intracytoplasmic membranes the initial activities of NADH oxidizing reactions increase only slightly (about 1.2 times) per protein, but the initial activities of succinate oxidizing reactions decrease (multiplied by a factor of 0.7). On a Bchl basis activities of NADH oxidizing reactions increase 3.4 times while activities of succinate dependent reactions increase 1.9 times. With isolated intracytoplasmic membranes activities of NADH as well as succinate dependent reactions increase to a comparable extent on a Bchl basis (about 1.8 times) and stay nearly constant on a protein basis. Cytochrome c oxidase responds like succinate dependent reactions. The data indicate that in cells growing under the conditions applied NADH oxidizing electron transport systems are incorporated into both, cytoplasmic and intracytoplasmic membranes, while incorporation of succinate oxidizing systems is confined to intracytoplasmic membranes only.Activities of photophosphorylation and succinate dependent NAD⁺ reduction in the light increase per Bchl about 1.8 times. On a Bchl basis increases of the fast light induced on reactions at 422 nm and increases of soluble cytochrome c ₂ levels are comparable to increases of photophosphorylations and succinate dependent activities. But increases of slow light off reactions at 428 nm and of b-type cytochrome levels become three times greater then increases of cytochrome c ₂ reactions and levels. These results infer that although electrontransport reactions of intracytoplasmic membranes change correlated to each other, Bchl, cytochrome c ₂ and b-type cytochromes cellular levels are independent of each other. Furthermore, the data indicate that cytochrome c ₂ rather than b-type cytochrome is involved with steps rate limiting for photophosphorylation.Abbreviations Bchl bacteriochlorophyll - DCIP 2,6-dichlorophenolindophenol     

The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus

Whole cells of the methylotrophic bacteriumMethylophilus methylotrophus cultured under methanol-limited conditions contain approximately equal amounts of two majorc-type cytochromes,c _H andc _L. Virtually all of the cytochromec _H, and over one-third of the cytochromec _L, are loosely attached to the periplasmic surface of the respiratory membrane whence they can be released by sonication or by washing cells in ethylenediaminetetraacetate (EDTA). The latter causes inhibition of methanol oxidase activity and stimulation of ascorbate-N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity, neither of which effects are reversible by divalent metal ions. Kinetic analyses indicate that ascorbate-TMPD is oxidised via two routes, viz. a slow low-affinity pathway involving loosely membrane-boundc-type cytochromes plus cytochrome oxidaseaa ₃, and a faster higher-affinity pathway involving the firmly membrane-bound cytochrome oxidasec _L o complex; the former route predominates in the presence of divalent metal ions, and the latter route after exposure to EDTA. These and other results are discussed in terms of the spatial organisation of the terminal respiratory chain, and of the role ofc-type cytochromes in the oxidation of methanol and ascorbate-TMPD.Abbreviations EDTA Enthylenediaminetetraacetate - PMS Phenazinemethosulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - SDS Sodium dodecylsulphate - I₅₀ Concentration of inhibitor required to give 50% inhibition of enzyme activity - PQQ Pyrroloquinoline quinone     

The intramitochondrial location of cytochrome c peroxidase in wild-type and petite Saccharomyces cerevisiae

Cytochemical and ultrastructural analysis of wild-type cells of Saccharomyces cerevisiac, grown aerobically in a glucose-limited chemostat, shows that cytochrome c peroxidase is localized between the membranes of the cristae, that is, in the intracristal space. This enzyme is thus positioned appropriately within the organelle to act as an alternate terminal oxidase for the respiratory chain. The proximity of the peroxidase to major sites of generation of its two substrates may account for the small leakage of hydrogen peroxide from yeast mitochondria, as compared with the larger outflow from mammalian mitochondria.In the cytoplasmic petite mutant, gross distortion of promitochondrial membrane arrangement is evident. Nevertheless, cytochrome c peroxidase activity is present in the same amounts as is found in wildtype cell, and is localized predominantly within annuli of membrane which constitute the promitochondria in these cells.No unequivocal evidence was obtained for the localization of catalase in microbodies or other organelles in either wild-type or petite cells.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Activation studies by phospholipids on the purified cytochromec 4:o oxidase ofAzotobacter vinelandii

A modified procedure is described that was used to solubilize and purify the TMPD-dependent cytochromec ₄:o oxidase fromAzotobacter vinelandii. Two functional components (Fractions I and V) were obtained after DEAE-cellulose chromatography. Fraction V contained both cytochromec ₄ (3.6 nmol/mg protein) and cytochromeo (1.6 nmol/mg protein). This cytochrome oxidase complex oxidized TMPD at moderate rates. Fraction I, a clear greenish-yellow fraction, contained primarily phosphatidylethanolamine with some phosphatidylglycerol. Fraction I itself could not oxidize TMPD, but when it was preincubated with Fraction V, a 2–4-fold stimulation in TMPD oxidase activity occurred. Other authentic micellar phospholipids also readily activited TMPD oxidase activity in Fraction V. Themaximum activation effect obtained with Fraction I was in essence duplicated with purified phosphatidylethanolamine.Dedicated to the memory of David E. Green, a fine gentleman, an excellent scientist, and a true scholar. He will be missed by many of his former colleagues.