Dynamic control on the rate of the reduction of the b type cytochromes in submitochondrial particles.

1. In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first-order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of chtochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall-off in the rate of cytochrome b reduction. 2. The biphasic reduction of cytochrome b was observed over a wide temperature range (0--30 degrees C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat-heart mitochondria. With rat-heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p-trifluoromethoxyphenylhydrazone or oligomycin. 3. In both the rapid and the slow phases, the rate of reduction of cytochrome b-561 was equal to that of b-565. Thus both cytochromes b-561 and b-565 were affected by the mechanism which determined the reduction-rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase. 4. The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by teh experimental conditions. When antimycin was used as the only inhibitor, 96% of the b-type cytochromes were reduced in the rapid phase. If the c and a-type cytochromes were first reduced by ascorbate and tetramethyl-p-phenylenediamine in the presence of KCN and antimycin, all the b-type cytochromes were fully reduced at the slow-rate. 5. With succinate, the rate of the rapid phase depended on the activation level of the succinic-dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b). 6. It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone. 7. It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.     

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.     

Effects of selected inhibitors on electron transport in Neisseria gonorrhoeae.

The electron transport system of Neisseria gonorrhoeae was partially characterized by using spectrophotometric, spectroscopic, and oxygen consumption measurements. The effects of selected electron transport inhibitors (amytal, rotenone, 2-heptyl-4-hydroxyquinoline, antimycin A1, and potassium cyanide [KCN]) on electron transfer in whole-cell and sonically treated whole-cell preparations of N. gonorrhoeae were examined. The oxidation of reduced nicotinamide adenine dinucleotide, measured as a decrease in absorbance at 340 nm, was inhibited by each of the compounds tested. Oxygen consumption stimulated by reduced nicotinamide adenine dinucleotide was also inhibited, whereas oxygen uptake stimulated by succinate and malate was inhibited by KCN alone, suggesting the presence of a KCN-sensitive terminal oxidase. Room temperature optical difference spectra indicate an operational electron bypass around the amytal-rotenone-binding site. Difference spectra in the presence of 2-heptyl-4-hydroxyquinoline suggest a possible site of interaction of this compound at the substrate side of cytochrome b. Reduced-minus-oxidized spectra of ascorbate-tetramethyl-p-phenylenediamine suggest the participation of b-, a-, and d-type cytochromes in terminal oxidase activity. Hence, N. gonorrhoeae appears to have an electron transport chain containing cytochrome c, two b-type cytochromes (one of which has an oxidase function), and possibly a- and d-type cytochromes. An abbreviated chain exists through which succinate and malate can be oxidized directly by a KCN-sensitive component.     

Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. I. Biochemical characterization and electron transport of Ascaris microsomes.

Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25 M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suum. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components. The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424 nm. However, the alpha-peak at 560 nm was asymmetric with a shoulder at 555 nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.     

The participation of cytochromes in the reduction of N20 to N2 by a denitryfying bacterium.

The oxidation of cytochromes during the reduction of N2O to N2 by a denitrifying bacterium was studied spectrophotometrically. The reduced b- and c-type cytochromes are partially oxidized when N2O is added to intact cells reduced with lactate under anaerobic conditions. The oxidation of cytochromes is inhibited non-competitively by azide, cyanide, 2,4-dinitrophenol and CuSO4, which inhibit the reduction of N2O to N2. In the presence of each inhibitor at a high concentration, at which the reduction of N2O to N2 is perfectly inhibited, cytochromes are not oxidized by N2O, while when an adequate, low concentration of inhibitor is added, b-type cytochrome is partially oxidized but c-type cytochrome is apparently not oxidized. In cell-free extracts, prepared by the sonic disruption of cells, that have entirely lost their activity in the reduction of N2O to N2, cytochromes are not oxidized by N2O. From the above results, it was concluded that b-type and c-type cytochromes should participate in the electron transport mechanism of the reduction of N2O to N2.     

Regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex

We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.     

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.     

