Proc: Nati. Acad. Sci. USA Vol. 74, No. 9, pp. 3662-3666, September 1977 Biochemistry
Coupling in cytochrome c oxidase (ionophore transfer complex/electrogenic intrinsic ionophores/uncoupling combinations/respiratory control/electron-positive charge coupling)
R. J. KESSLER, G. A. BLONDIN, H. VANDE ZANDE, R. A. HAWORTH, AND D. E. GREEN Institute for Enzyme Research, University of Wisconsin, Madison, Madison, Wisconsin 53706
Contributed by David E. Green, June 6, 1977
ABSTRACT Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K+ when cytochrome oxidase is incorrated into liposomes containing acidic hospholipids or by the combination of lipid cytochrome c an bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome-oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid. In a previous communication (1) Kessler et al. established that uncouplers are mediators of cyclical cation transport and that this coupled transport takes precedence over all other coupled processes carried out by the mitochondrion. This action of uncouplers depended upon an interaction between uncoupler, cation, and an electrogenic ionophore intrinsic to the mitochondrion. The species thus formed mediated cyclical cation transport. Uncouplers in the presence of the appropriate cations are in effect diagnostic reagents for intrinsic electrogenic ionophores. Hunter et al. (2) in our laboratory have established that mitochondria can exist in either of two states, the N state, appropriate for oxidative phosphorylation, and the Ca2+ state, the evidence of which is uncoupled respiration. A control mechanism regulates the transition between these two states. Haworth (3) has shown that the uncoupled respiration of mitochondria in the Ca2+ state is an expression of coupled cyclical cation transport. Since cyclical cation transport is mediated by uncoupler, it follows that there must be a natural uncoupler present and operative in mitochondria in the Ca2+ state. These three studies provided the experimental framework for an examination of coupling in cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). It was reasonable to assume that uncoupled respiration in cytochrome oxidase was an expression of cyclical cation transport mediated by intrinsic electrogenic ionophores and by an uncoupler. If this were so, the experimental door would be opened not only to the eluci-
dation of the form in which electrogenic ionophores are contained within cytochrome oxidase (and presumably in other electron transfer systems of the mitochondrion), but also to the structural realities of electron-ionophore interactions in energy coupling. The present communication presents documentation for (i) the resolution of cytochrome oxidase into an electron transfer complex (ETC) and an ionophore transfer complex (ITC); (ii) the essentiality of the ITC for energy coupling in cytochrome oxidase; and (iii) the ionophoric components of the ITC that participate in energy coupling.
Resolution of cytochrome oxidase into an ETC and an ITC Cytochrome oxidase prepared by the procedure of Fowler et al. (4) and further purified by the method used by Kopaczyk et al. (5) has an a heme concentration of 10-12 nmol/mg of protein. When this preparation was dissolved in 80% methanol and exposed to 30 mM HCI at -10° for several minutes before neutralization (addition of acetate buffer to pH 5.0), it was separable into a colorless insoluble fraction and a soluble deep green fraction. [See the legend of Table 1 for the details of the method developed (6) to achieve this resolution. ] The colored fraction has an a heme and copper concentration of about 18 nmol/mg of protein, whereas the colorless fraction is essentially devoid of either of these two components. The green fraction is thus enriched in the oxidation-reduction components of the ETC in consequence of the removal of protein devoid of these components. The colorless fraction, as we shall show later, is an ITC that under the conditions of the resolution polymerizes to form a highly insoluble retrograde particle. By contrast, the resolved ETC was far more tractable in respect to dispersal in aqueous media than the ITC. The acid/methanol method (6) is highly effective in resolving cytochrome oxidase into its component complexes but it is less than satisfactory in respect to the retention of electron transfer activity. We have found a method that, even though it resolves cytochrome oxidase less completely than the method of Korman and Vande Zande nonetheless does so without inactivation of electron transfer activity. As shown in Table 1, the a heme concentration increased from an initial value of about 8 nmol to a final value of 13-16 nmol/mg of protein when cytochrome oxidase prepared by the method of Yonetani (7) was resolved by prolonged exposure to cholate at 0-5' in the presence of ammonium sulfate. The colorless fraction retained a significant proportion of its original content of phospholipid, and this made it possible to prepare highly dispersed suspensions of the resolved ITC.
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Abbreviations: ITC, ionophore transfer complex; ETC, electron transfer complex; ANS, aminonaphthalenesulfonic acid. 3662
Biochemistry: Kessler et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Resolution of cytochrome oxidase (C.O.) into ETC and ITC
Method
Starting preparation of C.O.*
Condition for resolution
1
12
83% MeOH/30 mM
2
9
2% cholate/25%
HCl
ETC*
ITC*
18
0
16 0 (NH4)2SO4 Method 1: Cytochrome oxidase prepared by the method of Fowler et al. (4) and purified by the procedure of Kopaczyk et al. (5), used to purify an electron transfer particle, was suspended at a concentration of 17.5 mg/ml in 50 mM Tris-HCl, pH 8.0. This suspension was mixed Vortex with 5 volumes of methanol, 30 mM in HCI, and cooled to -10°. The mixture was centrifuged in the cold room in a clinical centrifuge for 3 min. The supernatant fluid was decanted. The residue was washed with a small volume of methanol/HCl containing 16.7% water. The residue was resuspended in water or dilute Tris-HCI buffer (pH 8.0). The ETC in the supernatant fluid can be precipitated by neutralization of the acid/methanol solution to pH 5.5 with acetate buffer. Method 2: Cytochrome oxidase prepared by the method of Yonetani (7) was suspended at a concentration of 10 mg/ml in a medium 0.1 M in potassium phosphate (pH 7.4) and 2% in potassium cholate. The mixture, after addition of saturated (NH4)2SO4 to 25% saturation, was brought to pH 8.0 and kept at 04° for 3-5 days. The colorless insoluble fraction (ITC) was centrifuged off; the resolved ETC remained in the supernatant fluid. The a heme concentration was determined by the pyridine hemochromogen method (8) as well as by the usual spectrophotometric method. In the preparation of the ETC by Method 1, a portion of the a heme (20%) undergoes modification (shift of the peak from 587 to 555 nm); the contribution of the modified heme to the total a heme complement has been included. The same absorption-coefficient was assumed for the unmodified and modified a heme. The a heme:copper ratio of 1:1 in the ETC is unmodified in the resolution achieved by Method 1, a token of the preservation of the structural integrity of the complex. * nmol of a heme per mg of protein. on a
Electron transfer activity of the ETC resolved by extended exposure to cholate and ammonium sulfate When the electron transfer activity of cytochrome oxidase was evaluated before and after resolution by the method described in the legend of Table 2, the loss in electron transfer activity and the increase in the a heme concentration were parallel. Such parallelism means that the removal of ITC during the resolution of cytochrome oxidase led to a loss in coupling, as measured by a decline in the rate of oxygen uptake. When the electron transfer activity of resolved cytochrome Table 2. Restoration of respiratory activity in resolved cytochrome oxidase by addition of phospholipid (PL) and a high concentration of cytochrome c
State of oxidase
Heme a content of oxidase, nmol/mg protein
Unresolved Resolved
9.1 15.9
Rate of respiration, nmol O/min per nmol a heme +PL +PL* No PL low [c] high [ci high [ci 395 140
245 295
395 476
Cytochrome oxidase was prepared by Method 2 described in the legend of Table 1. The assay mixture was 13 mM in durohydroquinone, 0.2 mM in cytochrome c in the presence of phospholipid and 0.002 mM in the absence of phospholipid, 25 ,uM in tetramethylphenylene diamine, and 10 mM in potassium phosphate (pH 7.4). The concentrations of cytochrome oxidase and of phospholipid (asolectin) added to the assay mixture were, respectively, 30 /Ag/ml and 80 jg/ml. The rate of oxygen uptake was measured with an oxygen electrode at 300. * Corrected for inhibition by phospholipid.
