Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.     

Characterization of the component, which controls the transformation between the kinetic forms of the b cytochromes.

1. In the presence of KCN and a saturating concentration of antimycin the reduction of the b-type cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b:bA-the active form which was reduced in the rapid phase, and bS-the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the redox state of an unknown component, names "y", located between cytochromes b and c1. Pre-treatment with ascorbate plus N,N,N1,N1-tetramethyl-p-phenylenediamine transforms all the b-type cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name "dynamic control mechanism" was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Biochem. 52, 107-116] 2. Increasing concentrations of antimycin (0-2 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of "y" and thus permits the observation of the biphasic phenomen, but that it is not essential for the operation of this dynamic control mechanism.     

Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.     

Triphasic reduction of cytochrome b and the protonmotive Q cycle pathway of electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain

Reduction of cytochrome b in isolated succinate-cytochrome c reductase is a triphasic reaction. Initially, there is a relatively rapid, partial reduction of the cytochrome b, the rate of which matches the rate of reduction of cytochrome c1. This is followed by partial or complete reoxidation of the b, which is then followed by slow rereduction. At very low concentrations of succinate, the initial partial reduction of b is followed by reoxidation, but the third (rereduction) phase is absent, owing to insufficient substrate to rereduce the cytochromes. If antimycin is added at various times during the triphasic reaction, it inhibits the reoxidation and also inhibits the rereduction phase. Antimycin does not inhibit the initial phase of b reduction and, if added before or during this phase, it causes reduction of b to proceed to completion as a monophasic reaction. Myxothiazol inhibits the first phase of b reduction and the subsequent reoxidation, but does not inhibit the third, slow phase of b reduction. The resulting monophasic reduction of b which is observed in the presence of myxothiazol is slower than that in the presence of antimycin. The combination of both inhibitors, whether added together or successively during the triphasic reaction, completely inhibits b reduction. The triphasic reduction of cytochrome b is consistent with electron transfer by a protonmotive Q cycle in which there are two pathways for cytochrome b reduction. One pathway allows the initial phase of cytochrome b reduction by a myxothiazol-sensitive reaction in which reduction of b by ubisemiquinone is linked to reduction of iron-sulfur protein and cytochrome c1 by ubiquinol. In the second phase of the triphasic reaction, the b cytochromes are reoxidized by ubiquinone or ubisemiquinone through an antimycin-sensitive reaction. If oxidation of ubiquinol by iron-sulfur protein is blocked, either by myxothiazol or by reduction of iron-sulfur protein and cytochrome c1, the b cytochromes can be reduced by reversal of the antimycin-sensitive pathway, thus accounting for the third phase of b reduction.     

Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b(560) was reduced; the dark oxidation of cytochrome b(560) was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b(560), another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b(560). This pigment, tentatively identified as cytochrome b(565), was also detected in spectra at 77 degrees k, after brief illumination at room temperature; the maxima at 77 degrees k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b(565) and an oxidation of cytochrome b(560). Dark oxidation of b(565) was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77 degrees k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77 degrees k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.     

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b.

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reoxidation is observed in the wild type in the present of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steady-state reduction; reduction in the presence of substrate, cyanide and oxygen; the 'red shift' and lowering of E'-o of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH-2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycin-binding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Coenzyme Q analogues reconstitute electron transport and proton ejection but not the antimycin-induced "red shift" in mitochondria from coenzyme Q deficient mutants of the yeast Saccharomyces cerevisiae

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase activity but contained normal amounts of cytochromes b and c1 by spectral analysis. Addition of the exogenous coenzyme Q derivatives including Q2, Q6, and the decyl analogue (DB) restored the rate of antimycin- and myxothiazole-sensitive cytochrome c reductase with both substrates to that observed with reduced DBH2. Similarly, addition of these coenzyme Q analogues increased 2-3-fold the rate of cytochrome c reduction in mitochondria from wild-type cells, suggesting that the pool of coenzyme Q in the membrane is limiting for electron transport in the respiratory chain. Preincubation of mitochondria from the Q-deficient yeast cells with DBH2 at 25 degrees C restored electrogenic proton ejection, resulting in a H+/2e- ratio of 3.35 as compared to a ratio of 3.22 observed in mitochondria from the wild-type cell. Addition of succinate and either coenzyme Q6 or DB to mitochondria from the Q-deficient yeast cells resulted in the initial reduction of cytochrome b followed by a slow reduction of cytochrome c1 with a reoxidation of cytochrome b. The subsequent addition of antimycin resulted in the oxidant-induced extrareduction of cytochrome b and concomitant oxidation of cytochrome c1 without the "red" shift observed in the wild-type mitochondria. Similarly, addition of antimycin to dithionite-reduced mitochondria from the mutant cells did not result in a red shift in the absorption maximum of cytochrome b as was observed in the wild-type mitochondria in the presence or absence of exogenous coenzyme Q analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

Function of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions of the mitochondrial respiratory chain

