QcrCAB operon of a nocardia-form actinomycete Rhodococcus rhodochrous encodes cytochrome reductase complex with diheme cytochrome cc subunit

Structural genes encoding quinol-cytochrome c reductase (QcR) were cloned and sequenced from nocardia-form actinomycete Rhodococcus rhodochrous. QcrC and qcrA encode diheme cytochrome cc and the Rieske Fe-S protein, respectively, while the qcrB product is a diheme cytochrome b. These amino acid sequences are similar to those of Corynebacterium and Mycobacterium, the members of high G+C content firmicutes. The presence of diheme cytochrome cc subunit as a sole c-type cytochrome in these organisms suggests the direct elecron transfer to cytochrome c oxidase. The N-terminal half of the Rieske Fe-S proteins of these bacteria has a unique structure with three transmembrane helices, while the C-terminal half sequence is conserved. A phylogenetic tree using the latter region showed that high G+C firmicutes form a clear clade with Thermus, but not with low G+C firmicutes.     

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Study of the Hansenula anomala yeast flavocytochrome-b2--cytochrome-c complex 1. Characterization of fluorescent Zn(II)-substituted cytochrome c

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn-cytochrome c appears to be in the form of a stable though non-covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal-free porphyrin-cytochrome c, the precursor of Zn-cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate-reduced state showed a simple binding equilibrium (Kd = 0.1 microM at I = 0.03 M, 10 degrees C) with a stoichiometry of one Zn-cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn-porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.     

Influence of increased environmental temperature on oxidation processes in rat liver mitochondria

Exposure of rats to elevated temperature of 28 degrees C or 35 degrees C for 3 days six hours daily resulted in a decreased rate of oxidation with succinate or glutamate + malate as substrates, by the mitochondria of liver. The higher decrease was observed in environment temperature of 35 degrees C. There was no change in ADP/O ratio. The activities of NADH: cytochrome c reductase and cytochrome oxidase were stimulated but activities of succinate dehydrogenase and succinate cytochrome reductase were decreased.     

The role of c-type cytochromes in the photosynthetic electron transport pathway of Rhodobacter capsulatus

(1) Short flash excitation of membrane vesicles of a cytochrome-c2-deficient mutant of Rhodobacter capsulatus (strain MT-G4/S4) led to rapid oxidation of a c-type cytochrome. In redox titrations, the photooxidation of c-type cytochrome was attenuated with a midpoint of approx. +360 mV. Vesicles from a control strain, MT1131, gave similar results. These findings are consistent with those of Prince et al. (Prince, R.C., Davidson, E., Haith, L.E. and Daldal, F. (1986) Biochemistry 25, 5208-5214). (2) In anaerobic intact cells the extent of rapid re-reduction of c-type cytochrome oxidised after a flash was less in MT-G/S4 than in MT1131. Cytochrome c reduction in both strains was inhibited by myxothiazol. The myxothiazol-sensitive component of the electrochromic absorbance change in cells indicated that rapid charge separation through the cytochrome bc1 complex was less extensive after a flash in MT-G4/S4 than in MT 1131. (3) In anaerobic intact cells and in chromatophores of Rb. capsulatus strain MT-GS18, a mutant deficient in both cytochrome c1 and cytochrome c2, flash excitation led to the oxidation of c-type cytochrome. Redox titrations and spectra of chromatophores suggested that this is the same cytochrome as was photooxidized in vesicles of MT-G4/S4 and MT1131. This result is in contrast with earlier findings (Prince, R.C. and Daldal, F. (1987) Biochim. Biophys, Acta 894, 370-378) in which it was reported that no photooxidation of c-type cytochrome occurred in the absence of c1 and c2, and argues against the possibility that cytochrome c1 can rapidly and directly donate electrons to the reaction centre. (4) It is proposed that a previously uncharacterized, membrane-bound c-type cytochrome (Em7 approximately +360 mV) is present in Rb-capsulatus MT1131, in the c2-deficient mutant MT-G4/34 and in the c1/c2-deficient mutant MTGS18. This cytochrome and cytochrome c2 are alternative electron donors to the reaction centre in strain MT1131.     

Photoreduction of cytochrome c1.