3663
Table 3. Synergism of zwitterionic phospholipids and deoxycholate in the partition of Ca2+ in a two-phase system nmol Ca2+/ml in organic phase Additions PE DOC PE + DOC PC PC + DOC
21 0 224 4 200
The aqueous phase (1 ml) was 0.25 M in tetramethylammonium piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) (pH 7.4) and contained 1.25 Mmol of deoxycholate and 10 ,mol of 45CaC12. The
organic phase was a mixture of toluene/butanol (98:2) and contained 0.75 mg of phospholipid (PE or PC). PE, phosphatidyl ethanolamine; DOC, deoxycholate; PC, phosphatidyl choline.
oxidase was measured at a low concentration of cytochrome c and in the absence of added phospholipid, the decline in respiratory activity in consequence of elimination of the ITC was demonstrable. However, when electron transfer activity was measured at a relatively high concentration of cytochrome c and in the presence of added phospholipid, the original electron transfer activity of unresolved cytochrome oxidase (activity per unit of a heme) could be re-established (Table 2). This means that resolution of cytochrome oxidase did not involve inactivation of the ETC but rather loss of the ITC. When the ITC complex was replaced by an uncoupling combination (the combination of bile acids, phospholipid, and cytochrome c at relatively high concentration), then the full electron-transfer potential of the resolved ETC was once more expressed. In effect, the respiratory control of resolved ETC was relieved by the uncoupling combination of lipid c and bile acid. Uncoupling action of lipid c-bile salt combinations In the assay of cytochrome oxidase it is essential to distinguish between coupling of electron flow to a positively charged ionophoric species provided by the ITC and coupling of electron flow to a positively charged ionophoric species provided by the reagents used in the assay system. The experimental conditions used to assay the electron transfer activity of resolved cytochrome oxidase (legend of Table -2) are appropriate for measuring coupling between the electron transfer complex and the ITC (low cytochrome c and no added phospholipid). However, when both phospholipid and cytochrome c at a relatively high concentration are added to the assay medium, this combination is equivalent to adding an uncoupler combination and such a combination fully restores the electron transfer activity lost by removal of the ITC. It may not be obvious why the combination of cytochrome c at high concentration and phospholipid should act as uncoupler. Cytochrome c forms a complex with phospholipid that is soluble in organic solvents (9). This complex is referred to as lipid c (10). Tyson et al. (11) have shown that the ionophoric capability of lipid c tested in a Pressman cell is an order of magnitude greater than that of the same phospholipid in the absence of cytochrome c. In the preparation of cytochrome oxidase, both before and after resolution, bile salts are used. It can be shown that the combination of bile salts and phospholipids greatly enhance the ionophoric activity of phospholipids (Table 3). Bile acids such as cholate and deoxycholate can act as uncouplers in the same fashion as fatty acids. The combination of an electrogenic ionophore and an uncoupler will of course induce uncoupling via cyclical cation transport. It is this uncoupling action of lipid c in conjunction with bile acids that provides the rationale for the use of phospholipids and high
3664
Biochemistry: Kessler et al. ETP
MiOChondria (uncoupler)
Proc. Natl. Acad. Sci. USA 74 (1977) Cylchrom Oxidks. (with K+) (wihout K+)
Proton
Spike
02 Pulse
K+ Spike
FIG. 1. Proton and K+ spikes manifested on energization of mitochondria, electron transfer particles (ETPH), and cytochrome oxidase by introduction of an oxygen pulse. The spikes were measured with a pH electrode and a K+-sensitive electrode, respectively. The K+-free medium used for the experiment with cytochrome oxidase had a final volume of 6.5 ml and contained 65 ,mol of tetramethylammonium phosphate (pH 7.5), 5 mg of cytochrome c, 0.1 ml of saturated durohydroquinone, 0.25 Mmol of tetramethylphenylene diamine, 0.4 mg of catalase, and 5 mg of cytochrome oxidase. The K+-containing medium was identical except that 65 gmol of potassium phosphate replaced tetramethylammonium phosphate. The measurements with ETPH were done in a medium (7 ml final volume) 0.25 M in sucrose, 1 mM in Tris-HCl (pH 7.4), 50 mM in KCl, and 20 mM in potassium succinate that contained 0.4 mg of catalase, 17.5 mg of ETPH, and 17 gg of oligomycin and rotenone. The experiments with mitochondria were done in a medium (7 ml final volume) 0.25 M in sucrose, 1 mM in Tris-HCl (pH 7.5), and 50 mM in KCl and that contained 21 mg of heavy beef heart mitochondria, 21 Mg of rotenone, 0.4 mg of catalase, 42 nmol of carbonylcyanide-p-trifluoromethoxyphenylhydrazone and 25 Mmol of succinate. In all the experiments, 2 Mmol of H202 was introduced into the cell after anaerobiosis had developed. The two vertical lines for each experiment shown in the figure denote the time of introduction of H202 (left line) and the complete removal of oxygen (right line). The peaks for 100% uptake or release of H+ or K+ have been set at the same height to facilitate comparison of the time relations.
