C.P. Lee

High-energy forms of cytochrome b. I. The effect of ATP and antimycin on cytochrome b in phosphorylating sub-mitochondrial particles

Abstract 1. As in non-phosphorylating particles, antimycin causes a shift of the wavelength of maximum absorption of ferrocytochrome b in phosphorylating particles to the red by 2 nm. The antimycin-effect curve is sigmoidal. 2. ATP also causes a red shift, less than that brought about by antimycin and additive to the effect of the latter. 3. In addition to a red shift ATP brings about an oxidation of cytochrome b . 4. The ATP-induced red shift also occurs in the presence of rotenone when NADH is electron donor and in the presence of malonate or 4,4,4-trifluoro-1-(2-thionyl)-1,3-butadione when succinate is the donor. 5. The ATP-induced oxidation of cytochrome b occurs in the presence of both rotenone and antimycin. The oxidant is probably ubiquinone. 6. It is proposed that mitochondria contain two species of cytochrome b in equal amounts, one ( b i ) affected by antimycin and the other ( b ) not affected. Antimycin reacts preferentially with a form of b i present in high concentrations in energized particles. 7. It is proposed that the ATP-induced red shift is due to the formation of b 2+ ∼ X, a low-potential cytochrome b ( b 2+ ∼ X ag b 3+ + X + e ). 8. It is proposed that b i 3+ ∼ X is a high-potential cytochrome b ( b i 2+ + X ag b i 3+ ∼ X + e ). 9. A mechanism of Site-II phosphorylation is proposed in which ATP synthesis is linked with intramolecular electron transfer within the dimer b 2+ ∼ X · b i 3+ ∼ X.

Effect of ion-transporting antibiotics on the energy-linked reactions of submitochondrial particles☆

Abstract The effects of ion-transporting antibiotics on the energy-linked reactions of submitochondrial particles were studied. It was found that valinomycin and nigericin in the presence of K+ led to the release of the oligomycin-induced respiratory control, and to a virtually complete inhibition of the four energy-linked reactions: pyridine nucleotide transhydrogenation, BTB and ANS responses, and reversal of electron transfer. Valinomycin alone in the presence of K+ did not release the oligomycin-induced control, but inhibited the energy-linked reactions by approximately 50%. Nigericin alone in the presence of K+ stimulated the oligomycin-inhibited respiration by approximately 50%, but did not affect the energy-linked reactions. The results are discussed in terms of current hypotheses of energy coupling in oxidative and photosynthetic phosphorylation.

Fractionation by sucrose density gradient centrifugation of membrane fragments derived by sonic disruption of beef heart mitochondria

Abstract 1. 1. Fractionation of beef heart submitochondrial membrane fragments derived by sonic disruption was achieved by linear sucrose density gradient centrifugation. Three distinct membrane fractions were obtained: a pellet (P), a middle (X) and an upper (Y) zone. 2. 2. Electron micrographs revealed that the pellet contained large single pieces and aggregates of membrane fragments. The X zone contained rough membrane fragments with protruding subunits (F1) and the Y zone contained relatively small fragments without subunits. 3. 3. In comparison with the Y zone, the X zone exhibited higher activities of NADH oxidase and ATPase, higher oligomycin-induced respiratory control and cytochrome content, but lower activities of NADH:cytochrome c reductase (rote-none-sensitive) and cytochrome oxidase with externally-added cytochrome c as the substrate. Lubrol greatly enhanced the cytochrome oxidase activity in the X zone but not in the Y zone. NADH oxidase was greatly stimulated by externally added cytochrome c in the Y zone but not in the X zone. In general, the pellet exhibited varying activities falling between those of the X zone and the Y zone. 4. 4. The relationship between these membrane fractions and the conventional EDTA particle preparation is discussed.

Comparison of fluorescence probe and light-scattering readout of structural states of mitochondrial membrane fragments.

The possible functional relationship between energy conservation reactions and mitochondrial structure as observed electron microscopically [l] or by nonspecific light scattering changes (NSLS) is obscured by kinetic discrepancies between the energy coupling reaction as indicated by cytochrome responses [2], by NSLS [3] and by the “orthodox-condensed” matrix transition of intact mitochondria [4]. Submitochondrial particles afford a simpler system for studying conformational changes as read out by NSLS since they have lost matrix material. In these particles, energy coupling reactions, as read out by the membrane-bound fluorochrome, 8-anilino-1-naphthalene sulfonic acid (ANS) [5] , are closely identified with the energy conservation reactions of membrane fragments, as indicated by observations of reversed electron transfer [6] and by titrations with uncoupling agents [7] . Packer has reported energy-dependent NSLS changes in digitonin particles [8] but such preparations have a “sidedness” identical to intact mitochondria [9]. In this communication, well-defined membrane fragments [lo] have been employed, in which the subunits of the inner membrane, identified as the coupling factor F, [ 1 l] project outwards. pH-induced changes of light-scattering, optical rotatory dispersion, and ANS fluorescence have recently been correlated by Packer [ 121. In the studies reported here, the response of NSLS as an indicator of membrane conformation is compared with the response of ANS fluorescence as an indicator of energy coupling in transitions between the non-energized and energized states of the membrane. We find that the two