Kinetics of cytochrome b reduction in submitochondrial particles

(1) In agreement with Eisenbach and Gutman (Eisenbach, M. and Gutman, M. (1975) Eur. J. Biochem. 52, 107–116) the reduction of cytochrome b in beef-heart submitochondrial particles by succinate in the presence of antimycin was found to be biphasic, the relative amounts of fast and slow phases being dependent on the redox state of a component located on the oxygen side of the antimycin block. (2) HQNO in a concentration sufficiently large to saturate the specific antimycin- and HQNO-binding sites can substitute for antimycin in these experiments. (3) The rate of the slow phase of the reduction of cytochrome b is decreased under anaerobic conditions and after pretreatment with 2,3-dimercaptopropanol (BAL). (4) In the presence of antimycin and cyanide, cytochrome b-562 is, to some extent, preferentially reduced in the rapid phase and b-566 in the slow phase. (5) The previously proposed regulatory effects of redox-sensitive components X and Y on the redox level and reduction kinetics, respectively, of cytochrome b are ascribed to the role of the Fe-S protein, when it is oxidized, in producing the reductant of cytochrome b by oxidation of QH₂, and by the fact that when QH₂ is bound to it, the reduced Fe-S protein cannot be oxidized by its natural oxidant, cytochrome c₁.     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

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.     

Heterogeneous pools of cytochrome c2 in photo-denitrifying cells of Rhodobacter sphaeroides forma sp. denitrificans

When cells of the denitrifying phototrophic bacterium Rhodobacter sphaeroides forma sp. denitrificans were grown anaerobically under illumination in the presence of nitrate, the content of photosynthetic reaction centers per cellular protein was less than that in cells grown photosynthetically without nitrate under the same light intensity. The contents of cytochromes c1 and c2, which work in both photosynthetic and denitrifying electron transport systems, were almost constant, being independent of the presence of nitrate during growth. Consequently, the ratio of cytochromes c1 and c2 to the reaction center was more than three in the photo-denitrifying cells, whereas it was close to one in the photosynthetic cells under light-limiting conditions. In spite of the excess of cytochromes c1 + c2 over the reaction center in the photo-denitrifying cells, all cytochromes c1 + c2 were oxidized by illumination within hundreds of milliseconds in the presence of antimycin. When glycerol was added to increase the viscosity in the periplasm, biphasic oxidation of cytochromes c1 + c2 was apparent in the photo-denitrifying cells with repetitive flashes. The fast phase oxidation, which took place instantaneously (less than 1 ms) after the first and second flashes, showed a similar pattern to the oxidation in the light-limiting photosynthetic cells. The rate of the slow phase oxidation was sensitive to viscosity and was thought to reflect a diffusion-controlled second-order reaction between cytochrome c2 and the reaction center. The biphasic oxidation of cytochromes c1 + c2 suggests that these cytochromes exist in the photo-denitrifying cells as two different pools in relation to the reaction center.     

Reduction of cytochromes with menaquinol and sulfide in membranes from green sulfur bacteria

Reduction of cytochromes in chlorosome-free membranes of Chlorobia was studied anaerobically, with an LED array spectrophotometer. For Chlorobium tepidum these membranes contained 0.2 moles cytochrome per mole of bacteriochlorophyll a. The observed change upon complete reduction of oxidized membranes with dithionite could be satisfactorily fitted with three cytochrome components having absorption peaks at 553 (cyt c), 558 and 563 nm (cyt b), in relative amounts of 5:1:2. About 20% of total cytochrome 553 were reducible by ascorbate. Menaquinol reduced all of the 553-component, and this reduction was sensitive to stigmatellin, NQNO and antimycin A. The reduction was insensitive to KCN. However, it was transient at low concentrations of menaquinol in the absence of KCN, but permanent in its presence, demonstrating that electron transport into an oxidation pool was blocked. The 563-component was only slightly reduced by menaquinol unless NQNO or antimycin were present. The stimulation of cytochrome 563-reduction by these inhibitors was more pronounced in the presence of ferricyanide. This phenomenon reflects oxidant-induced reduction of cytochrome b and demonstrates that a Q-cycle is operative in Chlorobia. Also, sulfide fully reduced cytochrome 553, but more slowly than menaquinol. KCN inhibited in this case, as did stigmatellin, NQNO and antimycin A. NQNO was a better inhibitor than antimycin A. Cytochrome 563 again was hardly reduced unless antimycin A was added. The effect was more difficult to observe with NQNO. This supports the conclusion that sulfide oxidation proceeds via the quinone pool and the cytochrome bc-complex in green sulfur bacteria.Abbreviations BChl bacteriochlorophyll - cyt cytochrome - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - SQR sulfide-quinone reductase Dedicated to Prof. Dr. Aloys Wild on occasion of his 65th birthday.     