levels of cytochrome c in the routine assay of cytochrome oxidase activity. Just as we have to distinguish between an electrogenic ionophore intrinsic to the mitochondrion or added exogenously, so we have to make a similar distinction in respect to uncoupler. The fact that bile acids added exogeneously provide a source of uncoupler does not rule out the presence of an intrinsic uncoupler in the preparation of cytochrome oxidase. Cyclical transport of cations in cytochrome oxidase Kessler et al. (1) have demonstrated that uncoupled respiration in mitochondria is an expression of coupled cyclical cation transport mediated by an uncoupler and an electrogenic ionophore. It was readily demonstrable by the oxygen pulse technique that the respiration of cytochrome oxidase under the assay conditions described in the legends for Tables 1 and 2 was coupled to cyclical cation transport. The bile acids used in the preparation of cytochrome oxidase can act as uncouplers, and the electrogenic ionophore is provided either by the ITC or by the phospholipid added exogeneously. Fig. 1 shows the spike of K+-dependent proton uptake and release when oxygen was introduced into an anaerobic suspension of cytochrome oxidase. Note that the spike terminates before oxygen was exhausted. This spike is the hallmark of coupled cyclical cation transport. Unless the rates of inflow and outflow of ions are perfectly balanced from the onset of energization, there will be a short period of imbalance during which cations will be taken up and then released (or protons will be released and then taken up). The spike is the visible expression of this imbalance and thus constitutes direct evidence that the respiration involves electron
Table 4. Cation-dependent augmentation af ANS fluorescence by electrogenic ionophores Relative ANS fluorescence in presence of 0.1 mM ionophore Ionophore system Ca2+ K+ ITC (cytochrome oxidase)* 1930 528 10,000-Dalton ionophoroproteint 1360 580 Valinomycin 10 Beauvericin 24 26 12 Phosphatidyl ethanolamine 8 Triton X-100 22 13 * The molecular weight of the ITC is estimated to be 66,000 on the basis of a 3:2 weight ratio between the ITC and ETC of cytochrome oxidase; the molecular weight of the ETC is 100,000. It is assumed in the estimate of molarity that there is one molecule of a Ca2+ ionophore and one molecule of a K+ ionophore for 66,000 daltons of the ITC. t This protein was prepared by extracting beef heart mitochondria with a neutral mixture of chloroform/methanol (2:1) and precipitating the protein several times with ether, removing insoluble protein after each cycle of precipitation and resolubilization. Fluorescence measurements were made in a Perkin-Elmer MPF-3 Fluorescence Spectrophotometer at 200 with an excitation wavelength of 380 nm. The sample (4 ml) was 10 mM in Tris-HCl (pH 7.5), 50MM in ANS, and 0.2 M in the chloride salt of either Ca2+ or K+. The slit width for the control (without ionophore) and the experimental sample (with ionophore) was maintained constant. flow coupled to cyclical cation transport. That this spike is not
due merely to membrane leakiness is attested to by the exact parallelism in time between the introduction of oxygen into the system and the initiation of the spike. Ionophoric capability of the ITC Electrogenic ionophores such as valinomycin, beauvericin, and Triton X-100 induce a large increment in fluorescence in aqueous solutions of aminonaphthalenesulfonic acid (ANS) at neutral pH, but only in the presence of the cations acted upon by these ionophores. The equilibrium for this interaction in aqueous media is highly unfavorable; relatively large concentrations both of electrogenic ionophore and the salts of the cation are required to observe these fluorescence changes (12). Ionophoroproteins such as those of the ITC of cytochrome oxidase by contrast are some two orders of magnitude more efficient than free ionophores on a molar basis with respect to this induction of ANS fluorescence (Table 4). Under the conditions used for this measurement, there would appear to be quantitative interaction of the ionophoroprotein with ANS. We base this conclusion on the fact that increase of the salt concentration from 0.2 M to 2 M does not increase the extent of fluorescence. The fluorescence change in the absence of added salt is less than 10% of the change in the presence of salt. Uncoupler completely eliminates the increment in ANS fluorescence induced by the proteins of the ITC. The possibility that residual phospholipid in the ITC preparation could account for the observed fluorescence change in ANS has been examined. The fluorescence changes induced by phospholipids (neutral or acidic) in amounts equivalent to the estimated phospholipid concentration present in the ITC preparation were determined and shown to be negligible by comparison with the fluorescence changes induced by the proteins of the ITC preparation. When the ionophoroproteins of the ITC of cytochrome oxidase are subjected to tryptic digestion, free ionophore can be
Biochemistry: Kessler et al. Table 5. Induction of mitochondrial swelling by the free ionophore fraction compared to the ionophoropeptide fraction of the ITC Mitochondrial swelling, O.D. units/min per mg protein Test system K+ Ca2+ Ionophoropeptide 335 852 Free ionophore fraction 25,900 67,000 The swelling of beef heart mitochondria was followed spectrophotometrically at room temperature either in a K+-containing medium or in a Ca2+-containing medium. The K+-containing medium was 0.15 M in KNO3, 0.01 M in Tris-nitrate (pH 7.5) and contained 0.5 ,ug of antimycin and rotenone per ml. The Ca2+-containing medium was 0.1 M in calcium acetate, 0.01 M in Tris-acetate (pH 7.5) and contained 0.5 ,g of antimycin and rotenone per ml. The ionophores were introduced in ethanolic solutions.
released. The free ionophore, after isolation by chromatography on alumina, can induce swelling of mitochondria in decimolar solutions of Ca(NO3)2 or KNO3 (Table 5). On a molar basis the free ionophore is some two orders of magnitude more efficient in this induction than the ionophoropeptides obtained by tryptic
degradation of the ionophoroproteins. We describe the isolation and characterization of the ionophores released from the ionophoroproteins of the ITC of cytochrome oxidase oxidase in ref. 13. Extraction of ionophoroproteins The ionophoroproteins of unresolved cytochrome oxidase were extractable into chloroform/methanol (2:1) essentially quantitatively when the mixture was acidified with 2% glacial acetic acid. The protein thus extracted accounts for some 50% of the total protein of cytochrome oxidase. The same proteins were found in the extract obtained by extraction of cytochrome oxidase with acidified chloroform/methanol (2:1) and in the ITC obtained from cytochrome oxidase by resolution either by the cholate/ammonium sulfate or the methanol/HCl procedure. Unresolved cytochrome oxidase prepared by the procedure of Kopaczyk et al. (5) contains six proteins of molecular weights 41,000-43,000, 22,000-25,000 (a doublet), 15,500, 14,000, and 10,000, as determined by electrophoresis in 10% acrylamide gel containing 5% sodium dodecyl sulfate. When cytochrome oxidase was resolved by the method of Korman and Vande Zande (6), the first three protein components were found in the ITC and the last three in the ETC. Thus, the ITC and ETC have two entirely different sets of proteins. Respiratory control in cytochrome oxidase Respiratory control is an inherent property of all coupled particles, and when observed in cytochrome oxidase it reflects the fact that electron flow is limited by the unavailability of a coupling combination. The question is why respiratory control in cytochrome oxidase is demonstrable only under special conditions. The absence of respiratory control in cytochrome oxidase (RC index of 1.0) means that electron flow can be maximally coupled to cyclical cation transport mediated by an electrogenic ionophore and an uncoupler both present and operative in a cytochrome oxidase preparation. We have already demonstrated that unresolved cytochrome oxidase carries out cyclical cation transport; the rate of this transport is not increased by addition of exogeneous uncoupler. This would necessarily imply that unresolved cytochrome oxidase contains all the components required for maximal release of respiration. These components would be an electrogenic ionophore (pro-
Proc. Natl. Acad. Sci. USA 74 (1977)
3665
Table 6. Emergence of respiratory control with incorporation of cytochrome oxidase into liposomes of whole mitochondrial phospholipid
Respiratory Test system
Original cytochrome oxidase suspension Incorporated into liposomes of whole
control index 1.0
mitochondrial P.L.* 7-8 Incorporated into liposomes of neutral mitochondrial P.L.t 1.4 The assay conditions were as described in the legend of Table 2. A low level of cytochrome c (0.002 mM) was used in the assay mixture. The method of Hinkle et al. (16) was followed for the incorporation of cytochrome oxidase into the liposomes. Cytochrome oxidase was prepared by the method of Yonetani (7). P.L., phospholipid. * Chloroform/methanol (2:1) extract of mitochondria (contains the full complement of phospholipids and neutral lipid). Acetone extract (90%) of mitochondria (deficient in cardiolipin and neutral lipids).
