The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.     

Light-inducible Cytochrome Reduction in Membrane Preparations from Corn Coleoptiles: I. STABILIZATION AND SPECTRAL CHARACTERIZATION OF THE REACTION

Conditions for obtaining reproducible light-induced reduction of a b-type cytochrome in membrane fractions from coleoptiles of dark-grown Zea mays L. include a glucose-glucose oxidase system that lowers O₂ tension and generates H₂O₂, substrate amounts of ethylenediaminetetraacetic acid which, in some manner, facilitates photoreduction by both added flavin and the endogenous photoreceptor and a sample temperature below 10 C. Cytochrome reduction could be obtained by photoexcitation of either a tightly bound endogenous receptor, which is probably a flavin, or added riboflavin, flavin mononucleotide, or flavin adenine dinucleotide. The latter flavin was the least effective. The endogenous photoreceptor appears to be rather firmly bound to the membranes, suggesting that this association may also exist in vivo. When any of the above four photoreceptors or methylene blue were used to sensitize the reaction, a cytochrome with a reduced α-band near 560 nanometers and a Soret difference peak near 429 nanometers was the electron acceptor. This cytochrome could be clearly distinguished spectrally from other cytochromes that predominated in the membrane preparations.     

Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase.

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH----FAD----cytochrome b-245----O2.     

Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa

We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b₅ or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.

Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum.

     

FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg⁻¹ protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min⁻¹·mg⁻¹ protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.     

Electron transport in glyoxysomal membranes

Glyoxysomes were isolated from germinating castor bean endosperm by equilibrium density gradient centrifugation in a vertical rotor. To recover the membranes, glyoxysome ghosts were prepared by osmotic shock and then subjected to differential centrifugation. The glyoxysomal membranes and the endoplasmic reticulum (ER), isolated by the same methods, were assayed for electron transport components. Both organelles contained NADH ferricyanide reductase, NADH cytochrome c reductase, and cytochromes b₅ and P-420. The ER also contained cytochrome P-450. Pyridine hemochrome derivatives of the organelle membranes and hemin produced coincident difference spectra, indicating that only b-type cytochromes are present in glyoxysomal and ER membranes. The maximal activities of ferricyanide reductase and cytochrome c reductase in glyoxysomes, 2.19 and 0.33 μmol min^?1 mg membrane protein^?1, respectively, represent 30 and 18% of the activities in the ER. The cytochrome b₅ content of the glyoxysomal membrane is 0.108 nmol mg^?1, 31% of the level found in ER. The reductases from both organelles were resistant to solubilization by salt (0.2 m KCl) and were easily solubilized by detergent (1% Triton X-100). Flavin analysis of the organelles from germinating castor beam endosperm confirmed spectral evidence that the flavin content of glyoxysomes is quite high, 100 pmol mg protein^?1, more than twice that of mitochondria. Three-quarters of the glyoxysomal flavin was solubilized by KCl, but even after salt treatment the glyoxysomal membrane flavin content, 98 pmol mg membrane protein^?1, is three times greater than that of the ER.     

Identification and characterization of the flavin:NADH reductase (PrnF) involved in a novel two-component arylamine oxygenase

Two-component oxygenases catalyze a wide variety of important oxidation reactions. Recently we characterized a novel arylamine N-oxygenase (PrnD), a new member of the two-component oxygenase family (J. Lee et al., J. Biol. Chem. 280:36719-36728, 2005). Although arylamine N-oxygenases are widespread in nature, aminopyrrolnitrin N-oxygenase (PrnD) represents the only biochemically and mechanistically characterized arylamine N-oxygenase to date. Here we report the use of bioinformatic and biochemical tools to identify and characterize the reductase component (PrnF) involved in the PrnD-catalyzed unusual arylamine oxidation. The prnF gene was identified via sequence analysis of the whole genome of Pseudomonas fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. The purified PrnF protein catalyzes reduction of flavin adenine dinucleotide (FAD) by NADH with a k_cat of 65 s⁻¹ (K_m = 3.2 μM for FAD and 43.1 μM for NADH) and supplies reduced FAD to the PrnD oxygenase component. Unlike other known reductases in two-component oxygenase systems, PrnF strictly requires NADH as an electron donor to reduce FAD and requires unusual protein-protein interaction with the PrnD component for the efficient transfer of reduced FAD. This PrnF enzyme represents the first cloned and characterized flavin reductase component in a novel two-component arylamine oxygenase system.     

