Aeropyrum pernix K1, a strictly aerobic and hyperthermophilic archaeon,has two terminal oxidases,cytochrome ba3 and cytochrome aa3

Aeropyrum pernix K1 is a strictly aerobic and hyperthermophilic archaeon that thrives even at 100 degrees C. The archaeon is quite interesting with respect to the evolution of aerobic electron transport systems and the thermal stability of the respiratory components. An isolated membrane fraction was found to oxidize bovine cytochrome c.The activity was solubilized in the presence of detergents and separated into two fractions by successive chromatography. Two cytochrome oxidases, designated as CO-1 and CO-2, were further purified. CO-1 was a ba(3)-type cytochrome containing at least two subunits. Chemically digested fragments of CO-1 revealed a peptide with a sequence identical to a part of a putative cytochrome oxidase subunit I encoded by the gene ape1623. CO-2, an aa(3)-type cytochrome, was present in lower amounts than CO-1 and was immunologically identified as a product of aoxABC gene (DDBJ accession no. AB020482). Both cytochromes reacted with carbon monoxide. The apparent K(m) values of CO-1 and CO-2 for oxygen were 5.5 and 32 micro M, respectively, at 25 degrees C. The terminal oxidases CO-1 and CO-2 phylogenetically correspond to the SoxB and SoxM branches, respectively, of the heme-copper oxidase tree.     

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.     

Repression and inhibition of cephalosporin synthetases in Streptomyces clavuligerus by inorganic phosphate

Cephalosporin production by growing cells of Streptomyces clavuligerus was reduced by 100 mM inorganic phosphate. Resting cell production was repressed by prior growth in high phosphate and inhibited by phosphate. The cell-free activity of desacetoxycephalosporin C synthetase (ring expansion activity) was repressed by prior growth in high phosphate and inhibited by phosphate. Isopenicillin N synthetase (cyclase) was inhibited but not repressed. Penicillin epimerase was neither inhibited nor repressed by phosphate.Abbreviations DCW dry cell weight - MOPS 3-(N-morpholino) propane-sulfonic acid     

Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

The general mitochondrial processing peptidase from wheat is integrated into the cytochrome bc 1-complex of the respiratory chain

The bc ₁-complex (EC 1.10.2.2.) from Triticum aestivum L. was purified by cytochrome-c affinity chromatography and gel filtration using either etiolated seedlings or wheat-germ extract as starting material. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated enzyme revealed ten bands, which were analysed by immunoblotting and direct amino-acid sequencing. The enzyme from wheat is the first bc ₁-complex that is reported to contain four core proteins (55.5, 55.0, 51.5 and 51.0 kDa). In addition, the wheat bc ₁-complex comprises cytochrome b (35 kDa), cytochrome c ₁ (33 kDa) the Rieske iron-sulphur protein (25 kDa) and three small subunits < 15 kDa. This composition differs from the one reported in fungi, mammals and potato. Partial sequence determination of the large subunits suggests that the 55.5 and 55.0-kDa-proteins represent the -subunit of the general mitochondrial processing peptidase, and the 51.5 and 51.0-kDa proteins the -subunit of this enzyme. The bc ₁-complex from wheat efficiently processes mitochondrial precursor proteins as shown in an in-vitro processing assay. In control experiments the isolated bc ₁-complexes from potato, yeast, Neurospora and beef, all purified by the same isolation procedure, were also tested for processing activity. Only the protein complexes from plants contain the general mitochondrial processing peptidase. The composition of the wheat bc ₁-complex sheds new light on the co-evolution of the processing peptidase and the middle segment of the respiratory chain.Abbreviations MPP mitochondrial processing peptidase We wish to thank Prof. G. Schatz, Biozentrum Basel, Switzerland and Prof. H. Weiss, Universität Düsseldorf, Germany for providing antibodies against the repiratory subunits of the bc ₁-complex from yeast and Neurospora and to H. Mentzel, A. Leisse, R. Breitfeld and B. Hidde for excellent technical assistance. Thanks are also due to Prof. M. Boutry, Université de Louvaine-la-Neuve, Belgium for providing a plasmid containing the -subunit of ATPase from tobacco. This research was supported by the Deutsche Forschungsgemeinschalft and the Bundesministerium für Forschung und Technologie.     

Characterization of pbpA and pbp2 Encoding Penicillin-binding Proteins Located on the Downstream of Clavulanic Acid Gene Cluster in Streptomyces clavuligerus

Two genes, pbpA (orf18) and pbp2 (orf19) located on the downstream of clavulanic acid (CA) gene cluster of Streptomyces clavuligerus were cloned into pET-28a(+), and confirmed to encode a family of high molecular-weight penicillin-binding proteins (PBPs). Both genes were amplified from genomic DNA by PCR and expressed in E. coli BL21 (DE3). Hydropathy plots of the proteins revealed a single stretch of hydrophobic amino acids indicating them to be transmembrane proteins. Pbp2 had lower affinity to penicillin G compared to PbpA, and was essential to the cell growth in contrast to PbpA. Revisions requested 3 November 2005; Revisions received 13 December 2005     

Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus

The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for β-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M _r of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protein was purified. The cloned gene was used to construct a plasmid containing a hom disruption cassette which was then transformed into S. clavuligerus. A hom mutant of S. clavuligerus was obtained by insertional inactivation via double crossover, and the effect of hom gene disruption on cephamycin C yield was investigated by comparing antibiotic levels in culture broths of this mutant and in the parental strain. Disruption of hom gene resulted in up to 4.3-fold and twofold increases in intracellular free l-lysine concentration and specific cephamycin C production, respectively, during stationary phase in chemically defined medium.     