vided by the ITC) and an uncoupler (provided by the bile acid) added in the course of isolating cytochrome oxidase. Why then can cytochrome oxidase show essentially complete respiratory control when incorporated into phospholipid liposome? The important point is that this effect of incorporating cytochrome oxidase into liposomes is not general for all phospholipids (14). It is only a particular blend of phospholipids that imposes respiratory control (Table 6). For example, whole mitochondrial lipid was highly effective in inducing respiratory control whereas mitochondrial lipid extracted with a solvent mixture that leaves behind cardiolipin and neutral lipids was relatively ineffective. The fact that respiratory control, when imposed, was released by addition of valinomycin + nigericin (an ionophore combination that mediates cyclical cation transport) provides the clearest evidence that the ITC intrinsic to cytochrome oxidase is inoperative under conditions in which respiratory control is imposed and operative under conditions in which respiratory control is not imposed. Why should the blend of phospholipids in the liposomes determine whether respiratory control is imposed or not? We have established a correlation between the geometric character of the liposomes, the nature of the phospholipid, and the extent of respiratory control. The phospholipids that impose respiratory control induced formation of relatively small liposomal vesicles (possibly tubular membranes), whereas the phospholipids that do not impose respiratory control induced formation of relatively large liposomal vesicles (Fig. 2). There appears to be a close correlation between the degree of curvature of the liposomal vesicles and the emergence of respiratory control. The ITC in cytochrome oxidase apparently is inoperative when the liposome in which it is incorporated has a high degree of curvature and operative when the curvature is less extreme (large vesicles). It would appear that the phospholipid blend determines the geometry of the liposomal vesicles and that the geometry goes parallel with the polarity of the membrane. On prolonged standing, the liposomal particles that showed initially high respiratory control with incorporated cytochrome oxidase progressively lose this control, and this loss goes parallel with the conversion of small vesicles to increasingly larger vesicles. Thus, even with the same phospholipid blend, the liposomal vesicles can undergo progressive changes in geometry and corresponding changes in the degree of respiratory control. Another feature of the liposome methodology developed by E. Racker and his colleagues (15-17) is that respiratory control in liposomes in which cyto-
Biochemistry: Kessler et al.
3666
Proc. Natl. Acad. Sci. USA 74 (1977)
A
B
FIG. 2.
Electron
micrographs of liposomal vesicles
in which cy-
tochrome oxidase has been incorporated. For details of the preparation of the was
phospholipids, see legend
of
TableS5. Cytochrome oxidase
incorp'orated into the vesicles by the method of Hinkle et
a!.
(16).
The
specimens used for electron microscopy were negatively stained with 2% ammonium molybdate (X100,000.) (A) Vesicles with respiratory control obtained by incorporation of cytochrome oxidase into of whole mitochondrial phospholipid. (B) Vesicles without respiratory control obtained by incorporation of cytochrome oxidase into liposomes containing only the neutral phospholipids of mito-
liposomes
chondria chrome oxidase has been
ejection
during
incorporated goes parallel with proton
energization,
control in liposomes in
whereas the lack of
which
respiratory
cytochrome oxidase
has been
has been extensively examined by Racker and his colleagues (14-17) in a series of experimental studies. They rationalize this emergence in terms of a correlation between random orientations of the units of cytochrome oxidase in the membrane, leading to loss of respiratory control, and uniform orientation of the units, leading to the emergence of respiratory control. Thus, the lack of respiratory control in the standard preparations of cytochrome oxidase is attributed to random orientation, and the emergence of respiratory control when such preparations are incorporated into suitable liposomal vesicles is attributed to uniform orientation. This assumption is untenable. Our oxygen pulse studies (see Fig. 1) show that cyclical cation transport (uncoupled respiration) can be observed, regardless of whether the orientation leads to proton uptake (cation release) or proton release (cation uptake). In other words, cyclical cation transport can proceed in either direction, leading to proton release as in mitochondria (in the presence of added uncoupler) or to proton uptake as in ETP or cytochrome oxidase (in the presence of intrinsic uncoupler). We can induce respiratory control in cytochrome oxidase merely by resolving the ETC. Then addition of lipid c in combination with the bile acids used in the resolution can release respiratory control. No reorientation of the electron transfer units in the membrane has to be invoked in such a case. This investigation was supported in part by Program Project Grant GM 12847 of the National Institute of General Medical Sciences.
incorporated goes parallel with proton uptake during energization. This means that the orientation of cytochrome oxidase in the minivesicles is the opposite of that in the large yesicles.
Discussion
The studies described in the present communication permit the following conclusions:
(i) cytochrome oxidase
is a duplex of
an
when cytochrome oxidase is stripped of its complement of ITC, it loses the capacity for electron flow; ETC
and
an
ITC; (ii)
(Mi) when the activity of the ITC is suppressed, the electron transport activity of cytochrome oxidase is depressed to comparable degree; (iv) the ITC contains complement of
a
a
ionophores transport;
sufficient
and
for the
mediation
of
cyclical
cation
(v) cyclical cation transport is directly demon-
cytochrome oxidase by the oxygen pulse technique. interrelationships and correlations we can further in cytochrome oxidase depends upon
strable in From these conclude that coupling
interaction between moving electron in an ETC positively charged ionophoric species in ITC. In earlier studies from this laboratory Kopaczyk et al. (5) demonstrated that the four complexes of the electron transfer chain, as well as the tripartite repeating unit, could be resolved into what these authors referred to as a catalytic protein fraction and a structural protein fraction. It is now apparent that the catalytic protein fraction corresponds to the ETC and the structural protein fraction to the ITC. Once these identities were recognized it was possible to draw upon the extensive studies of structural protein and deduce from these studies the duplex nature of each of the four complexes of the electron transfer chain as well as of the tripartite repeating unit, and the invariant linkage on the one hand between ETCs and ITCs and on the other hand between the F1 system and its associated ITC. The emergence of respiratory control in cytochrome oxidase
electrostatic and a
a
an
1. Kessler, R. J., Vande Zande, H., Tyson, C. A., Blondin, G. A., Fairfield, J., Glasser, P. & Green, D. E. (1977) Proc. Natl. Acad.
Sci. USA 74,2241-2245. 2. Hunter, D. R., Haworth, R. A. & Southard, J. H. (1976) J. Biol. Chem. 251, 5069-5077. 3. Haworth, R. A. (1977) Biophysical Society Annual Meeting, New Orleans, LA, February, 1977, Abstract. 4. Fowler, L. R., Richardson, S. H. & Hatefi, Y. (1962) Biochim. Biophys. Acta 64, 170-173. 5. Kopaczyk, K., Perdue, J. & Green, D. E. (1966) Arch. Biochem.
Biophys. 115,215-225. 6. Korman, E. F. & Vande Zande, H. (1968) Fed. Proc. 27, Abstract 1737. 7. Yonetani, T. (1967) in Methods in Enzymology, eds. Estabrook, R. W. & Pullman, M. E. (Academic Press, New York), Vol. 10, pp. 332-5. 8. Drabkin, D. L. (1942) J. Biol. Chem. 146,605-617. 9. Widmer, C. & Crane, F. L. (1958) Biochim. Biophys. Acta 27, 203-204. 10. Green, D. E. & Fleischer, S. (1963) Biochim. Biophys. Acta 70, 554-582. 11. Tyson, C. A., Vande Zande, H. & Green, D. E. (1976) J. Biol.
Chem. 251, 1326-1331. 12. Feinstein, M. B. & Felsenfeld, H. (1971) Proc. Natl. Acad. Sci. USA 68, 2037-2041. 13. Blondin, G. A., Kessler, R. J. & Green, D. E. (1977) Proc. Nat!. Acad. Sci. USA 74,3667-3671. 14. Eytan, G. D., Matheson, M. J. & Racker, E. (1976) J. Biol. Chem.
261,6831-6837.
15. Racker, E. (1972) in Molecular Basis of Electron Transport, eds. Schultz, J. & Cameron, B. F. (Academic Press, New York and London), p. 45. 16. Hinkle, P. C., Kim, J. J. & Racker, E. (1972) J. Biol. Chem. 247, 1338-1339. 17. Kagawa, Y., Johnson, L. W. & Racker, E. J. (1973) Biochem. Biophys. Res. Commun. 50,245-251.