Resolution and reconstitution has been used to examine the involvement of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions in this region of the mitochondrial respiratory chain. The iron-sulfur protein is required for electron transfer from succinate and from ubiquinol to cytochrome c1. It is not required for reduction of cytochrome b under these conditions, but it is required for oxidation of cytochrome b by cytochrome c plus cytochrome c oxidase. Removal of the iron-sulfur protein from the b-c1 complex prevents reduction of both cytochromes b and c1 by succinate or ubiquinol if antimycin is added to the depleted complex. As increasing amounts of iron-sulfur protein are reconstituted to the depleted complex, the amounts of cytochromes b and c1 reduced by succinate in the presence of antimycin increase and closely parallel the amounts of ubiquinol-cytochrome c reductase activity restored to the reconstituted complex, measured before addition of antimycin. The function of the iron-sulfur protein in these oxidation-reduction reactions is consistent with a cyclic pathway of electron transfer through the cytochrome b-c1 complex, in which the iron-sulfur protein functions as a ubiquinol-cytochrome c1/ubisemiquinone-cytochrome b oxidoreductase.     

Respiratory Chain of Antimycin A-producing Streptomyces antibioticus

An active respiratory chain system was demonstrated in sonically treated mycelium of Streptomyces antibioticus, the producer of antimycin A. The respiratory electron transfer from substrate to oxygen proceeded successively through flavoprotein(s), b-, c-, and a-type cytochromes, and terminated with the cyanide-sensitive cytochrome oxidase. The cytochrome composition of the culture was not affected by the age of the mycelium, the intensity of antimycin A production, or differences in the media. Slater factor, coenzyme Q, and vitamin K were not interposed as hydrogen carriers in the respiratory chain between flavoproteins and cytochromes. The oxidation of reduced nicotinamide adenine dinucleotide and succinate was unaffected by antimycin A. Evidence is presented in support of the absence of the antimycin A-sensitive site from the electron transport system of S. antibioticus.     

The pathway of electrons through OH2:cytochrome c oxidoreductase studied by pre-steady -state kinetics

The kinetic behaviour of the prosthetic groups and the semiquinones in in QH2:cytochrome c oxidoreductase has been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) In the absence of antimycin, cytochrome b-562 is reduced in two phases separated by a lag time. The initial very rapid reduction phase, that coincides with the formation of the antimycin-sensitive Qin, is ascribed to high-potential cytochrome b-562 and the slow phase to low-potential cytochrome b-562. the two cytochromes are present in a 1:1 molar ratio. The lag time between the two reduction phases decreases with increasing pH. Both the [2 Fe-2S] clusters and cytochrome c1 are reduced monophasically under these conditions, but at a rate lower than that of the initial rapid reduction of cytochrome b-562. (3) In the presence of antimycin and absence of oxidant, cytochrome b-562 is still reduced biphasically, but there is no lag between the two phases. No Qin is formed and both the Fe-S clusters and cytochrome c1 are reduced biphasically, one-half being reduced at the same rate as in the absence of antimycin and the other half 10-times slower. (4) In the presence of antimycin and oxidant, the recently described antimycin-insensitive species of semiquinone anion, Qout (De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) J. Biol. Chem. 256, 11996-11998) is formed at the same rate as that of the reduction of all species of cytochrome b. In this case cytochrome b is reduced in a single phase. (5) The reversible change of the line shape of the EPR spectrum of the [2Fe-2S] cluster 1 is caused by ubiquinone bound in the vicinity of this cluster. (6) The experimental results are consistent with the basic principles of the Q cycle. Because of the multiplicity, stoicheiometry and heterogeneous kinetics of the prosthetic groups, a Q cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase is proposed.     

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.     

Multiple forms of cytochrome b in Mycobacterium phlei: kinetics of reduction.

The kinetics of reduction of the b-type cytochromes in the electron transport particles (ETP) from Mycobacterium phlei were studied with nicotinamide adenine dinucleotide, reduced form (NADH) or succinate as electron donors. There appeared to be three active cytochromes b in the ETP,bS563 and bS559, which were reducible by either substrate, and bN563, which was reducible by NADH but not by succinate. In the presence of adenosine 5'-triphosphate, a substantial increase in b563 reduction was observed with succinate at anaerobiosis. This was followed by a decrease in absorption. Adenosine 5'-triphosphate did not effect an increase in cytochrome b563 reduction at transition with NADH, but the occurrence of a secondary decrease in absorption was reflected in a decrease in total enzymatic reduction. The adenosine 5'-triphosphate effect was altered in trypsin-treated ETP, and abolished by uncoupling agents or by removal of the coupling factor-latent adenosine triphosphatase. In the presence of a supernatant fraction obtained during the preparation of the ETP, b563 reduction with succinate was greatly increased. A smaller increase was observed with NADH. Cytochrome b reduction was also studied in ETP inhibited by 2-n-nonylhydroxyquinoline-N-oxide, which appears to inhibit at bS563. On the basis of these data the interrelationships among the b-type cytochromes can be described in relation to the M. phlei electron transport chain.     