Structural and functional organization of the mitochondrial respiratory chain: A dynamic super-assembly

The structural organization of the mitochondrial oxidative phosphorylation (OXPHOS) system has received large attention in the past and most investigations led to the conclusion that the respiratory enzymatic complexes are randomly dispersed in the lipid bilayer of the inner membrane and functionally connected by fast diffusion of smaller redox components, Coenzyme Q and cytochrome c. More recent investigations by native gel electrophoresis, however, have shown the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis has demonstrated that Complexes I and III in mammalian mitochondria and Complexes I, III, and IV in plant mitochondria kinetically behave as single units with control coefficients approaching unity for each single component, suggesting the existence of substrate channelling within the supercomplexes. The reasons why the presence of substrate channelling for Coenzyme Q and cytochrome c was overlooked in the past are analytically discussed. The review also discusses the forces and the conditions responsible for the formation of the supramolecular units. The function of the supercomplexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes and in preventing excess oxygen radical formation. Finally, there is increasing evidence that disruption of the supercomplex organization leads to functional derangements responsible for pathological changes.     

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     

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus

A high concentration of potassium phosphate (75–100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholino)-propane sulfonate (MOPS) or 2-(N-morpholino)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.List of Non-Common Abbreviations MOPS 3-(N-morpholino)propane sulfonic acid - MES 2-(N-morpholino)ethane sulfonic acid     

The cytochromec reductase/oxidase respiratory pathway ofParacoccus denitrificans: Genetic and functional studies

Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec ₅₅₂, and thea-type terminal oxidase. Deletion mutants in thebc ₁ and theaa ₃ complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec ₅₅₂ in electron transport between complex III and IV is presented.     

The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis

The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between two HQNO inhibition sites, or more probably in a Q- or b-cycle.Abbreviation HQNO= 2-n-heptyl-4-hydroxy-quinoline-N-oxide     

A structural model for the adduct between cytochrome c and cytochrome c oxidase

An ensemble of structural models of the adduct between cytochrome c and cytochrome c oxidase from Paracoccus denitrificans has been calculated based on the experimental data from site-directed mutagenesis and NMR experiments that have accumulated over the last years of research on this system. The residues from each protein that are at the protein–protein interface have been identified by the above experimental work, and this information has been converted in a series of restraints explicitly used in calculations. It is found that a single static structural model cannot satisfy all experimental data simultaneously. Therefore, it is proposed that the adduct exists as a dynamic ensemble of different orientations in equilibrium, and may be represented by a combination or average of the various limiting conformations calculated here. The equilibrium involves both conformations that are competent for electron transfer and conformations that are not. Long-range recognition of the partners is driven by non-specific electrostatic interactions, while at shorter distances hydrophobic contacts tune the reciprocal orientation. Electron transfer from cytochrome bc ₁ to cytochrome c oxidase is mediated through cytochrome c experiencing multiple encounters with both of its partners, only part of which are productive. The number of encounters, and thus the electron transfer rate, may be increased by the formation of a cytochrome bc ₁–cytochrome c oxidase supercomplex and/or (in human) by increasing the concentration of the two enzymes in the membrane space. Protein Data Bank Accession numbers The coordinates of the five best structural models for each of the four clusters have been deposited in the Protein Data Bank (PDB ID 1ZYY).     

Designing a whole-cell biotransformation system in <Emphasis Type="Italic">Escherichia coli</Emphasis> using cytochrome P450 from <Emphasis Type="Italic">Streptomyces peucetius</Emphasis>

A biotransformation system was designed to co-express CYP107P3 (CSP4), cytochrome P450, from Streptomyces peuceticus, along with CamA (putidaredoxin reductase) and CamB (putidaredoxin) from Pseudomonas putida, the necessary reducing equivalents, in a class I type electron-transfer system in E. coli BL21 (DE3). This was carried out using two plasmids with different selection markers and compatible origins of replication. The study results showed that this biotransformation system was able to mediate the O-dealkylation of 7-ethoxycumarin.     

The cytochrome composition of the meat spoilage bacterium Brochothrix thermosphacta: identification of cytochrome a 3- and d-type terminal oxidases under various conditions

Brochothrix thermosphacta, grown in batch culture in a yeast-dextrose broth, at temperatures from 30 °C to 10 °C, contained diverse membrane-bound respiratory cytochromes. Under conditions of moderate aeration, cytochromes of the a-, b- and d-type were detected at all growth temperatures, but the proportions changed as a function of temperature, with the spectra of cells grown at 10 or 15 °C being dominated by a-type cytochrome(s). Cytochrome a ₃ was detected by its reactions with CO and cyanide in cells from all growth conditions. An additional cytochrome a, which was not cyanide-reactive, was also detected, suggesting the presence of an aa ₃ oxidase complex. Cytochrome d was cyanide- and CO-reactive, but not detectable in photodissociation spectra, presumably because of the very rapid recombination of CO at the sub-zero temperatures used. Decreasing the oxygen transfer rates to batch cultures resulted in enhanced expression of cytochrome d and changed the proportion of the aa ₃-type oxidase that could be attributed to ligand-binding cytochrome a ₃; at the lowest oxygen transfer rates, no cytochrome a was detected, suggesting the presence of a cytochrome ba ₃ terminal oxidase complex. Intact cells showed no evidence of a c-type cytochrome and no haem C was detected in membrane preparations. After growth at 10°C, the cytochrome composition of B. campestris was essentially identical to that of B. thermosphacta. The multiplicity of putative terminal oxidases in B. thermosphacta is discussed.     

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b₅, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b₅ than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450_PB purified from rabbit liver, addition of cytochrome b₅ to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b₅ enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

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     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.