Funiculosin: an antibiotic with antimycin-like inhibitory properties.

The antibiotic funiculosin mimics the action of antimycin in several ways. It inhibits the oxidation of NADH and succinate, but not TMPD+ascorbate. The titer for maximal inhibition in Mg2+-ATP particles (0.4-0.6 nmol/mg protein) is close to the concentrations of cytochromes b and cc1. Funiculosin also induces the oxidation of cytochromes cc1 and an extra reduction of cytochrome b in the aerobic steady state, and it inhibits duroquinol-cytochrome c reductase activity in isolated Complex III. The location of the funiculosin binding site is clearly similar to that of antimycin. In addition, funiculosin, like antimycin, prevents electron transport from duroquinol to cytochrome b in isolated Complex III if the complex is pre-reduced with ascorbate. Funiculosin and antimycin differ, however, in the manner in which they modulate the reduction of cytochrome b by ascorbate+TMPD.     

Changes in oxidation-reduction potential of cytochrome b observed in the presence of antimycin A

The cytochrome b of sonic particles of mitochondria or the isolated segment of the respiratory chain containing cytochromes b and c₁ (Complex III) was 80–95% reducible with Q₁H₂ (ubiquinol-5) in the presence of antimycin plus selected electron acceptors added externally (i.e., oxidants which reacted preferentially with respiratory components on the oxygen side of the point of inhibition by antimycin) such as oxygen or ferricyanide depending on whether sonic particles or isolated Complex III was used. In contrast, less than 40% of the cytochrome b was reduced by Q₁H₂ in the absence of either antimycin or the external electron acceptor. In the presence of antimycin ascorbate or mercaptoethanol, which behaved as mild reducing agents, completely inhibited the reduction of cytochrome b by Q₁H₂.     

Cytochrome b reduction by hexaammineruthenium in mitochondria and submitochondrial particles: Evidence for heme b-562 localization at the M-side of the mitochondrial membrane

In the presence of ascorbate, hexaamineruthenium mediates rapid reduction of cytochrome b-562 in submitochondrial particles but not in mitochondria. The reaction is obsreved in the combined presence of antimycin (or funiculosin) and myxothiazol, which implies direct interaction of Ru(NH₃)²⁺₆ with b cytochrome(s). We assume that contrary to previous conclusions (Case and Leigh (1976) Biochem. J., 160, 769-783) redox centre of at least one of the oxidized cytochromes b, most probably of b-562, is exposed to the M-aqueous phase.     

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.     

The interaction of yeast Complex III with some respiratory inhibitors

We have examined the effects of eight inhibitors of the bovine-heart mitochondrial Complex III on the catalytic activity of the analogous complex from yeast mitochondria. All eight compounds were inhibitory, with potent inhibition being obtained with antimycin, myxothiazol and UHDBT (5-N-undecyl-6-hydroxy-4,7-dioxobenzothiazole). These three inhibitors, and also funiculosin, have been further studied by characterizing their effects on the visible absorbance, magnetic circular dichroism and EPR spectra of the complex and also on the potentiometric properties of the individual metal centers present in the complex. All four inhibitors had little or no effect on either the absorbance or magnetic circular dichroism spectra. Funiculosin produced a change in the EPR lineshape of the iron-sulfur cluster; EPR spectra recorded at 12 K also revealed complete reduction of cytochrome b-562 by ascorbate. UHDBT also changed the lineshape of the iron-sulfur cluster and this change could be partially reversed by myxothiazol. Neither antimycin nor myxothiazol affected the iron-sulfur cluster and produced only small changes in the EPR absorption envelope of the b cytochromes. Both funiculosin and UHDBT raised the midpoint potential of the iron-sulfur cluster, by about 150 and 70 mV, respectively. Only UHDBT changed the potential of c1, lowering it by about 30 mV. Funiculosin raised the potential of b-562 by about 30 mV, while myxothiazol had no effect; the other two compounds produced only small changes. All four compounds had only small effects on the midpoint potential of b-566. The relative contributions of the two b cytochromes to the magnetic circular dichroism amplitudes could be changed by the addition of inhibitors, even though the absolute magnetic circular dichroism spectra of oxidized and reduced complex were unaffected.     

