The control properties of phosphofructokinase in relation to the respiratory climacteric in banana fruit

Glucose 6-phosphate, fructose 6-phosphate, fructose 1, 6-diphosphate, and triose phosphates, and the enzymes phosphofructokinase, aldolase, and glucose 6-phosphate dehydrogenase were extracted from banana fruit (Musa cavendishii, Lambert var. Valery) at the (a) preclimacteric, (b) climacteric rise, (c) climacteric peak, and (d) postclimacteric stages of ripening. The level of fructose 1, 6-diphosphate increased 20-fold whereas the concentration of other intermediates changed no more than 2.5-fold between stages a and c. For these same extracts, phosphofructokinase activity increased 2.5-fold whereas the activity of glucose 6-phosphate dehydrogenase and aldolase changed only fractionally. Substrate saturation studies (fructose 6-phosphate) of phosphofructokinase activity showed a decrease in the [S]_0.5 from 5.6 to 1.7 mM betwen stages a and c. The enzyme from both sources seems to be regulated by a negative cooperative effect with the control being more stringent in the enzyme from stage a. The difference in enzyme activity is consistent with the increase in respiratory activity between the two stages.     

The oxidation mechanisms of thiosulphate and sulphide in Chlorobium thiosulphatophilum: Roles of cytochrome c-551 and cytochrome c-553

A thiosulphate-cytochrome c reductase was highly purified from Chlorobium thiosulphatophilum and its properties were studied. The enzyme catalyses reduction with Na₂S₂O₃ of c cytochromes, including cytochrome c-551 of the bacterium. Cytochrome c (555, C. thiosulphatophilum) does not react directly with the enzyme at an appreciable rate but stimulates greatly the reduction by the enzyme of cytochrome c-551 with Na₂S₂O₃. The reduction of c cytochromes catalysed by the enzyme is strongly inhibited by cyanide and sulphite.Cytochrome c (553, C. thiosulphatophilum), a c-type cytochrome with covalently bound flavin, was found to catalyse reduction with sulphide of c cytochromes, including cytochrome c-555. The reaction is strongly inhibited by cyanide. Cyanide seems to combine strongly with cytochrome c-553 probably at the flavin moiety. Thus, the absorption spectrum attributable to flavin of the haemoprotein is changed on addition of cyanide, and neither the original spectrum nor the activity reappears even after the cyanide-treated cytochrome has been subjected to gel filtration with a Sephadex G-25 column or to isoelectric focusing.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles

1. Beef heart mitochondria have a cytochrome c₁ : c : aa₃ ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have a ratio of 0.65 : 0.4 : 1.0. More than 50% of the submitochondrial particle membrane is in the ‘inverted’ configuration, shielding the catalytically active cytochrome c. The ‘endogenous’ cytochrome c of particles turns over at a maximal rate between 450 and 550 s^?1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300–400 s^?1, at 28° – 30°C, pH 7.4.2. Ascorbate plus N,N,N′,N′-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c₁ and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively.3. Cytochrome c₁ is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate+N,N,N′,N′-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c₁ and c in the aerobic steady state, with a rate constant for the c₁ → c reduction step greater than 10³ s^?1.4. The greater apparent response of the $\text{c}\text{aa}{}_3$ electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the $\text{c}{}_1\text{c}$ step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa₃ units in situ. Cytochrome c₁ can either reduce the cytochrome c-cytochrome aa₃ complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Evidence for a structural interacti on betwen ATP synthetase and cytochrome c oxidase in mitochondria

The reaction of cyanide with the oxidized form of cytochrome c oxidase in mitochondria is strongly inhibited by adenosine triphosphate (ATP). This inhibition is strictly dependent on the ATP concentration and is insensitive to changes in the concentrations of adenosine diphosphate (ADP) and orthophosphate. It is completely prevented by oligomycin or uncouplers of oxidative phosphorylation. The ATP is kinetically competitive with respect to cyanide and has a measured inhibitor constant of less than 2 μm The stoichiometry is one ATP/cyanide. This ATP effect is proposed to result from a structural interaction of ATP synthetase with cytochrome c oxidase, such that the formation of an ATP complex of the synthetase results in a decrease in the affinity of the oxidized form of cytochrome c oxidase for cyanide in the formation of an intermediate in the overall measured cyanide reaction.     

Aryl hydrocarbon hydroxylase activity in the fungus Cunninghamella bainieri: evidence for the presence of cytochrome P-450.