Electron transport in a Park-Williams strain of Corynebacterium diphtheriae

1. The electron-transport mechanism was examined in the ;particulate' and ;supernatant' fractions of disintegrated cells of a Park-Williams strain of Corynebacterium diphtheriae. 2. Succinate-oxidase activity was found mainly in the ;particulate' fraction, and NADH(2) oxidase mainly in the ;supernatant', which was devoid of cytochromes and menaquinone. 3. The sum of the activities of particles and supernatant fractions, with respect to both succinate oxidase and NADH(2) oxidase, was substantially less than that of the crude cell extract from which they were obtained. Full activity was restored on recombining ;particles' and ;supernatant'. The characteristics of this reassembled system were investigated. 4. The strain of organism (CN2000) examined contained cytochromes corresponding spectroscopically to ;a', ;b' and ;c' types. All three were reduced by succinate, lactate or NADH(2); but a portion of the cytochrome b, susceptible to reduction by dithionite, could not be reduced by the substrates. 5. Triton X-100 inhibits oxidation of succinate by particulate fraction; on adding succinate, the reduction of cytochrome b is not affected but that of cytochromes a and c is delayed. 6. Irradiation at 360mmu completely destroys menaquinone in the particle fraction. Succinate oxidation is severely decreased; succinate dehydrogenase and NADH(2) oxidation are little affected. Certain menaquinones will restore succinate oxidation in the irradiated material. 7. On adding succinate to irradiated particulate material cytochrome b is partially reduced at once, but reduction of cytochromes a and c is much delayed. A portion of the cytochrome b remains not reduced, but reduction occurs rapidly on the addition of menaquinone (MK-2).     

Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing 相似文献   

The utilization of iron and its complexes by mammalian mitochondria

Sonicated mitochondria catalyse the reduction of ferric salts, and the subsequent incorporation of Fe(2+) into haem, when provided with a reducing substrate such as succinate or NADH. The rate of haem synthesis was low under aerobic conditions and, after a short lag period, accelerated once anaerobic conditions were achieved; it was insensitive to antimycin A. The lag period was decreased by preincubating the mitochondria with NADH and Fe(3+). Newly formed Fe(2+) was autoxidized rapidly and the consequent O(2) uptake was measured with an oxygen electrode to determine the rate of enzymic formation of Fe(2+) from FeCl(3); this reaction was rapid in sonicated mitochondria provided with NADH or succinate and was insensitive to antimycin A. The reaction was very slow in intact mitochondria, suggesting a permeability barrier to Fe(3+) ions. This system was used to test the permeability of the mitochondrial membrane to various iron complexes of biological importance. Of the compounds tested only ferrioxamine G appeared to penetrate readily and the iron of this complex was reduced when intact mitochondria were supplied with succinate or NADH-linked substrates. The reduction was insensitive to rotenone or antimycin A. Both ferrioxamine G and ferrioxamine B were, however, reduced by particles. The membrane fraction of sonicated mitochondria was necessary for the reduction. The rate of ferrioxamine B reduction by sonicated mitochondria was measured by a dual-wavelength spectrophotometric assay and was found to be stimulated in conditions where the Fe(2+) produced was utilized for haem synthesis. The addition of FeCl(3) to anaerobic particles caused an oxidation of cytochrome b when this region of the respiratory chain was isolated by treatment with rotenone and antimycin A. These results suggest that the reduction of ferric iron and its complexes occurs inside the inner mitochondrial membrane in proximity to ferrochelatase. Possible sites for this reduction are the flavoproteins, succinate and NADH dehydrogenase.     

Respiratory Mechanisms in the Flexibacteriaceae: Terminal Oxidase Systems of Saprospira grandis and Vitreoscilla Species

Particles from both Saprospira grandis and Vitreoscilla species, obtained by high-pressure extrusion and sonic treatment, respectively, actively catalyze the oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate with O(2). These activities are inhibited by cyanide but not by antimycin; Saprospira is also amytal- and rotenone-insensitive. Vitreoscilla preparations were unable to oxidize mammalian ferrocytochrome c and reduced tetramethyl-p-phenylenediamine, whereas the Saprospira preparations did so actively. Low-temperature (77 K) difference spectroscopy of Vitreoscilla cells and particles indicates the presence of three maxima in the cytochrome alpha-region at 554, 558, and 562 nm. All three cytochromes are active in NADH and succinate oxidation, but none is ascorbate reducible. Cytochrome o is the only CO-binding pigment present and is probably the terminal oxidase; it has properties similar to the cytochrome o isolated in solubilized form from this organism. Saprospira cells and membranes exhibit four cytochrome absorption bands whose maxima are at 550, 554, 558, and 603 nm at 77 K. The latter component has not been noted previously. NADH and succinate reduce all four cytochromes, but ascorbate reduces only the 550- and 603-nm pigments. CO spectra indicate the presence of cytochrome a,a(3) which is probably the oxidase. A second CO-binding pigment is present which is not a peroxidase but may be a cytochrome.     

THE ROLE OF THE QUINONE POOL IN THE CYCLIC ELECTRON-TRANSFER CHAIN OF RHODOPSEUDOMONAS SPHAEROIDES: A MODIFIED Q-CYCLE MECHANISM

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c(2) oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c(1) and c(2), the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c(1) and c(2), and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c(2) oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c(1) and c(2) is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.