1. Ferricytochrome c1 solution was reduced completely between pH 7 and 10 by illumination under anaerobic conditions. Photoreduction was not affected by the ionic strength of the medium. However, it did not take place at pH lower than 6 or higher than 10, or in the presence of p-hydroxymercuric benzoate. The ferricyanide-reoxidized photoreduced c1 was not further reduced upon illumination. The reductant was most probably a specific sulfhydryl group in the subunit containing the heme of the cytochrome since this subunit contained one less p-HMB-titratable group in the photoreduced sample than in the untreated preparation. 2. The photoreduced cytochrome c1 showed the same spectra as the native cytochrome, and was not reactive with carbon monoxide. The equilibrium constant of the reaction c12+ + c3+ equilibrium c13+ + c2+ for the photoreduced c1 was found to be slightly lower (Keq = 2.6) than that for the native c1 (Keq = 3.5). The antimycin A-sensitive electron acceptor activity of ferricyanide-reoxidized photoreduced c13+ catalyzed by succinate-cytochrome c reductase was about 80% of that of the native c1. 3. A somewhat simplified method for isolation of cytochrome c1 was developed. Anaerobic ammonium sulfate fractionation and calcium phosphate gel chromatography were still used in order to achieve the purity level of about 25 nmol of heme/mg of protein. The cytochrome c1 prepared by this procedure showed the same properties tested as that by the beta-mercaptoethanol method (Yu, C.A., Yu, L., and King, T.E. (1972) J. Biol. Chem. 247, 1012-1019).     

Modulation of cytochrome biosynthesis in yeast by antimetabolite action of levulinic acid.

Levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, was used to inhibit cytochrome biosynthesis in growing yeast cells. In Saccharomyces cerevisiae the antimetabolite acts by inhibiting delta-aminolevulinic acid dehydratase in vivo, causing an accumulation of intracellular delta-aminolevulinic acid and simultaneous decreases in all classes of mitochondrial cytochromes. Changes in cellular cytochrome content with increasing levulinic acid concentration suggested the existence of different regulatory patterns in S. cerevisiae and Candida utilis. In C. utilis, cytochrome a.a3 formation is very resistant to the antimetabolite action of levulinic acid. In this aerobic yeast, cytochrome c+c1 is the most sensitive to levulinic acid, and cytochrome b exhibits intermediate sensitivity.     

Studies on NADPH-cytochrome c reductase. II. Steady-state kinetic properties of the crystalline enzyme from ale yeast

From a study of the steady-state kinetics (at pH 7.6, 30 degrees C) of the reduction of cytochrome c, a 'ping-pong' mechanism may be postulated for the crystalline NADPH-cytochrome c reductase from ale yeast, Saccharomyces cerevisiae [1], a result derivable from a three-substrate ordered system with a rapid equilibrium random sequence in substrates, NADPH and FAD, followed by reactions of the third substrate, Cyt C3+. On this basis, estimates for the kinetic parameters were made together with the inhibitor dissociation constants for NADP+ (competitive with respect to NADPH as variable substrate, but noncompetitive with respect to cytochrome c3+ as the variable substrate). A noncompetitive type of inhibition was also found for cytochrome c2+ with NADPH as variable substrate, in confirmation of the proposed mechanism. With 2,6-dichloroindophenol as the acceptor, in place of cytochrome c3+, a value for KNADPH could be estimated which agreed with that estimated above, with cytochrome c3+ as the acceptor, again, in confirmation of the postulated mechanism. The reactions with molecular O2 catalyzed by the enzyme with NADPH as the reductant have been studied polarographically, and its Km for O2 estimated to be about 0.15 mmol/l at pH 7.6, 25 degrees C. The product of the reaction appears to be H2O2, which acts as a noncompetitive inhibitor for NADPH (Ki = 0.5 mmol/l), and tentatively an enzyme ternary complex containing oxygen and FADoh (semiquinone of FAD) may be assumed to be the kinetically important intermediate, which may be postulated to be in quasi-equilibrium with an enzyme ternary complex containing Oo2 (superoxide) and FAD.     

Redox potentials in hydro-organic media at normal and subzero temperatures. Ferro-ferricyanide and cytochrome c as models.

Redox potentials of ferro-ferricyanide and cytochrome c were measured in water/ethylene glycol and water/dimethylsulfoxide (volume ratio from 100/0 to 50/50) between 25 and -25 degrees C. For both systems, the midpoint potential decreases in the presence of organic solvents and increases by cooling. The magnitude of these variations is larger in dimethylsulfoxide than in ethylene glycol; moreover in the same solvent mixture it is larger with ferro-ferricyanide than with cytochrome c, so that the difference between the redox potentials of these two systems can be strongly affected and even reversed. While in pure water (cacodylate buffer pH 7.0, NaCl 0.1 M) they are respectively +388 and +265 mV, in 50% dimethylsulfoxide at 25 degrees C they decrease to +112 and +208 mV. Reduction of cytochrome c by ferro-ferricyanide, in this mixture, is then expected and was indeed observed. On the other hand, as (deltaE/deltaT)T, (E being the redox potential) is higher for ferro-ferricyanide than for cytochrome c, the oxidative power of the former for the latter is expected to increase as temperature decreases. This effect was observed in 50% ethylene glycol at -16 degrees C. Organic solvents and large temperature variations appear then as powerful perturbants of redox reactions. Their effects should be taken into account in studies of redox reactions carried out in cooled hydro-organic media.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a3 2+ . CO.