Differences in brain cytochrome responses to carbon monoxide and cyanide in vivo

Cytochrome oxidation-reduction responses to two mitochondrial electron transport inhibitors, carbon monoxide (CO) and cyanide (CN), were studied in the intact brains of fluorocarbon-circulated rats. In vivo reflectance spectrophotometry indicated that cortical b-type cytochromes (564 nm) were highly resistant to reduction by CN in the presence of O2 but showed reduction responses to the administration of 1-5% CO in 90% O2. In contrast, cyanide-sensitive cytochromes aa3 (605 nm) and c + c1 (551 nm) did not increase their reduction levels during exposure to 5% CO in 90% O2. The in vivo CO-mediated b-cytochrome reduction responses did not occur after pretreatment with the cytochrome b inhibitor, antimycin A. Transmission spectrophotometry of superfused hemoglobin-free rat brain slices confirmed cortical b-type cytochromes to be CN-resistant in the presence of O2. Another cytochrome absorbing at 445 nm also was resistant to reduction by 1-mM cyanide in vitro, but it could be reduced anaerobically. The reduced 445-nm cytochrome bound CO in the presence of cyanide. We postulate that this CN-resistant CO binding component might account for in vivo cytochrome aa3-CO interactions and directly or indirectly modulate cytochrome b reduction responses to CO. In any event, the spectral data indicate different primary tissue target sites for CO and CN in living rat brain and also suggest different bioenergetic consequences of exposure to the two agents.     

Kinetic characterization of the membrane-bound cytochromes of Escherichia coli grown under a variety of conditions by using a stopped-flow dual-wavelength spectrophotometer.

A study was made of the rapid oxidation kinetics of the cytochromes of Escherichia coli. The b-type cytochromes were kinetically heterogeneous, with one species (presumably cytochrome o) oxidized so rapidly that it could fully support observed oxidation rates. Cytochrome d but not cytochrome a1 was also kinetically competent to support respiration. However, in cells grown anaerobically in the presence of NO3-, cytochrome d exhibited slow oxidation kinetics and a red-shift in its reduced-minus-oxidized difference spectrum.     

The oxidation of glucose by Acinetobacter calcoaceticus: interaction of the quinoprotein glucose dehydrogenase with the electron transport chain

The coupling of the quinoprotein glucose dehydrogenase to the electron transport chain has been investigated in Acinetobacter calcoaceticus. No evidence was obtained to support a previous suggestion that the soluble form of the dehydrogenase and the soluble cytochrome b associated with it are involved in the oxidation of glucose. Analysis of cytochrome content, and of reduction of cytochromes in membranes by substrates, and of sensitivity to cyanide indicated that glucose, succinate and NADH are all oxidized by way of the same b-type cytochrome(s) and cytochrome oxidases (cytochrome o and cytochrome d). Mixed inhibition studies [with KCN and hydroxyquinoline N-oxide (HQNO)] showed that the b-type cytochrome(s) formed a binary complex with the o-type oxidase and that there was thus no communication between the electron transport chains at the cytochrome level. Measurements of the reduction of ubiquinone-9 by glucose and NADH, and inhibitor studies using HQNO, indicated that the ubiquinone mediates electron transport from both the glucose and NADH dehydrogenases. In some conditions the quinone pool facilitated communication between the 'glucose oxidase' and 'NADH oxidase' electron transport chains, but in normal conditions these chains were kinetically distinct.     

Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol.

Methanosarcina strain G?1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain G?1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.     

Ubiquinone at center N is responsible for triphasic reduction of cytochrome b in the cytochrome bc(1) complex.

We have examined the pre-steady state reduction kinetics of the Saccharomyces cerevisiae cytochrome bc(1) complex by menaquinol in the presence and absence of endogenous ubiquinone to elucidate the mechanism of triphasic cytochrome b reduction. With cytochrome bc(1) complex from wild type yeast, cytochrome b reduction was triphasic, consisting of a rapid partial reduction phase, an apparent partial reoxidation phase, and a slow rereduction phase. Absorbance spectra taken by rapid scanning spectroscopy at 1-ms intervals before, during, and after the apparent reoxidation phase showed that this was caused by a bona fide reoxidation of cytochrome b and not by any negative spectral contribution from cytochrome c(1). With cytochrome bc(1) complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduction by either menaquinol or ubiquinol was rapid and monophasic. Addition of ubiquinone restored triphasic cytochrome b reduction, and the duration of the reoxidation phase increased as the ubiquinone concentration increased. When reduction of the cytochrome bc(1) complex through center P was blocked, cytochrome b reduction through center N was biphasic and was slowed by the addition of exogenous ubiquinone. These results show that ubiquinone residing at center N in the oxidized cytochrome bc(1) complex is responsible for the triphasic reduction of cytochrome b.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

Cytochromes and hydrogen-oxidizing activity in the thermophilic hydrogen-oxidizing bacteria related to the genus Hydrogenobacter

The highly thermophilic, hydrogen-oxidizing aerobic bacteria related to Hydrogenobacter possess a respiratory chain comprising a quinone and b-type (alpha band at 556 nm and 562 nm) and c-type (alpha band at 552 nm) cytochromes. They have no aa₃-type cytochromes and their terminal oxidase is an o-type cytochrome. A polarographic method with an oxygen electrode was used for the measurement of the hydrogen-oxidizing activity. This activity was strongly inhibited by HQNO (2-N-heptyl-4-hydroxyquinoline N-oxide), an inhibitor of the respiratory chain in the quinone-cytochrome b region, and by KCN, an inhibitor of the terminal cytochrome oxidase. This study shows that the electrons released from hydrogen oxidation by the membrane-bound hydrogenase probably enter the respiratory chain at the level of the quinone-cytochrome b region.Abbreviations HQNO 2-N-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N',N'-tetramethyl-p-phenylenediamine - DW dry weight     

Kinetic indication for multiple sites of ubiquinol-1 interaction in ubiquinol-cytochrome c reductase in bovine heart mitochondria

We have assayed the ubiquinol-cytochrome c reductase activity either in situ or in different mitochondrial fractions, including the isolated bc₁ complex, employing ubiquinol-1 and exogenous cytochrome c as substrates. A clear biphasic behavior of both the time courses and the initial rates of cytochrome c reduction have been observed. Two K_m values have been found, one of 1–7 × 10^?6m ubiquinol-1, and another varying from 0.6 to 4.6 × 10^?5m ubiquinol-1, depending on the cytochrome c concentration and the type of mitochondrial fraction used. Either the kinetic phase with the lower K_m or the kinetic phase with the higher K_m exhibits an almost identical antimycin sensitivity. We have also monitored the rapid reduction of endogenous b cytochromes in the presence of antimycin, and the initial rates are again biphasic as a function of ubiquinol-1 concentration. These findings indicate that the steps conferring the biphasic kinetics to the ubiquinol-cytochrome c reductase activity involve the redox equilibria between exogenous ubiquinol-1 and the b cytochromes, and suggest that two redox pathways may be present in the electron transfer from ubiquinol to cytochrome c through the bc₁ segment of the mammalian respiratory chain.