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3^−/− mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3^−/− progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-X_L rescued survival; however, Bcl-X_L-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3^−/− cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-X_L permitted differentiation of G6PC3^−/− cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation and survival thus underlies neutropenia in G6PC3^−/− deficiency, both originating from a reduced ability to utilize glucose. Hoxb8-dependent cells are a model to study differentiation and survival of the neutrophil lineage.     

Composition of partially purified NADPH oxidase from pig neutrophils.

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.     

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b₅, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c reductases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b₅. In addition, cytochrome b₅ fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with M_r values in the range 10 000–13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

Topography of human cytochrome b5/cytochrome b5 reductase interacting domain and redox alterations upon complex formation

Cytochrome b₅ is the main electron acceptor of cytochrome b₅ reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b₅ reductase flavin autofluorescence that was dependent upon the presence of cytochrome b_5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5 ± 0.1 μM and a 1:1 stoichiometry for the complex formation. In addition, a 30 mV negative shift of cytochrome b₅ reductase redox potential in presence of cytochrome b₅ was also measured. These experiments suggest that the FAD group of cytochrome b₅ reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.     

ACTH- and Cortisol-Associated Neutrophil Modulation in Coronary Artery Disease Patients Undergoing Stent Implantation

Background

Psychosocial stress and activation of neutrophil granulocytes are increasingly recognized as major risk factors of coronary artery disease (CAD), but the possible relationship of these two factors in CAD patients is largely unexplored. Activation of neutrophils was reported to be associated with stenting; however, the issue of neutrophil state in connection with percutaneous coronary intervention (PCI) is incompletely understood from the aspect of stress and its hypothalamic-pituitary-adrenal axis (HPA) background. Thus, we aimed to study cortisol- and ACTH-associated changes in granulocyte activation in patients undergoing PCI.

Methodology/Principal Findings

Blood samples of 21 stable angina pectoris (SAP) and 20 acute coronary syndrome (ACS) patients were collected directly before (pre-PCI), after (post-PCI) and on the following day of PCI (1d-PCI). Granulocyte surface L-selectin, CD15 and (neutrophil-specific) lactoferrin were analysed by flow cytometry. Plasma cortisol, ACTH, and lactoferrin, IL-6 were also assayed. In both groups, pre- and post-PCI ratios of lactoferrin-bearing neutrophils were relatively high, these percentages decreased substantially next day; similarly, 1d-PCI plasma lactoferrin was about half of the post-PCI value (all p≤0.0001). Post-PCI ACTH was reduced markedly next day, especially in ACS group (SAP: p<0.01, ACS: p≤0.0001). In ACS, elevated pre-PCI cortisol decreased considerably a day after stenting (p<0.01); in pre-PCI samples, cortisol correlated with plasma lactoferrin (r∼0.5, p<0.05). In 1d-PCI samples of both groups, ACTH showed negative associations with the ratio of lactoferrin-bearing neutrophils (SAP: r = −0.601, p<0.005; ACS: r = −0.541, p<0.05) and with plasma lactoferrin (SAP: r = −0.435, p<0.05; ACS: r = −0.609, p<0.005).

Conclusions/Significance

Pre- and post-PCI states were associated with increased percentage of activated/degranulated neutrophils indicated by elevated lactoferrin parameters, the 1d-PCI declines of which were associated with plasma ACTH in both groups. The correlation of plasma cortisol with plasma lactoferrin in the extremely stressed ACS before stenting, however, suggests an association of cortisol with neutrophil activation.     