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity. Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a3 in the mixed-valence enzyme after photodissociation. Upon addition of CO to partially reduced formate cytochrome c oxidase (a2+a3 3+ . HCOOH) the cytochrome a3 2+. CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a3 and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a3 3+ . HCOOH compound is dissociated slowly with a concomitant formation of the a3 2+ . CO compound and oxidation of cytochrome a. When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a3 and the a2+ 3 . CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain.

1. Formate inhibits cytochrome c oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome aa3. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species. 2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome aa3 (a3 + a33+) and in the half-reduced species (a2 + a33+). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the aa3 spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of a32+ minus a33+, free of the difficulties with cyanide (which induces marked high leads to low spin spectral shifts in cytochrome a33+) and azide (which induces peak shifts of cytochrome a2+ towards the blue in both alpha- and Soret regions). 3. The rate of formate dissociation from cytochrome a2+ a33+ -HCOOH is faster than its rate of dissociation from a3+ a33+ -HCOOH, especially in the presence of cytochrome c. The Ki for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 degrees C. 4. Succinate-cytochrome c reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2. 5. Formate inhibition of ascorbate plus N, N, N', N'-tetramethyl-p-phenylenediamine oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in 'on' or 'off' inhibition rates were observed when intact mitochondria were compared with submitochondrial particles. 6. NADH-cytochrome c reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome aa3 level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Characterization of the reaction between ferrocytochrome c and cytochrome c oxidase.

The reaction between cytochrome c oxidase and ferrocytochrome c has been investigated by the stopped-flow method. It has been found that only one electron acceptor, a heme group, in the oxidase is rapidly reduced by cytochrome c. The presence of N3- does not affect the reduction of the acceptor, which supports the hypothesis that this is identical with cytochrome a. The results are consistent with the existence of a simple equilibrium between cytochrome a and cytochrome c: c-2 + a-3+ in equilibrium c-3+ + a-2+ with an equilibrium constant corresponding to an oxidation-reduction potential of cytochrome a 30 mV higher than that for cytochrome c at pH 7.4. The oxidation-reduction potential of the a-3+ /a-2+ couple, 285 mV (based on a potential of 255 mV for cytochrome c), and the optical properties of the reduced form indicate that it is identical with neither of the reduced hemes seen in potentiometric titrations. The oxidase species resulting from the rapid reduction of cytochrome a by cytochrome c is proposed to represent a metastable intermediate state which, under anaerobic conditions, eventually is transformed into a more stable state characterized by a reduced high-potential heme.     

Stepwise reduction of cytochromes b-562, b-566 and b-558 in rat liver mitochondria.

1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria with out addition of substrate caused reduction of cytochromes b-562 (having an alpha-band at 562 nm at room temperature), c + c1, and a + a3. The effect of KCN on cytochrome b-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromes c+c1 and a+a3 was not reversed by this uncoupler.2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 muM KCN under conditions were cytochromes b-562, c+c1 and a+a3 were reduced, caused reduction of cytochrome b-566. The absorbance spectrum of cytochrome b-566 had an alpha-band at 565.5 nm, a beta-band at 538 nm and a gamma-band at 431 nm, but no shoulder around 558 nm at room temperature. 3. Addition of succinate to rotenone-KCN-inhibited and ATP-treated rat liver mitochondria under conditions where cytochromes b-566, b-562, c+c1 and a+a3 were already fully reduced, caused reduction of cytochrome b-558 (having an alpha-band at 558 nm, a beta-band at 527 nm and a gamma-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochrome b-566). 4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochrome b-566, but is due to a different species from cytochrome b-566.     

Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.     

Calcium induced release of mitochondrial cytochrome c by different mechanisms selective for brain versus liver.

Increased mitochondrial Ca2+ accumulation is a trigger for the release of cytochrome c from the mitochondrial intermembrane space into the cytosol where it can activate caspases and lead to apoptosis. This study tested the hypothesis that Ca2+-induced release of cytochrome c in vitro can occur by membrane permeability transition (MPT)-dependent and independent mechanisms, depending on the tissue from which mitochondria are isolated. Mitochondria were isolated from rat liver and brain and suspended at 37 degrees C in a K+-based medium containing oxidizable substrates, ATP, and Mg2+. Measurements of changes in mitochondrial volume (via light scattering and electron microscopy), membrane potential and the medium free [Ca2+] indicated that the addition of 0.3 - 3.2 micromol Ca2+ mg-1 protein induced the MPT in liver but not brain mitochondria. Under these conditions, a Ca2+ dose-dependent release of cytochrome c was observed with both types of mitochondria; however, the MPT inhibitor cyclosporin A was only capable of inhibiting this release from liver mitochondria. Therefore, the MPT is responsible for cytochrome c release from liver mitochondria, whereas an MPT-independent mechanism is responsible for release from brain mitochondria.     