Spectrophotometric and kinetic studies of the reconstituted succinate oxidase system

Summary Some spectral and kinetic properties of the reconstituted succinate oxidase system of Keilin and King have been studied. 1. With the reconstituted enzyme system the total rate of succinate oxidation was not affected by dilution. 2. The distinct absorption peaks that appeared at 603, 562, 552 and 442 m μ , are essentially the same as those observed for the untreated heart-muscle preparation except for a slight shift of the α -peak of cytochrome a from 604 to 603 m μ . 3. A partial inactivation and/or destruction of the cytochromes may have occurred during the alkali treatment of heart-muscle preparation as indicated by the decrease in the extent of enzymic reduction by succinate as well as their low turnover number. 4. The velocity constants for the sequential reactions of succinate oxidase have been calculated. The magnitudes of these constants are quite similar to those obtained for the untreated heart-muscle preparation. 5. Kinetic evidence indicates that the soluble fraction derived from the alkali treatment of the heart-muscle preparation has a competitive inhibitory effect in relation to the active soluble succinate dehydrogenase (succinate:(acceptor) oxido-reductase, EC 1.3.99.1) on the reduction of cytochrome b by succinate. 6. The molecular relationship between succinate dehydrogenase and cytochrome b as well as the participation of cross-linked respiratory chain assemblies during succinate oxidation are discussed.

Circular dichroism of cytochrome oxidase, cytochrome b566, and cytochrome c in beef heart mitochondrial membrane fragments

Abstract 1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found. 2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced- minus -oxidized difference molar ellipticity at 445 nm, Δ [ θ ] 445 is 3.0·10 5 degree·cm −2 ·dmole −1 heme a for membrane-bound oxidase compared to 1.6·10 5 degree·cm −2 ·dmole −1 heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase. 3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase. 4. 4. Cytochrome c , which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c 1 or from cytochrome b 561 in either the oxidized or the reduced form. 5. 5. Cytochrome b 566 in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b 566 reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline- N -oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b 566 is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b 566 on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochromes midpoint redox potential.

L-3-glycerol phosphate oxidation with energy coupling in submitochondrial particles from skeletal muscle mitochondria

Abstract Submitochondrial particles (SMP) from rabbit skeletal muscle mitochondria (RMM) oxidize NADH, succinate and L-3-glycerol phosphate (L-3-GP); NADH is not oxidized by RMM unless cholate is added. Ferricyanide has complete access to succinic and L-3-GP dehydrogenases in SMP, but access only to L-3-GP dehydrogenase in RMM. Energy conservation in SMP was demonstrated by the criteria of respiratory control and uncoupler-induced oxidation of cytochrome b with all three substrates. L-3-GP dehydrogenase is on the outer, while NADH and succinic dehydrogenases are on the inner side of the RMM inner membrane which is impermeable to NADH, L-3-GP and ferricyanide. Since SMP are effectively permeable to these three substrates, they must be open membrane fragments capable of conserving energy in the absence of transmembrane electrochemical potentials.

Further characterization of gradient-fractionated sub-mitochondrial 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.

Energy-linked swelling of EDTA submitochondrial particles

The osmotic properties of EDTA submitochondrial particles have been studied by means of light-scattering measurements and radioisotopic determination of water distribution. It is shown that EDTA particles exhibit a respiration-linked swelling which: (i) requires oligomycin and NO3−; (ii) is promoted by nigericin and inhibited by valinomycin in the presence of K+ but not in the presence of Na+; and (iii) is reversed by FCCP. It is concluded that the energy-linked swelling of EDTA particles is caused by energy-linked influx of salts.

Uncoupling of submitochondrial particles by gramicidin

The gramicidins A, B, and C are linear polypeptide units with the following generic formula: N-formylpentadecapeptide ethanolamine [5] . These ion-transporting antibiotics show little ionic discrimination, the spectrum of selectivity being K+, Rb+, CY, Nh+, Na+, Li+, MeNH3+, as well as H+ [5,6] . Its ion transporting properties have been studied in mitochondria [7,8], chloroplasts [9, lo] , chromatophores [ 11, 12, 131, erythrocytes [6,7,14] phospholid micelles [6,7, 141, black lipid membranes [ 151, and Streptococcus faecalis [ 161. Previous studies from this laboratory [2,3,4] have explored the effects of ion-transporting antibiotics [cf. l] on the energy-linked functions and ion-translocation properties of submitochondrial particles (SMP)‘. As a continuation of these studies, in the present communication we wish to report the effects of gramioidin on SMP and discuss the results in terms of the permeability-modifier action of gramicidin.