Coupling in cytochrome c oxidase (ionophore transfer complex/electrogenic intrinsic ionophores/uncoupling combinations/respiratory control/electron-positive charge coupling)
R. J. KESSLER, G. A. BLONDIN, H. VANDE ZANDE, R. A. HAWORTH, AND D. E. GREEN Institute for Enzyme Research, University of Wisconsin, Madison, Madison, Wisconsin 53706
Contributed by David E. Green, June 6, 1977
ABSTRACT Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K+ when cytochrome oxidase is incorrated into liposomes containing acidic hospholipids or by the combination of lipid cytochrome c an bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome-oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid. In a previous communication (1) Kessler et al. established that uncouplers are mediators of cyclical cation transport and that this coupled transport takes precedence over all other coupled processes carried out by the mitochondrion. This action of uncouplers depended upon an interaction between uncoupler, cation, and an electrogenic ionophore intrinsic to the mitochondrion. The species thus formed mediated cyclical cation transport. Uncouplers in the presence of the appropriate cations are in effect diagnostic reagents for intrinsic electrogenic ionophores. Hunter et al. (2) in our laboratory have established that mitochondria can exist in either of two states, the N state, appropriate for oxidative phosphorylation, and the Ca2+ state, the evidence of which is uncoupled respiration. A control mechanism regulates the transition between these two states. Haworth (3) has shown that the uncoupled respiration of mitochondria in the Ca2+ state is an expression of coupled cyclical cation transport. Since cyclical cation transport is mediated by uncoupler, it follows that there must be a natural uncoupler present and operative in mitochondria in the Ca2+ state. These three studies provided the experimental framework for an examination of coupling in cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). It was reasonable to assume that uncoupled respiration in cytochrome oxidase was an expression of cyclical cation transport mediated by intrinsic electrogenic ionophores and by an uncoupler. If this were so, the experimental door would be opened not only to the eluci-
dation of the form in which electrogenic ionophores are contained within cytochrome oxidase (and presumably in other electron transfer systems of the mitochondrion), but also to the structural realities of electron-ionophore interactions in energy coupling. The present communication presents documentation for (i) the resolution of cytochrome oxidase into an electron transfer complex (ETC) and an ionophore transfer complex (ITC); (ii) the essentiality of the ITC for energy coupling in cytochrome oxidase; and (iii) the ionophoric components of the ITC that participate in energy coupling.
Resolution of cytochrome oxidase into an ETC and an ITC Cytochrome oxidase prepared by the procedure of Fowler et al. (4) and further purified by the method used by Kopaczyk et al. (5) has an a heme concentration of 10-12 nmol/mg of protein. When this preparation was dissolved in 80% methanol and exposed to 30 mM HCI at -10° for several minutes before neutralization (addition of acetate buffer to pH 5.0), it was separable into a colorless insoluble fraction and a soluble deep green fraction. [See the legend of Table 1 for the details of the method developed (6) to achieve this resolution. ] The colored fraction has an a heme and copper concentration of about 18 nmol/mg of protein, whereas the colorless fraction is essentially devoid of either of these two components. The green fraction is thus enriched in the oxidation-reduction components of the ETC in consequence of the removal of protein devoid of these components. The colorless fraction, as we shall show later, is an ITC that under the conditions of the resolution polymerizes to form a highly insoluble retrograde particle. By contrast, the resolved ETC was far more tractable in respect to dispersal in aqueous media than the ITC. The acid/methanol method (6) is highly effective in resolving cytochrome oxidase into its component complexes but it is less than satisfactory in respect to the retention of electron transfer activity. We have found a method that, even though it resolves cytochrome oxidase less completely than the method of Korman and Vande Zande nonetheless does so without inactivation of electron transfer activity. As shown in Table 1, the a heme concentration increased from an initial value of about 8 nmol to a final value of 13-16 nmol/mg of protein when cytochrome oxidase prepared by the method of Yonetani (7) was resolved by prolonged exposure to cholate at 0-5' in the presence of ammonium sulfate. The colorless fraction retained a significant proportion of its original content of phospholipid, and this made it possible to prepare highly dispersed suspensions of the resolved ITC.
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Abbreviations: ITC, ionophore transfer complex; ETC, electron transfer complex; ANS, aminonaphthalenesulfonic acid. 3662
Biochemistry: Kessler et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Resolution of cytochrome oxidase (C.O.) into ETC and ITC
Method
Starting preparation of C.O.*
Condition for resolution
1
12
83% MeOH/30 mM
2
9
2% cholate/25%
HCl
ETC*
ITC*
18
0
16 0 (NH4)2SO4 Method 1: Cytochrome oxidase prepared by the method of Fowler et al. (4) and purified by the procedure of Kopaczyk et al. (5), used to purify an electron transfer particle, was suspended at a concentration of 17.5 mg/ml in 50 mM Tris-HCl, pH 8.0. This suspension was mixed Vortex with 5 volumes of methanol, 30 mM in HCI, and cooled to -10°. The mixture was centrifuged in the cold room in a clinical centrifuge for 3 min. The supernatant fluid was decanted. The residue was washed with a small volume of methanol/HCl containing 16.7% water. The residue was resuspended in water or dilute Tris-HCI buffer (pH 8.0). The ETC in the supernatant fluid can be precipitated by neutralization of the acid/methanol solution to pH 5.5 with acetate buffer. Method 2: Cytochrome oxidase prepared by the method of Yonetani (7) was suspended at a concentration of 10 mg/ml in a medium 0.1 M in potassium phosphate (pH 7.4) and 2% in potassium cholate. The mixture, after addition of saturated (NH4)2SO4 to 25% saturation, was brought to pH 8.0 and kept at 04° for 3-5 days. The colorless insoluble fraction (ITC) was centrifuged off; the resolved ETC remained in the supernatant fluid. The a heme concentration was determined by the pyridine hemochromogen method (8) as well as by the usual spectrophotometric method. In the preparation of the ETC by Method 1, a portion of the a heme (20%) undergoes modification (shift of the peak from 587 to 555 nm); the contribution of the modified heme to the total a heme complement has been included. The same absorption-coefficient was assumed for the unmodified and modified a heme. The a heme:copper ratio of 1:1 in the ETC is unmodified in the resolution achieved by Method 1, a token of the preservation of the structural integrity of the complex. * nmol of a heme per mg of protein. on a
Electron transfer activity of the ETC resolved by extended exposure to cholate and ammonium sulfate When the electron transfer activity of cytochrome oxidase was evaluated before and after resolution by the method described in the legend of Table 2, the loss in electron transfer activity and the increase in the a heme concentration were parallel. Such parallelism means that the removal of ITC during the resolution of cytochrome oxidase led to a loss in coupling, as measured by a decline in the rate of oxygen uptake. When the electron transfer activity of resolved cytochrome Table 2. Restoration of respiratory activity in resolved cytochrome oxidase by addition of phospholipid (PL) and a high concentration of cytochrome c
State of oxidase
Heme a content of oxidase, nmol/mg protein
Unresolved Resolved
9.1 15.9
Rate of respiration, nmol O/min per nmol a heme +PL +PL* No PL low [c] high [ci high [ci 395 140
245 295
395 476
Cytochrome oxidase was prepared by Method 2 described in the legend of Table 1. The assay mixture was 13 mM in durohydroquinone, 0.2 mM in cytochrome c in the presence of phospholipid and 0.002 mM in the absence of phospholipid, 25 ,uM in tetramethylphenylene diamine, and 10 mM in potassium phosphate (pH 7.4). The concentrations of cytochrome oxidase and of phospholipid (asolectin) added to the assay mixture were, respectively, 30 /Ag/ml and 80 jg/ml. The rate of oxygen uptake was measured with an oxygen electrode at 300. * Corrected for inhibition by phospholipid.