The aryl hydrocarbon hydroxylase (AHH) enzyme from the fungus Cunninghamella bainieri has been characterized. It is NADPH dependent and exhibits a pH optimum near 7.8. It is inhibited by CO, SKF 525-A, and metyrapone, but cyanide shows no inhibitory effect. These data, together with the pattern of inhibition and stimulation shown by metal ions, suggest that the fungal AHH activity is due to a cytochrome P-450. About 25% of the hydroxylase activity remains in the supernatant while the remainder precipitates after centrifugation at 100,00g for 2.5 h. The 100,000g supernatant was further fractionated by (NH₄)₂SO₄ precipitation. A NADPH-dependent cytochrome c reductase is concentrated mainly in the 100,000g supernatant, and a cytochrome c oxidase is present mainly in the 100,000g pellet. The cytochrome c reductase is essential for AHH activity as shown by the inhibition of AHH activity with cytochrome c and dichloroindophenol. Solubilization of a portion of the 100,000g pellet in aqueous digitonin followed by dithionite reduction and addition of CO resulted in the observation of a maximum absorbance at 450 nm characteristic of cytochrome P-450.     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.     

Inhibition by cyanide of the respiratory chain oxidases of Escherichia coli

The kinetics of inhibition by KCN of NADH oxidation in respiratory particles from Escherichia coli could be related to the relative amounts of cytochromes d and o which were present. Particles which contained higher levels of cytochrome d relative to cytochrome o were less sensitive to inhibition by cyanide. When cyanide reacted with the respiratory particles, the absorption bands of reduced cytochrome d at 442 and 628 nm in the reduced plus cyanide minus reduced difference spectrum were eliminated, as also were the bands at 423, 428, and 555 nm of b- and/or c-type cytochromes.Cyanide appeared to react with the oxidized form of cytochrome d to eliminate its α-band absorption with a second-order rate constant of 0.011 m^?1 sec^?1 for the rate of formation of cyanocytochrome d in the absence of added substrate. Under turnover conditions using NADH as substrate, the rate constant was 0.58 m^?1 sec^?1. This value is close to that determined from cyanide inhibition of NADH oxidase activity. The magnitude of the second-order rate constant for the formation of cyanocytochrome d was directly related to the rate of electron flux through cytochrome d. It is suggested that an intermediate species formed during the normal oxidation-reduction cycle of cytochrome d reacts with cyanide.     

Effect of cardiolipin on the enzymatic activity of Nitrobacter agilis cytochrome c oxidase

Effects of cardiolipin on the reaction rates of Nitrobacter agilis cytochrome c oxidase with cytochrome c were studied at various concentrations of phosphate buffer. Cardiolipin stimulated greatly the oxidation by the enzyme of horse and yeast ferrocytochromes c, especially at higher ionic strengths. However, the oxidation by the enzyme of N. agilis ferrocytochrome c-550, the physiological electron donor for the oxidase, was not accelerated by addition of cardiolipin. Analysis of the lipid compositions showed that neither the cell membranes of N. agilis nor the enzyme preparation contained cardiolipin. These results suggest that cardiolipin is not necessary for the reaction of N. agilis cytochrome c oxidase with N. agilis cytochrome c-550. On the basis of these results, the difference in the reactivity with cytochrome c of cytochrome c oxidase between the bacterial and mitochondrial enzymes is discussed.     

Nitrate Reductase and Soluble Cytochrome c Reductase(s) in Higher Plants

The 4S cytochrome c (Cyt c) reductase activity of several plant species was markedly stimulated by cyanide and ferrocyanide but those of the 8S nitrate reductase component and other particulate components of the maize (Zea mays L.) scutellum by comparison, were increased only slightly. The effect of cyanide and ferrocyanide was not due to elimination of cytochrome oxidase interference but resulted from the stimulation of NADH-dependent reduction of Cyt c. A 4S Cyt c reductase component which could be isolated by ammonium sulfate fractionation and diethyl-aminoethyl-cellulose chromatography was found to be stimulated markedly by cyanide and ferrocyanide. The remaining 4S Cyt c reductase, which was insensitive to cyanide and ferrocyanide, was also fractionated with ammonium sulfate into two components. One of these, like the 8S Cyt c reductase, was sensitive to a protease from the maize roots which is relatively specific for nitrate reductase. This 4S Cyt c reductase species could be a subunit of nitrate reductase.     