The respiratory chain components of higher plant mitochondria

Tightly coupled mitochondria have been prepared from a variety of plant sources: white potato (Solanum tuberosum), Jerusalem artichoke (Heliantus tuberosus), cauliflower buds (Brassica oleracea), and mung bean hypocotyls (Phaseolus aureus). Mitochondria with no appreciable coupling were also prepared from skunk cabbage spadices (Symplocarpus foetidus).

Room temperature difference spectra show that these mitochondria are very similar in the qualitative and quantitative composition of their electron carriers. The different cytochromes are present in the amounts of 0.1 to 0.3 mμmole per mg of mitochondrial protein. The molar ratios of the different electron carriers are, on the average: 0.7:0.7:1.0:3 to 4:10 to 15 respectively for cytochrome aa₃, cytochromes b, cytochromes c, flavoproteins, and pyridine nucleotides.

From low temperature difference spectra carried out under particular experimental conditions, it can be deduced that these mitochondria contain 3 b cytochromes whose α bands are located at 552, 557, and 561 mμ, and 2 c cytochromes, one of which, a c₁-like cytochrome, is firmly bound to the mitochondrial membrane. Cytochrome oxidase can be optically resolved into its 2 components a and a₃.

For all kinds of mitochondria, the rates of oxidation of succinate are similar as well as the turnover of cytochrome oxidase (50-70 sec⁻¹), regardless of the metabolic activities of the tissues. The number of mitochondria per cell appears to be the controlling factor of the intensity of tissue respiration.

     

Rapid incorporation of the human neutrophil plasma membrane cytochrome b into phagocytic vacuoles

Phagocytic vacuoles containing lgG coated latex particles were isolated from human neutrophils by floatation. The absorbance spectrum of the cytochrome $\text{b}$ was associated with the vacuoles within 10 sec of particle uptake and the vacuolar concentration increased little thereafter. In contrast, the cytoplasmic granule proteins myeloperoxidase and vitamin B₁₂ binding protein associate with the vacuoles more slowly. The addition of dithionite to intact cells rapidly reduces most of the cytochrome $\text{b}$, whereas only a small proportion of the myeloperoxidase, which is located intracellularly, is reduced in the absence of detergent. Most of the cytochrome $\text{b}$ appears to be localised in the neutrophil plasma membrane.     

b-Type Cytochromes in Higher Plant Plasma Membranes

The composition and characteristics of b-type cytochromes from higher plant plasma membranes, purified using aqueous two-phase partitioning, were investigated. At least three different cytochromes were identified by their wavelength maxima and redox midpoint potentials (E₀′). Cytochrome b-560.7 (E₀′ from + 110 to + 160 millivolts) was present in zucchini (Cucurbita pepo) hypocotyls and bean (Phaseolus vulgaris L.) hooks, although in different concentrations. The main component in cauliflower (Brassica oleracea L.) inflorescences (cytochrome b-558.8) is probably functionally similar to this cytochrome. The plasma membrane generally contains two to three cytochrome species. However, the occurrence and concentrations were species dependent. The high potential cytochrome can be reduced by ascorbate but not NADH, and may be involved in blue light perception.     

Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a₃ and cytochrome c+c₁ were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a₃ ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c₁/cytochrome a+a₃ ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.     

Energetic factors affecting carbon dioxide fixation in isolated chloroplasts

Light- and HCO₃⁻-saturated (10 millimolar) rates of O₂ evolution (120 to 220 micromoles O₂ per milligram chlorophyll per hour), obtained with intact spinach chloroplasts, are decreased up to 3-fold by changes in assay conditions such as omission of catalase from the medium, the use of high (≥1 millimolar) inorganic phosphate, inclusion of NO₂⁻ as an electron acceptor, or bright illumination at low partial pressures of O₂. These inhibitions may be reversed by addition of uncoupling levels of NH₄Cl or of antimycin concentrations that partially block cyclic electron transfer between cytochrome b₆ and cytochrome f. Measurements of the pH gradient across the thylakoid membrane with the fluorescent probe, 9-aminoacridine, indicate that changes in ΔpH are sufficient to account for both the inhibited and restored rates of electron transport. It follows that the rate of HCO₃⁻-saturated photosynthesis may be restricted by a proton gradient back pressure under these conditions.