The mode of action of stigmatellin,a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

The new antibiotic stigmatellin, obtained from the myxobacterium Stigmatella aurantiaca, was found to block the electron flow in the respiratory chain of bovine heart submitochondrial particles at the site of the cytochrome b-c₁ segment. Its inhibitory potency was identical with that of antimycin and myxothiazol, and like these antibiotics, stigmatellin caused a shift in the spectrum of reduced cytochrome b. Difference spectroscopic studies with the three inhibitors in various combinations indicated that the binding site of stigmatellin was different from that of antimycin, but more or less identical with that of myxothiazol. Experiments with 14 synthesized derivatives of stigmatellin showed that good inhibitory activity can be expected only if the side chain was kept relatively lipophilic, and the keto and the hydroxy groups of the chromone system were left intact.     

Electron transport systems of Candida utilis. Purification and properties of the respiratory chain-linked external NADH dehydrogenase+

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 · 10⁶. The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ₆ and CoQ₁ derivatives as acceptors. Rotenone (10^?5 M) and seconal (10^?3 M) do not inhibit enzymatic activity.     

Kinetics of ubiquinone reduction by the resolved succinate: Ubiquinone reductase

A significant lag in the thenoyltrifluoroacetone (TTFA)-sensitive succinate: ubiquinone reductase activity was observed when a ubiquinone-deficient resolved preparation of the enzyme was assayed in the presence of exogenous ubiquinone-2 (Q2) and 2,6-dichlorophenolindophenol. No such lag was seen when the free radical of N,N,N′,N′-tetramethyl-p-phenylenediamine (Wurster's Blue) was used as the terminal electron acceptor, or when the reduction of Q2 was directly measured. The apparent Km value for exogenous Q2 was determined in the Q2-mediated TTFA-sensitive succinate: Wurster's Blue reductase reaction. When the enzyme activity was measured directly by monitoring Q2 reduction without terminal acceptors, the time course of the reaction deviated from zero-order kinetics at Q2 concentrations which were much higher than those expected from the KQ2m value determined in the presence of Wurster's Blue. The time course of Q2 reduction fits a curve describing a competitive interrelationship between oxidized and reduced Q2 at the specific binding site. The data obtained are in agreement with the Q-pool behavior of ubiquinone in mitochondrial membranes and suggest that the rate of ubiquinone reduction by succinate is dependent on the ratio.     

Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c₁ segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. II. Kinetics of reoxidation of the reduced enzyme

The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1–4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6–6.5 for the reaction with ubiquinone.     

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

Identification of two different Q-binding sites in QH2-cytochrome c oxidoreductase,using the Q analogue n-heptadecylmercapto-6-hydroxy-5,8-quinolinequinone

The pK and mid-point redox potential of the Q-analogue 7-(n-heptadecyl)mercapto-6-hydroxy-5,8-quinolinequinone (HMHQQ) in aqueous medium are so low that under the experimental conditions used for studying the inhibition of electron transfer in submitochondrial particles only the oxidized, anionic form is present. The K_D of the analogue, determined by comparing its inhibitory effect with that of n-heptyl-4-hydroxyquinolineN-oxide, is (0.003+0.24×mg protein/ml) μM. The inhibition of succinate oxidation is pH dependent, due to a pH-dependent change in the overcapacity of the QH₂-oxidizing system above the Q-reducing system. If the terminal part of the respiratory chain is reduced with ascorbate, the analogue inhibits the reduction of cytochrome b by substrate in the presence of antimycin with a similar K_D value. In the absence of ascorbate the K_D value is 100-times higher. The reduction of cytochrome b by substrate in particles treated with 2,3-dimercaptopropanol (BAL)+O₂ is also sensitive to HMHQQ, with a K_D value in between the two values given above. It is concluded that the QH₂ oxidase system contains two different sites for interaction with ubiquinone. The site responsible for the inhibition of steady-state electron transfer is near the Fe-S cluster, as is shown by the sensitivity to the redox state of this cluster and by the effect of HMHQQ on the EPR signal of the reduced cluster. The second site, which is similar to the antimycin-binding site, is occupied only at higher concentrations of inhibitor. The affinity of HMHQQ for this site is not affected by the redox state of the Fe-S cluster.     