Conformational isomerism and effective redox geometry in the oxidation of heme proteins by alkyl halides, cytochrome c, and cytochrome oxidase.

In contrast to its lethargy at physiological pH, horse heart cytochrome c can be oxidized at room temperature by the axial inner sphere oxidant bromomalononitrile (BMN) at higher acidities. The following stoichiometry obtains: 2Fe11 c + BrCH(CN2) + H+ leads to 2FeIII c + CH2(CN)2 + Br-, and the rate law is given by: rate = k2(FeIIc)(BMN). At an ionic strength of 1.0 (KCl), second-order rate constants vary from 300 l. per mol per sec (pH 2-3) to 0(pH 9). Below pH 6 there is a noticeable increase in rate with ionic strength while there is no specific salt effect for the process. At pH 7.4 there is no influence of added salt (0.01-1.0 M) upon the slow rate of reaction. The vast changes in rate occur over a pH region (3-6) in which only very minor changes in the visible spectrum of the cytochrome are manifest. The results are interpreted in terms of a conformational isomerism of cytochrome c in which the effective redox geometry alters from a predominantly "short C" form (in which an axial position is available for substitution) at lower pH's to a predominantly "C" form (axial positions encumbered) in the physiological region. At 5 degrees, pH 7.4, both hemes of beef heart cytochrome oxidase are oxidized by the addition of BMN (k2 = 29 plus or minus 3 l. per mol per sec). However, the reaction is inhibited by potassium cyanide and the protein containing iron(II) cyt alpha along with the cyano adduct of iron(II) or iron(III) cyt alpha3 is inert. The results demonstrate cytochrome alpha3 as the site of reaction and that alpha reduces alpha3 in the process. Cytochrome oxidase does catalyze the oxidation of cytochrome c with BMN as substrate. Taken together the results provide additional support for a recent theory and they demonstrate BMN to be an efficient probe for the effective redox geometry of a hemoprotein in solution.     

Further characterization of gradient-fractionated submitochondrial membrane fragments from beef heart mitochondria.

We have recently reported that with a linear sucrose density gradient centrifugation two distinct types of membrane fragments, designated as X- and Y-fragments are obtained (Huang, C. H., Keyhani, E. and Lee, C. P. (1973) Biochim. Biophys. Acta 305, 455-473). Further characterization of these two membranes fragments is reported. (1) Potassium chloride at the concentration of 0.15 m extracts 7% and 30% of cytochrome c from the X- and Y-fragments, respectively. (2) When cytochrome c was added to the mitochondrial suspension prior to sonication, the cytochrome c content was increased by 6-8-fold in both X- and Y-fragments. Subsequently KC1 extraction resulted in loss of cytochrome c by 1/4 in the X- and by 2/3 in the Y-fragments. (3) With partially inhibitory concentrations of KCN, cytochrome c in either the X- or the KC1 extracted X-fragments showed uncoupler-sensitive, biphasic reduction kinetics upon the addition of NADH to the oligomycin-supplemented system. Under identical conditions rapid first order reduction kinetics were seen for cytochrome c in Y-fragments supplemented with either oligomycin or oligomycin + carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (4) When cytochrome c was added to the mitochondrial suspension after sonication, a significant amount of cytochrome c was bound to both X- and Y-fragments, but was readily removed with a high ionic strength medium. (5) Lubrol had little effect on the ATPase activity of the X- and the Y-fragments, suggesting a lack of membrane-buried ATPase. (6) Partial depletion of ATPase in X-fragments did not induce an increase in reactivity towards externally added cytochrome c. (7) Both the X- and the Y-fragments showed an energy-linked fluorescence enhancement of 8-anilinonaphthalene-1-sulfonate and an energy-linked fluorescence decrease of quinacrine. (8) In the presence of K-+ nigericin alone or in combination with valinomycin exhibited a stimulating effect on the rate of NADH oxidase of the oligomycinsupplemented X- and Y-fragments.     

A new carbon monoxide-induced complex of cytochrome c oxidase.

Cytochrome c oxidase isolated from ox heart forms a complex in the presence of millimolar concentrations of CO with absorption bands at 606, 565 and 435 nm (difference spectrum), distinct from both ferrocytochrome a and the classical 590nm carbon-monoxyferrocytochrome a3. This species, which closely resembles Compound C, the derivative formed on photolysis and oxygenation of mixed-valence cytochrome a3+a32+CO, may represent a cytochrome a32+CO complex in which the associated ('invisible') copper is still oxidized.