Characterization of the Proton-Translocating Cytochrome c Oxidase Activity in the Plasma Membrane of Intact Anacystis nidulans Spheroplasts

Intact spheroplasts of the cyanobacterium (blue-green alga) Anacystis nidulans oxidized various exogenous c-type cytochromes with concomitant outward proton translocation while exogenous ferricytochrome c was not reduced. The H⁺/e⁻ stoichiometry was close to 1 with each of the cytochromes and did not depend on the actual rate of the oxidase reaction. Observed proton ejections were abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Cyanide, azide, and carbon monoxide inhibited cytochrome c oxidation and proton extrusion in parallel while dicyclohexylcarbodiimide affected proton translocation more strongly than cytochrome c oxidation. The cytoplasmic membrane of A. nidulans appears to contain a proton-translocating cytochrome c oxidase similar to the one described for mitochondria.     

Isolation and properties of cytochrome c-553, cytochrome c-550, and cytochrome c-549, 554 from Nitrobacter agilis

Three c-type cytochromes isolated from Nitrobacter agilis were purified to apparent homogeneity: cytochrome c-553, cytochrome c-550 and cytochrome c-549, 554. Their amino acid composition and other properties were studied. Cytochrome c-553 was isolated as a partially reduced form and could not be oxidized by ferricyanide. The completely reduced form of the cytochrome had absorption maxima at 419, 524 and 553 nm. It had a molecular weight of 25 000 and dissociated into two polypeptides of equal size of 11 500 during SDS gel electrophoresis. The isoelectric point of cytochrome c-553 was pH 6.8. The ferricytochrome c-550 exhibited an absorption peak at 410 nm and the ferrocytochrome c showed peaks at 416, 521 and 550 nm. The molecular weight of the cytochrome estimated by gel filtration and by SDS gel electrophoresis was 12 500. It had an E_m(7) value of 0.27 V and isoelectric point pH 8.51. The N-terminal sequence of cytochrome c-550 showed a clear homology with the corresponding portions of the sequences of other c-type cytochromes. Cytochrome c-549, 554 possessed atypical absorption spectra with absorption peaks at 402 nm as oxidized form and at 419, 523, 549 and 554 nm when reduced with Na₂S₂O₄. Its molecular weight estimated by gel filtration and SDS polyacrylamide gel electrophoresis was 90 000 and 46 000, respectively. The cytochrome had an isoelectric point of pH 5.6. Cytochrome c-549, 554 was highly autoxidizable.     

Recurrence of the cytochrome fold in a nitrate-respiring bacterium

The crystal structure of cytochrome c₅₅₀ (formerly MDC) from the oxygen or nitrate-respiring bacterium Micrococcus denitrificans has been determined to a resolution of four Ångstrom units. By comparison with the structures of eukaryotic cytochrome c and photosynthetic Rhodospirillum rubrumc₂, the polypeptide chain path can be followed from one end of the protein to the other. Many aromatic side chains can be identified even at 4 Å. The same “cytochrome fold” is seen in all three redox proteins. Cytochrome c₅₅₀ has the same insertions on the left-hand side of the molecule as c₂ (in comparison with c), plus three more external additions and a changed right-hand side hairpin bend, which together account for approximately 25 residues. In terms of gross chain folding, c₅₅₀ is more like c₂ than c, but in its arrangement of aromatic residues in the righthand side channel, the two respiratory cytochromes c and c₅₅₀ are most nearly alike. The similarities and differences in these three proteins support earlier suggestions concerning important and unimportant zones on the molecular surface, and lead to testable hypotheses about evolutionary descent. The cytochrome fold is the oldest common structure found to date among diverse proteins and may be a useful diagnostic tool in studying the process of protein evolution.     

Reevaluation of assay methods and establishment of kit for superoxide dismutase activity

Superoxide dismutase (SOD) activity was measured by seven assay methods. The nitrite method was found to be the best for our SOD assay kit. This method was then modified to give better sensitivity and minimize interference by coexisting protein, a factor which has been previously ignored. Hydroxylamine or its O-sulfonic acid, xanthine oxidase, hypoxanthine, EDTA, and the sample were incubated with or without KCN at pH 8.2, 37°C, for 30 min. Diazo dye-forming reagent was added and the absorption was measured at 550 nm. Human plasma and erythrocyte lysate from healthy adults and Down's syndrome patients were assayed by this SOD kit and by the cytochrome c method. Our kit gave 8.5 times higher sensitivity than the cytochrome c method. This high sensitivity allowed the use of a simple spectrophotometer and, moreover, only one dilution was needed to determine the SOD unit with the help of our formulas. Good recovery, reproducibility, and stability of reagents were demonstrated.     