Electron transport in mitochondria isolated from the flagellate Polytomella caeca

1. Mitochondria isolated from Polytomella caeca contain cytochromes b, c+c₁ and a+a₃ and several flavoprotein species. 2. Electron transport is inhibited by antimycin A, rotenone, piericidin A and cyanide. 3. Spectral data indicate that antimycin A inhibits the reoxidation of reduced cytochrome b. 4. Various types of flavoprotein are characterized by simultaneous spectrophotometric and fluorimetric measurements on antimycin A-inhibited preparations and also by their absorption and fluorescence-emission spectra. 5. The rotenone-sensitive site lies between the two flavoproteins of the respiratory chain, designated FpD1 and FpD2. 6. Other flavoprotein species detected include those involved in the oxidation of succinate and externally added NADH; a large proportion of mitochondrial flavine is reduced by dithionite but not by known respiratory substrates. 7. The kinetics of flavoprotein and cytochrome reactions were studied.     

The kinetics of reoxidation of yeast complex III. An evaluation of the Q-cycle

The reoxidation of reduced yeast Complex III by oxidants believed to react with cytochrome c1 exhibited multiple phases for both cytochrome c1 and the cytochromes b; the reoxidation of cytochrome b, but not cytochrome c1, was markedly slowed by the presence of antimycin. The data are consistent with the Q-cycle or any other scheme which proposes a branched path for electron transport between the cytochrome b centers and the endogenous Q6, provided certain constraints are relaxed. The reoxidation of the endogenous quinone proceeded at a rate comparable to that of the rapidly reacting cytochrome b and appeared to be complete within 100 ms. Removal of the endogenous quinone did not change the rate or extent of reoxidation of any of the heme centers, demonstrating that quinone is not required for electron transport between cytochromes b and the iron-sulfur cluster. This result is inconsistent with the requirements of the Q-cycle. Funiculosin completely inhibited the reoxidation of cytochrome b whereas the reoxidation of cytochrome c1 exhibited simple first-order kinetics in the presence of this inhibitor, implying that the iron-sulfur cluster is on the direct path of electron transfer from cytochrome b to cytochrome c1. Potent inhibition of cytochrome b oxidation was also observed with myxothiazol and mucidin. The reaction of reduced Complex III with Q1 also exhibited multiple phases in the oxidation of the cytochrome b centers; these phases were unaffected by the presence of myxothiazol. Addition of antimycin, or removal of the endogenous quinone, eliminated the rapid phases; only one of the cytochrome b centers was oxidized under these conditions. Epr showed that it is the low-potential cytochrome b that is the species rapidly oxidized.     

Action de l'antimycine a sur l'activité succinoxydase des mitochondries du tubercule de Pomme de Terre,et comportement des cytochromes de type b

A kinetic study of the effect of antimycin A on succinate oxidase from plant mitochondria produced sigmoidal curves for the reduction of cytochromes b₅₆₀ and b₅₈₅ and for the inhibition of succinate oxidase. In the stationary state the interaction of the various components of the respiratory chain (flavins, ubiquinone, cytochromes…) occurs in a sequential mode which allows the application of simple equations utilizing rate constants and cytochrome concentrations. In these equations, it is assumed that there exists an excess of ubiquinone relative to other components of the respiratory chain as suggested by Kröger & Klingenberg (1970) and that the reoxidation of b cytochromes is fast. The inhibition by antimycin A, characterized by non-linear inhibition curves for succin-oxidase and inhibitor fixation in complex III on a component other than cytochrome c₁, is interpreted in terms of this model. This hypothesis presupposes the existence of an F factor between cytochrome b₅₆₀ and cytochrome c₁ as suggested by other authors. Utilizing these equations, theoretical curves for the inhibition of the reduction of cytochrome b₅₆₀ have been constructed and the results agree with the experimental data. The kinetic behavior of cytochrome b₅₆₆ during the induction of anaerobiosis suggests that it is not directly involved in the electron transfer chain but rather is either in thermodynamic equilibrium with cytochrome b₅₆₀ or in a shunt between cytochrome b₅₆₀ and factor F. From the experimental data, an equation is derived for the inhibition of the reduction of cytochrome b₅₆₆ by antimycin A. The actual effects of ATP and mClCCP on succinoxidase agree well with those predicted by the model.     

Fuscin,an inhibitor of respiration and oxidative phosphorylation in ox-neck muscle mitochondria

1. The effect of fuscin on the mitochondrial oxidation of pyruvate plus malate, of succinate and of ascorbate plus tetramethyl-p-phenylenediamine (TMPD) and on the redox changes of succinate-reducible cytochromes b and c was investigated using tightly-coupled ox-neck muscle mitochondria.     

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc₁ complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q_i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q_o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A₃ in a fashion similar to the Q_o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q_i and the Q_o sites of the fungal cytochrome bc₁ complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc₁ complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q_o and Q_i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc₁ complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q_i and Q_o sites. Moreover, crystal structure of chicken cytochrome bc₁ complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q_i and Q_o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc₁ inhibitor that acts at both of the active sites of this enzyme.     

Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex: ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE*

We previously proposed that the dimeric cytochrome bc₁ complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc₁ monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289–22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc₁ complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10–16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c₁ by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

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.     

Differential efficacy of inhibition of mitochondrial and bacterial cytochrome bc1 complexes by center N inhibitors antimycin, ilicicolin H and funiculosin

We have compared the efficacy of inhibition of the cytochrome bc₁ complexes from yeast and bovine heart mitochondria and Paracoccus denitrificans by antimycin, ilicicolin H, and funiculosin, three inhibitors that act at the quinone reduction site at center N of the enzyme. Although the three inhibitors have some structural features in common, they differ significantly in their patterns of inhibition. Also, while the overall folding pattern of cytochrome b around center N is similar in the enzymes from the three species, amino acid sequence differences create sufficient structural differences so that there are striking differences in the inhibitors binding to the three enzymes. Antimycin is the most tightly bound of the three inhibitors, and binds stoichiometrically to the isolated enzymes from all three species under the cytochrome c reductase assay conditions. Ilicicolin H also binds stoichiometrically to the yeast enzyme, but binds approximately 2 orders of magnitude less tightly to the bovine enzyme and is essentially non-inhibitory to the Paracoccus enzyme. Funiculosin on the other hand inhibits the yeast and bovine enzymes similarly, with IC₅₀ ∼ 10 nM, while the IC₅₀ for the Paracoccus enzyme is more than 10-fold higher. Similar differences in inhibitor efficacy were noted in bc₁ complexes from yeast mutants with single amino acid substitutions at the center N site, although the binding affinity of quinone and quinol substrates were not perturbed to a degree that impaired catalytic function in the variant enzymes. These results reveal a high degree of specificity in the determinants of ligand-binding at center N, accompanied by sufficient structural plasticity for substrate binding as to not compromise center N function. The results also demonstrate that, in principle, it should be possible to design novel inhibitors targeted toward center N of the bc₁ complex with appropriate species selectivity to allow their use as drugs against pathogenic fungi and parasites.     

Electron transport in the membrane of lutoids from the latex ofHevea brasiliensis

1. An antimycin-insensitive NADH-cytochromec oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex ofHevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH.2. Twob-type cytochromes are present in the membranes. Cytochromeb₅₆₃ is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 °K. A second cytochrome, cytochromeb₅₆₁, seems to be reducible by hydrosulfite only.3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochromeP-450 could not be demonstrated.4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

13th International Congress of Biochemistry

Cytochrome caa₃ (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa₃ develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a₃ centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated Cu_A atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa₃ complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.     

The Respiratory Chain of Plant Mitochondria: XVI. Interaction of Cytochrome b(562) with the Respiratory Chain of Coupled and Uncoupled Mung Bean Mitochondria: Evidence for Its Exclusion from the Main Sequence of the Chain

Cytochromes b₅₅₃, b₅₅₇, and b₅₆₂ of mung bean (Phaseolus aureus) mitochondria become partially reduced with endogenous substrate on addition of antimycin A to the aerobic mitochondrial suspension. Addition of ATP causes partial reoxidation of the three cytochromes. This partial oxidation by ATP is inhibited by oligomycin and reversed by uncoupler. Ubiquinone does not appear to act as electron acceptor for the oxidation reaction, but a nonfluorescent flavoprotein, or possibly ironsulfur protein, component does appear to act as acceptor. This is consistent with reverse electron transport driven by ATP across the first site of energy conservation of the respiratory chain. Endogenous pyridine nucleotide and the fluorescent flavoprotein with E_m7.2 = −155mv (midpoint potential at pH 7.2, referred to normal hydrogen electrode) in uncoupled mitochondria become reduced in anaerobiosis attained by oxidation of succinate in the absence of respiratory inhibitors of the cytochrome chain, provided that Pi and ATP are present. Under these same conditions, cytochrome b₅₅₇ is completely reduced but cytochrome b₅₆₂ remains nearly completely oxidized. There is no equilibration across the first site of energy conservation between the carriers on the low potential side and cytochrome b₅₆₂ with E_m7.2 = −77mv on the high potential side. It is concluded that cytochrome b₅₆₂ is not a part of the main sequence of electron transport carriers of the mitochondrial respiratory chain of plants; it can participate in redox reactions with the respiratory chain in coupled mitochondria but not in uncoupled mitochondria unless antimycin A is present.