The effect of respiratory inhibitors during growth and development of Dictyostelium discoideum

Two major pathways of electron transport to oxygen were identified in intact cells of Dictyostelium discoideum, a cyanide-sensitive pathway and a cyanide-insensitive, salicylhydroxamic-acid-sensitive pathway. The extent to which each pathway contributed to the total respiratory activity was shown to change during exponential growth and throughout development. During exponential growth both pathways appear to be utilized to varying degrees dependent on culture age, during late exponential growth the activity of the salicylhydroxamic-acid-sensitive pathway would seem to be almost totally lost. During development the cyanide-sensitive pathway appears to be dominant up to the aggregation stage, but both pathways are active in pseudoplasmodial cells. It is also suggested that the presence of iron in the growth medium may be essential directly or indirectly, for the maintained activity of the salicylhydroxamic-acid-sensitive pathway late in exponential growth.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A₂₇₈/A₄₉₀ is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids.

The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile sulfur groups and either four (ferredoxin II) or six (ferredoxin I) cysteine residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A₃₈₅/A₂₈₃ absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively.

The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.

The physiological activity of ferredoxins I and II appears to be similar as far as the electron transfer in the phosphoroclastic reaction is concerned.     

A kinetic model structure for delayed fluorescence from plants

In this research, we demonstrated that the plastoquinone-related electron-transport kinetics in photosynthesis could be sufficiently described with as few as three state variables, Q(A)(-), Q(B)(-), and Q(B)(2-). A third-order kinetic model structure was developed with delayed fluorescence as the measurable output. Delayed fluorescence emissions from drought-stressed, DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] treated, or healthy plants were measured with a photon-counting system and used to verify the model structure through nonlinear least-squares optimization. While there were no visible differences between the healthy and the stressed plants, the model showed an obvious decrease of Q(A) reduction rate in the drought-stressed samples and a clear decline of functional Q(A)Q(B) pairs in the DCMU-treated samples. The changes were consistent with the known mechanisms by which water and DCMU affect electron transport in photosynthetic plants. The results proved that the three-state formulation was a compact and practically useful model structure for describing delayed fluorescence from plants.     

Identification of three distinct spectral species of yeast mitochondrial cytochrome b using a combination of respiratory inhibitors

Appropriate combination of specific inhibitors of electron transport in the cytochrome bc₁ segment of the respiratory chain of Saccharomyces cerevisiae allows the rapid resolution of three spectral forms of mitochondrial cytochrome b. (1) Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to aerobic yeast submitochondrial particles preincubated with cyanide and mucidin in the presence of NADH reveals cytochrome b-561.5. (2) Addition of funiculosin to aerobic yeast submitochondrial particles preincubated with cyanide, mucidin and n-heptylhydroxyquinolineN-oxide in the presence of NADH reveals cytochrome b-558 independently of cytochrome b-561.5 and cytochrome b-565. (3) Specific resolution of cytochrome b-565 can be obtained either by addition of mucidin to aerobic submitochondrial particles preincubated with cyanide, DCMU and NADH, or by addition of antimycin plus an oxygen pulse to NADH-reduced particles, preincubated with cyanide, in the presence of ascorbate plus TMPD, or by addition of antimycin A in the presence of oxidized TMPD to aerobically NADH-reduced particles.     

Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli

Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed. Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway. Cytochromes with α-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl-4-hydroxyquinoneN-oxide. CuSO₄ inhibits the reoxidation by TMAO of the first two of these cytochromes. This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates → cytochromes 548,552 → cytochromes 554,557 → trimethylamine-N-oxide reductase → TMAO. These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells. This suggests that the cytochromes of the TMAO reduction pathway and / or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E. coli.     

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.