3663
Table 3. Synergism of zwitterionic phospholipids and deoxycholate in the partition of Ca2+ in a two-phase system nmol Ca2+/ml in organic phase Additions PE DOC PE + DOC PC PC + DOC
21 0 224 4 200
The aqueous phase (1 ml) was 0.25 M in tetramethylammonium piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) (pH 7.4) and contained 1.25 Mmol of deoxycholate and 10 ,mol of 45CaC12. The
organic phase was a mixture of toluene/butanol (98:2) and contained 0.75 mg of phospholipid (PE or PC). PE, phosphatidyl ethanolamine; DOC, deoxycholate; PC, phosphatidyl choline.
oxidase was measured at a low concentration of cytochrome c and in the absence of added phospholipid, the decline in respiratory activity in consequence of elimination of the ITC was demonstrable. However, when electron transfer activity was measured at a relatively high concentration of cytochrome c and in the presence of added phospholipid, the original electron transfer activity of unresolved cytochrome oxidase (activity per unit of a heme) could be re-established (Table 2). This means that resolution of cytochrome oxidase did not involve inactivation of the ETC but rather loss of the ITC. When the ITC complex was replaced by an uncoupling combination (the combination of bile acids, phospholipid, and cytochrome c at relatively high concentration), then the full electron-transfer potential of the resolved ETC was once more expressed. In effect, the respiratory control of resolved ETC was relieved by the uncoupling combination of lipid c and bile acid. Uncoupling action of lipid c-bile salt combinations In the assay of cytochrome oxidase it is essential to distinguish between coupling of electron flow to a positively charged ionophoric species provided by the ITC and coupling of electron flow to a positively charged ionophoric species provided by the reagents used in the assay system. The experimental conditions used to assay the electron transfer activity of resolved cytochrome oxidase (legend of Table -2) are appropriate for measuring coupling between the electron transfer complex and the ITC (low cytochrome c and no added phospholipid). However, when both phospholipid and cytochrome c at a relatively high concentration are added to the assay medium, this combination is equivalent to adding an uncoupler combination and such a combination fully restores the electron transfer activity lost by removal of the ITC. It may not be obvious why the combination of cytochrome c at high concentration and phospholipid should act as uncoupler. Cytochrome c forms a complex with phospholipid that is soluble in organic solvents (9). This complex is referred to as lipid c (10). Tyson et al. (11) have shown that the ionophoric capability of lipid c tested in a Pressman cell is an order of magnitude greater than that of the same phospholipid in the absence of cytochrome c. In the preparation of cytochrome oxidase, both before and after resolution, bile salts are used. It can be shown that the combination of bile salts and phospholipids greatly enhance the ionophoric activity of phospholipids (Table 3). Bile acids such as cholate and deoxycholate can act as uncouplers in the same fashion as fatty acids. The combination of an electrogenic ionophore and an uncoupler will of course induce uncoupling via cyclical cation transport. It is this uncoupling action of lipid c in conjunction with bile acids that provides the rationale for the use of phospholipids and high
3664
Biochemistry: Kessler et al. ETP
MiOChondria (uncoupler)
Proc. Natl. Acad. Sci. USA 74 (1977) Cylchrom Oxidks. (with K+) (wihout K+)
Proton
Spike
02 Pulse
K+ Spike
FIG. 1. Proton and K+ spikes manifested on energization of mitochondria, electron transfer particles (ETPH), and cytochrome oxidase by introduction of an oxygen pulse. The spikes were measured with a pH electrode and a K+-sensitive electrode, respectively. The K+-free medium used for the experiment with cytochrome oxidase had a final volume of 6.5 ml and contained 65 ,mol of tetramethylammonium phosphate (pH 7.5), 5 mg of cytochrome c, 0.1 ml of saturated durohydroquinone, 0.25 Mmol of tetramethylphenylene diamine, 0.4 mg of catalase, and 5 mg of cytochrome oxidase. The K+-containing medium was identical except that 65 gmol of potassium phosphate replaced tetramethylammonium phosphate. The measurements with ETPH were done in a medium (7 ml final volume) 0.25 M in sucrose, 1 mM in Tris-HCl (pH 7.4), 50 mM in KCl, and 20 mM in potassium succinate that contained 0.4 mg of catalase, 17.5 mg of ETPH, and 17 gg of oligomycin and rotenone. The experiments with mitochondria were done in a medium (7 ml final volume) 0.25 M in sucrose, 1 mM in Tris-HCl (pH 7.5), and 50 mM in KCl and that contained 21 mg of heavy beef heart mitochondria, 21 Mg of rotenone, 0.4 mg of catalase, 42 nmol of carbonylcyanide-p-trifluoromethoxyphenylhydrazone and 25 Mmol of succinate. In all the experiments, 2 Mmol of H202 was introduced into the cell after anaerobiosis had developed. The two vertical lines for each experiment shown in the figure denote the time of introduction of H202 (left line) and the complete removal of oxygen (right line). The peaks for 100% uptake or release of H+ or K+ have been set at the same height to facilitate comparison of the time relations.