Manganese (III) phthalocyanine-substituted cytochrome c

Manganese phthalocyanine-substituted cytochrome c has been prepared by the reaction of Mn(III) tetrasulfonated phthalocyanine with apocytochrome c in acetate buffer, pH 5.8. Its structure and properties have been investigated by difference spectroscopy, circular dichroism (cd), electron paramagnetic resonance (epr), electrophoresis, molecular weight estimation, and potentiometric measurements. The epr and spectroscopic data show that the manganese phthalocyanine-substituted cytochrome c represents the low spin, six-coordinated. Mn(Ill) complex with the metal ion in the plane of the phthalocyanine ring. The sixth ligand, which is coordinated axially to the metal ion, is probably the methionine-80. Electrophoresis and molecular weight studies show this complex to be a monomer. As is shown by cd experiments, Mn(III)L-apocyt has a more ordered structure than that of apocytochrome c. Its conformation is, however, significantly altered compared to native cytochrome c. The manganese(III)-phthalocyanine complex is able to combine with cyanide. The cyanide derivative gives a stable reduced form upon dithionite reduction. If, however, Mn(IlI)Lapocyt is reduced with dithionite before addition of cyanide, it loses its ability to coordinate with cyanide. Nitric oxide reacts with the manganese(III) complex to form, in all probability, the nitrosyl derivative. The half-reduction potential of Mn(IlI)L-apocyt is about +400 mV, and the complex is reduced by cytochrome c. Spectroscopic data suggest that the mechanism of this process is complicated.     

Characterization of cytochrome c-550 from cyanobacteria

Cytochrome c-550 has been purified from several cyanobacteria. It is a low-potential, auto-oxidizable cytochrome. This cytochrome should not be confused with a degradation product of cytochrome ? which may be formed during the isolation of the latter protein. Cytochromes c-550 are distinctive in size, amino-acid composition and N-terminal amino-acid sequence.     

Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Cytochrome c interaction with membranes. Formylated cytochrome c.

A single species of tryptophan-59 formylated cytochrome c with a half-reduction potential of 0.085 ± 0.01 V at pH 7.0 was used to study its catalytic and functional properties. The spectral properties of the modified cytochrome show that the 6th ligand position is open to reaction with azide, cyanide, and carbon monoxide. Formylated cytochrome c binds to cytochrome c depleted rat liver and pigeon heart mitochondria with the precise stoichiometry of two modified cytochrome c molecules per molecule of cytochrome a (K_D of approx 0.1 μm). Formylated cytochrome c was reducible by ascorbate and was readily oxidized by cytochrome c oxidase. The apparent K_m value of the oxidase for the formylated cytochrome c was six times higher than for the native cytochrome and the apparent V was smaller. Formylated cytochrome c does not restore the oxygen uptake in C-depleted mitochondria but inhibits, in a competitive manner, the oxygen uptake induced by the addition of native cytochrome c. Formylated cytochrome c was inactive in the reaction with mitochondrial NADH-cytochrome c reductase but was able to accept electrons through the microsomal NADPH-cytochrome c reductase.     

Enzyme co-localization in pea leaf chloroplasts: glyceraldehyde-3-P dehydrogenase,triose-P isomerase,aldolase and sedoheptulose bisphosphatase

Nearest neighbor analysis of immunocytolocalization experiments indicates that the enzymes glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase are located close to one another in the pea leaf chloroplast stroma, and that aldolase is located close to sedoheptulose bisphosphatase. Direct transfer of the triose phosphates between glyceraldehyde-3-P dehydrogenase and triose-P isomerase, and from glyceraldehyde-3-P dehydrogenase and triose-P isomerase to aldolase, is then a possibility, as is direct transfer of sedoheptulose bisphosphate from aldolase to sedoheptulose bisphosphatase. Spatial organization of these enzymes may be important for efficient CO₂ fixation in photosynthetic organisms. In contrast, there is no indication that fructose bisphosphatase is co-localized with aldolase, and direct transfer of fructose bisphosphate from aldolase to fructose bisphosphatase seems unlikely.