levels of cytochrome c in the routine assay of cytochrome oxidase activity. Just as we have to distinguish between an electrogenic ionophore intrinsic to the mitochondrion or added exogenously, so we have to make a similar distinction in respect to uncoupler. The fact that bile acids added exogeneously provide a source of uncoupler does not rule out the presence of an intrinsic uncoupler in the preparation of cytochrome oxidase. Cyclical transport of cations in cytochrome oxidase Kessler et al. (1) have demonstrated that uncoupled respiration in mitochondria is an expression of coupled cyclical cation transport mediated by an uncoupler and an electrogenic ionophore. It was readily demonstrable by the oxygen pulse technique that the respiration of cytochrome oxidase under the assay conditions described in the legends for Tables 1 and 2 was coupled to cyclical cation transport. The bile acids used in the preparation of cytochrome oxidase can act as uncouplers, and the electrogenic ionophore is provided either by the ITC or by the phospholipid added exogeneously. Fig. 1 shows the spike of K+-dependent proton uptake and release when oxygen was introduced into an anaerobic suspension of cytochrome oxidase. Note that the spike terminates before oxygen was exhausted. This spike is the hallmark of coupled cyclical cation transport. Unless the rates of inflow and outflow of ions are perfectly balanced from the onset of energization, there will be a short period of imbalance during which cations will be taken up and then released (or protons will be released and then taken up). The spike is the visible expression of this imbalance and thus constitutes direct evidence that the respiration involves electron
Table 4. Cation-dependent augmentation af ANS fluorescence by electrogenic ionophores Relative ANS fluorescence in presence of 0.1 mM ionophore Ionophore system Ca2+ K+ ITC (cytochrome oxidase)* 1930 528 10,000-Dalton ionophoroproteint 1360 580 Valinomycin 10 Beauvericin 24 26 12 Phosphatidyl ethanolamine 8 Triton X-100 22 13 * The molecular weight of the ITC is estimated to be 66,000 on the basis of a 3:2 weight ratio between the ITC and ETC of cytochrome oxidase; the molecular weight of the ETC is 100,000. It is assumed in the estimate of molarity that there is one molecule of a Ca2+ ionophore and one molecule of a K+ ionophore for 66,000 daltons of the ITC. t This protein was prepared by extracting beef heart mitochondria with a neutral mixture of chloroform/methanol (2:1) and precipitating the protein several times with ether, removing insoluble protein after each cycle of precipitation and resolubilization. Fluorescence measurements were made in a Perkin-Elmer MPF-3 Fluorescence Spectrophotometer at 200 with an excitation wavelength of 380 nm. The sample (4 ml) was 10 mM in Tris-HCl (pH 7.5), 50MM in ANS, and 0.2 M in the chloride salt of either Ca2+ or K+. The slit width for the control (without ionophore) and the experimental sample (with ionophore) was maintained constant. flow coupled to cyclical cation transport. That this spike is not
due merely to membrane leakiness is attested to by the exact parallelism in time between the introduction of oxygen into the system and the initiation of the spike. Ionophoric capability of the ITC Electrogenic ionophores such as valinomycin, beauvericin, and Triton X-100 induce a large increment in fluorescence in aqueous solutions of aminonaphthalenesulfonic acid (ANS) at neutral pH, but only in the presence of the cations acted upon by these ionophores. The equilibrium for this interaction in aqueous media is highly unfavorable; relatively large concentrations both of electrogenic ionophore and the salts of the cation are required to observe these fluorescence changes (12). Ionophoroproteins such as those of the ITC of cytochrome oxidase by contrast are some two orders of magnitude more efficient than free ionophores on a molar basis with respect to this induction of ANS fluorescence (Table 4). Under the conditions used for this measurement, there would appear to be quantitative interaction of the ionophoroprotein with ANS. We base this conclusion on the fact that increase of the salt concentration from 0.2 M to 2 M does not increase the extent of fluorescence. The fluorescence change in the absence of added salt is less than 10% of the change in the presence of salt. Uncoupler completely eliminates the increment in ANS fluorescence induced by the proteins of the ITC. The possibility that residual phospholipid in the ITC preparation could account for the observed fluorescence change in ANS has been examined. The fluorescence changes induced by phospholipids (neutral or acidic) in amounts equivalent to the estimated phospholipid concentration present in the ITC preparation were determined and shown to be negligible by comparison with the fluorescence changes induced by the proteins of the ITC preparation. When the ionophoroproteins of the ITC of cytochrome oxidase are subjected to tryptic digestion, free ionophore can be
Biochemistry: Kessler et al. Table 5. Induction of mitochondrial swelling by the free ionophore fraction compared to the ionophoropeptide fraction of the ITC Mitochondrial swelling, O.D. units/min per mg protein Test system K+ Ca2+ Ionophoropeptide 335 852 Free ionophore fraction 25,900 67,000 The swelling of beef heart mitochondria was followed spectrophotometrically at room temperature either in a K+-containing medium or in a Ca2+-containing medium. The K+-containing medium was 0.15 M in KNO3, 0.01 M in Tris-nitrate (pH 7.5) and contained 0.5 ,ug of antimycin and rotenone per ml. The Ca2+-containing medium was 0.1 M in calcium acetate, 0.01 M in Tris-acetate (pH 7.5) and contained 0.5 ,g of antimycin and rotenone per ml. The ionophores were introduced in ethanolic solutions.
released. The free ionophore, after isolation by chromatography on alumina, can induce swelling of mitochondria in decimolar solutions of Ca(NO3)2 or KNO3 (Table 5). On a molar basis the free ionophore is some two orders of magnitude more efficient in this induction than the ionophoropeptides obtained by tryptic
degradation of the ionophoroproteins. We describe the isolation and characterization of the ionophores released from the ionophoroproteins of the ITC of cytochrome oxidase oxidase in ref. 13. Extraction of ionophoroproteins The ionophoroproteins of unresolved cytochrome oxidase were extractable into chloroform/methanol (2:1) essentially quantitatively when the mixture was acidified with 2% glacial acetic acid. The protein thus extracted accounts for some 50% of the total protein of cytochrome oxidase. The same proteins were found in the extract obtained by extraction of cytochrome oxidase with acidified chloroform/methanol (2:1) and in the ITC obtained from cytochrome oxidase by resolution either by the cholate/ammonium sulfate or the methanol/HCl procedure. Unresolved cytochrome oxidase prepared by the procedure of Kopaczyk et al. (5) contains six proteins of molecular weights 41,000-43,000, 22,000-25,000 (a doublet), 15,500, 14,000, and 10,000, as determined by electrophoresis in 10% acrylamide gel containing 5% sodium dodecyl sulfate. When cytochrome oxidase was resolved by the method of Korman and Vande Zande (6), the first three protein components were found in the ITC and the last three in the ETC. Thus, the ITC and ETC have two entirely different sets of proteins. Respiratory control in cytochrome oxidase Respiratory control is an inherent property of all coupled particles, and when observed in cytochrome oxidase it reflects the fact that electron flow is limited by the unavailability of a coupling combination. The question is why respiratory control in cytochrome oxidase is demonstrable only under special conditions. The absence of respiratory control in cytochrome oxidase (RC index of 1.0) means that electron flow can be maximally coupled to cyclical cation transport mediated by an electrogenic ionophore and an uncoupler both present and operative in a cytochrome oxidase preparation. We have already demonstrated that unresolved cytochrome oxidase carries out cyclical cation transport; the rate of this transport is not increased by addition of exogeneous uncoupler. This would necessarily imply that unresolved cytochrome oxidase contains all the components required for maximal release of respiration. These components would be an electrogenic ionophore (pro-
Proc. Natl. Acad. Sci. USA 74 (1977)
3665
Table 6. Emergence of respiratory control with incorporation of cytochrome oxidase into liposomes of whole mitochondrial phospholipid
Respiratory Test system
Original cytochrome oxidase suspension Incorporated into liposomes of whole
control index 1.0
mitochondrial P.L.* 7-8 Incorporated into liposomes of neutral mitochondrial P.L.t 1.4 The assay conditions were as described in the legend of Table 2. A low level of cytochrome c (0.002 mM) was used in the assay mixture. The method of Hinkle et al. (16) was followed for the incorporation of cytochrome oxidase into the liposomes. Cytochrome oxidase was prepared by the method of Yonetani (7). P.L., phospholipid. * Chloroform/methanol (2:1) extract of mitochondria (contains the full complement of phospholipids and neutral lipid). Acetone extract (90%) of mitochondria (deficient in cardiolipin and neutral lipids).
vided by the ITC) and an uncoupler (provided by the bile acid) added in the course of isolating cytochrome oxidase. Why then can cytochrome oxidase show essentially complete respiratory control when incorporated into phospholipid liposome? The important point is that this effect of incorporating cytochrome oxidase into liposomes is not general for all phospholipids (14). It is only a particular blend of phospholipids that imposes respiratory control (Table 6). For example, whole mitochondrial lipid was highly effective in inducing respiratory control whereas mitochondrial lipid extracted with a solvent mixture that leaves behind cardiolipin and neutral lipids was relatively ineffective. The fact that respiratory control, when imposed, was released by addition of valinomycin + nigericin (an ionophore combination that mediates cyclical cation transport) provides the clearest evidence that the ITC intrinsic to cytochrome oxidase is inoperative under conditions in which respiratory control is imposed and operative under conditions in which respiratory control is not imposed. Why should the blend of phospholipids in the liposomes determine whether respiratory control is imposed or not? We have established a correlation between the geometric character of the liposomes, the nature of the phospholipid, and the extent of respiratory control. The phospholipids that impose respiratory control induced formation of relatively small liposomal vesicles (possibly tubular membranes), whereas the phospholipids that do not impose respiratory control induced formation of relatively large liposomal vesicles (Fig. 2). There appears to be a close correlation between the degree of curvature of the liposomal vesicles and the emergence of respiratory control. The ITC in cytochrome oxidase apparently is inoperative when the liposome in which it is incorporated has a high degree of curvature and operative when the curvature is less extreme (large vesicles). It would appear that the phospholipid blend determines the geometry of the liposomal vesicles and that the geometry goes parallel with the polarity of the membrane. On prolonged standing, the liposomal particles that showed initially high respiratory control with incorporated cytochrome oxidase progressively lose this control, and this loss goes parallel with the conversion of small vesicles to increasingly larger vesicles. Thus, even with the same phospholipid blend, the liposomal vesicles can undergo progressive changes in geometry and corresponding changes in the degree of respiratory control. Another feature of the liposome methodology developed by E. Racker and his colleagues (15-17) is that respiratory control in liposomes in which cyto-
Biochemistry: Kessler et al.
3666
Proc. Natl. Acad. Sci. USA 74 (1977)
A
B
FIG. 2.
Electron
micrographs of liposomal vesicles
in which cy-
tochrome oxidase has been incorporated. For details of the preparation of the was
phospholipids, see legend
of
TableS5. Cytochrome oxidase
incorp'orated into the vesicles by the method of Hinkle et
a!.
(16).
The
specimens used for electron microscopy were negatively stained with 2% ammonium molybdate (X100,000.) (A) Vesicles with respiratory control obtained by incorporation of cytochrome oxidase into of whole mitochondrial phospholipid. (B) Vesicles without respiratory control obtained by incorporation of cytochrome oxidase into liposomes containing only the neutral phospholipids of mito-
liposomes
chondria chrome oxidase has been
ejection
during
incorporated goes parallel with proton
energization,
control in liposomes in
whereas the lack of
which
respiratory
cytochrome oxidase
has been
has been extensively examined by Racker and his colleagues (14-17) in a series of experimental studies. They rationalize this emergence in terms of a correlation between random orientations of the units of cytochrome oxidase in the membrane, leading to loss of respiratory control, and uniform orientation of the units, leading to the emergence of respiratory control. Thus, the lack of respiratory control in the standard preparations of cytochrome oxidase is attributed to random orientation, and the emergence of respiratory control when such preparations are incorporated into suitable liposomal vesicles is attributed to uniform orientation. This assumption is untenable. Our oxygen pulse studies (see Fig. 1) show that cyclical cation transport (uncoupled respiration) can be observed, regardless of whether the orientation leads to proton uptake (cation release) or proton release (cation uptake). In other words, cyclical cation transport can proceed in either direction, leading to proton release as in mitochondria (in the presence of added uncoupler) or to proton uptake as in ETP or cytochrome oxidase (in the presence of intrinsic uncoupler). We can induce respiratory control in cytochrome oxidase merely by resolving the ETC. Then addition of lipid c in combination with the bile acids used in the resolution can release respiratory control. No reorientation of the electron transfer units in the membrane has to be invoked in such a case. This investigation was supported in part by Program Project Grant GM 12847 of the National Institute of General Medical Sciences.
incorporated goes parallel with proton uptake during energization. This means that the orientation of cytochrome oxidase in the minivesicles is the opposite of that in the large yesicles.
Discussion
The studies described in the present communication permit the following conclusions:
(i) cytochrome oxidase
is a duplex of
an
when cytochrome oxidase is stripped of its complement of ITC, it loses the capacity for electron flow; ETC
and
an
ITC; (ii)
(Mi) when the activity of the ITC is suppressed, the electron transport activity of cytochrome oxidase is depressed to comparable degree; (iv) the ITC contains complement of
a
a
ionophores transport;
sufficient
and
for the
mediation
of
cyclical
cation
(v) cyclical cation transport is directly demon-
cytochrome oxidase by the oxygen pulse technique. interrelationships and correlations we can further in cytochrome oxidase depends upon
strable in From these conclude that coupling
interaction between moving electron in an ETC positively charged ionophoric species in ITC. In earlier studies from this laboratory Kopaczyk et al. (5) demonstrated that the four complexes of the electron transfer chain, as well as the tripartite repeating unit, could be resolved into what these authors referred to as a catalytic protein fraction and a structural protein fraction. It is now apparent that the catalytic protein fraction corresponds to the ETC and the structural protein fraction to the ITC. Once these identities were recognized it was possible to draw upon the extensive studies of structural protein and deduce from these studies the duplex nature of each of the four complexes of the electron transfer chain as well as of the tripartite repeating unit, and the invariant linkage on the one hand between ETCs and ITCs and on the other hand between the F1 system and its associated ITC. The emergence of respiratory control in cytochrome oxidase
electrostatic and a
a
an
1. Kessler, R. J., Vande Zande, H., Tyson, C. A., Blondin, G. A., Fairfield, J., Glasser, P. & Green, D. E. (1977) Proc. Natl. Acad.
Sci. USA 74,2241-2245. 2. Hunter, D. R., Haworth, R. A. & Southard, J. H. (1976) J. Biol. Chem. 251, 5069-5077. 3. Haworth, R. A. (1977) Biophysical Society Annual Meeting, New Orleans, LA, February, 1977, Abstract. 4. Fowler, L. R., Richardson, S. H. & Hatefi, Y. (1962) Biochim. Biophys. Acta 64, 170-173. 5. Kopaczyk, K., Perdue, J. & Green, D. E. (1966) Arch. Biochem.
Biophys. 115,215-225. 6. Korman, E. F. & Vande Zande, H. (1968) Fed. Proc. 27, Abstract 1737. 7. Yonetani, T. (1967) in Methods in Enzymology, eds. Estabrook, R. W. & Pullman, M. E. (Academic Press, New York), Vol. 10, pp. 332-5. 8. Drabkin, D. L. (1942) J. Biol. Chem. 146,605-617. 9. Widmer, C. & Crane, F. L. (1958) Biochim. Biophys. Acta 27, 203-204. 10. Green, D. E. & Fleischer, S. (1963) Biochim. Biophys. Acta 70, 554-582. 11. Tyson, C. A., Vande Zande, H. & Green, D. E. (1976) J. Biol.
Chem. 251, 1326-1331. 12. Feinstein, M. B. & Felsenfeld, H. (1971) Proc. Natl. Acad. Sci. USA 68, 2037-2041. 13. Blondin, G. A., Kessler, R. J. & Green, D. E. (1977) Proc. Nat!. Acad. Sci. USA 74,3667-3671. 14. Eytan, G. D., Matheson, M. J. & Racker, E. (1976) J. Biol. Chem.
261,6831-6837.
15. Racker, E. (1972) in Molecular Basis of Electron Transport, eds. Schultz, J. & Cameron, B. F. (Academic Press, New York and London), p. 45. 16. Hinkle, P. C., Kim, J. J. & Racker, E. (1972) J. Biol. Chem. 247, 1338-1339. 17. Kagawa, Y., Johnson, L. W. & Racker, E. J. (1973) Biochem. Biophys. Res. Commun. 50,245-251.
