ARCHIVES
OF
BIOCHEMISTRY
Mitochondrial
AND
BIOPHYSICS
177,
Cytochrome Components
MARIA
ERECINSKA,
Department
of Biochemistry
133-143
(19’76)
b-c, Complex: Its Oxidation-Reduction and Their Stoichiometryl DAVID
F. WILSON,
and Biophysics, Philadelphia, Received
University Pennsylvania April
AND
YURIKO
of Pennsylvania
MIYATA Medical
School,
19, 1976
A cytochrome bx, complex was isolated from pigeon breast muscle mitochondria and purified to a content of 3 nmol of cytochrome c, per milligram of protein. Anaerobic suspensions of the preparation were titrated with reducing equivalents (NADH) and oxidizing equivalents (ferricyanide). The oxidation-reduction components of the complex were measured by the number of reducing equivalents accepted or donated per cytochrome cI and compared with the stoichiometries of the known redox components as measured by independent methods. The preparation accepts or donates 5.2 i 0.3 equivalents per cytochrome c I, while the measured content of cytochrome cl, cytochrome b,,, , cytochrome b,,,, Rieske iron-sulfur protein, ubiquinone, and succinate dehydrogenase accounts for 5.0 i 0.2 equivalents per cytochrome c,. It is concluded that there are no unknown redox components in the cytochrome bx, complex. The cytochrome bx, complex (energy transduction site 2) appears to be a structural unit containing equal amounts of cytochrome c,, cytochrome b,,,, cytochrome b,,,, and the Rieske iron-sulfur protein.
One of the major objectives in the study of mitochondrial oxidative phosphorylation is identification of the redox components of the respiratory chain, in particular those associated with each of the three phosphorylation sites and the description of their thermodynamic properties. Transduction of energy at site 3 occurs in cytochrome c oxidase and has been shown to involve four one-electron (n. = 1) redox components (1) while the reactions at site 2 (b-c, region of the respiratory chain) and site 1 (NADH dehydrogenase) are, on the other hand, less well characterized. Rieske and co-workers (2-5) established that after isolation, the cytochrome b-c, region of the respiratory chain (complex III) contains cytochromes b and c, (in a 2:l molar ratio), ncnheme iron, ubiquinone, and the antimycin A binding site. The results from titrations with ascorbate, however, raised the possibility that the complex contained GM
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by U.S.
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yet another electron acceptor in amounts stoichiometric with cytochrome c1 (6, 7). Similar suggestions were made by OrmeJohnson et al. (8) on the basis of titrations of isolated ubiquinone-cytochrome c reductase with dithionite and NADH and by several other authors (9-11) on the basis of indirect evidence. Cytochrome b-c, complex prepared using a Triton X-lOO-deoxycholate method (12, 13) retains full durohydroquinone cytochrome c reductase activity and the unique spectral properties of the two b cytochromes (b,,, and b,,,). This preparation contains high concentrations of the cytochromes and of the Rieske iron-sulfur protein, but is deficient in ubiquinone and in succinate dehydrogenase. In the present paper we will present evidence that the purified cytochrome b-c, complex contains only cytochrome bsG5, cytochrome bj6,, cytochrome c 1, and the Rieske iron-sulfur protein in stoichiometric amounts. Thus site 2, like site 3, is an organized complex
Grant 133
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
134
ERECINSKA,
containing four one-electron nents in equal amounts. MATERIALS
AND
WILSON
redox compo-
METHODS
The cytochrome b-c, complex was isolated from pigeon breast muscle mitochondria by the procedure published previously for the purification of a succinate-cytochrome c reductase (12, 13). During the repeated fractionation with ammonium sulfate (in the presence of Triton X-100 and deoxycholate) employed for the isolation, a continuing loss of succinate dehydrogenase was observed. In the preparation utilized in this study the total succinate dehydrogenase (active and inactive) was less than 10% of the cytochrome c, content. Therefore, the cytochrome bx, complex appears to be a more appropriate name for this preparation. Analytical procedures. Protein was determined by the biuret method (14) using crystalline bovine serum albumin as a standard. Cytochrome b and c, contents were determined after conversion of their heme moieties into the respective pyridine hemochromogens as described by Basford et al. (15). The millimolar extinction coefficients used for calculations were 34.1 (at 557 nm) for pyridine hemochromogen b and 19.1 (552 red-540 ox) for pyridine hemochromogen c Nonheme iron was determined by the orthophenanthroline method according to Brumby and Massey (161 using iron nitrate prepared from iron wire as a standard. Acid nonextractable flavin was assayed fluorometrically according to the method of Wilson and King (17). Coenzyme Q was estimated by the procedure of Kroger and Klingenberg (18). Spectral studies and titrations. Spectral measurements were carried out using a Johnson Foundation scanning dual wavelength spectrophotometer. This instrument is provided with a digital wavelength drive on one of the two monochromators while the other is being held constant at a reference wavelength. The absorption spectrum of either the fully oxidized or fully reduced sample (or at any intermediate level of reduction) is measured and stored in the memory of a digital computer. The stored spectrum is then subtracted from any other subsequently measured and the trace which is recorded represents the difference between the two. The operation of this system is described in detail in (12, 19). Titrations were carried out in an anaerobic cuvette designed by Dutton (201 equipped with two micropipette titrators, one containing a standard solution of lo-20 mM ferricyanide, the other containing a standard solution of 5-12 mM NADH. Rapid interaction between NADH and the components of the cytochrome b-c, complex was ensured by the presence of diaphorase (20 units A/ml) and 80 FM flavin mononucleotide. At the end of each titration the concentra-
AND
MIYATA
tions of the standard solutions were estimated spectrophotometrically. The ferricyanide was measured at 420 nm using a millimolar extinction coefficient of 1.0 and the NADH was measured spectrophotometrically at 340 nm in the presence of pyruvate before and after the addition of lactate dehydrogenase (A emM = 6.23). The titrations were carried out either by measuring the absorbance changes at the wavelength pair 564-552 nm (Fig. 1) or by scanning the entire spectral region between 500 and 630 nm (Figs. 3-5). In each case, sufficient time was required to allow for the equilibration between the reductant (or the oxidant) and the respiratory chain components. Materials. Sodium deoxycholate, Triton X-100, NADH, flavin mononucleotide, and diaphorase (type II from Cl. kluyueri) were obtained from Sigma Chemical Company (St. Louis, MO.). RESULTS
Stoichiometry of the Electron Carriers in the Cytochrome b-c, Complex from Pigeon Breast Mitochondria Isolated Using Triton X-100 Deoxycholate Procedure The composition of three different preparations of the cytochrome b-c, complex isolated using a combination of nonionic (Triton X-100) and ionic (deoxycholate) detergents is shown in Table I. The preparations contain heme c, heme b, nonheme
FIG. 1. Spectrophotometric recording of the titration of an anaerobic reduced preparation of the cytochrome b-c, complex with ferricyanide. The cytochrome bx, complex was suspended at 15.4 /AM heme c in 50 mM phosphate buffer, pH 7.2 and preequilibrated with ultrapure argon gas to remove most of the dissolved oxygen. Sufficient dithionite solution was added to reduce the complex and remove the remaining oxygen. The reoxidation was carried out by stepwise addition of ferricyanide (the concentrations are given in the figure). Any excess of dithionite was destroyed by the initial addition of ferricyanide. Thereafter, at least three oxidative (with ferricyanide) and reductive (with NADH; Fig. 5) titrations were carried out on each preparation. Thus there is no dithionite remaining in the incubation mixture at any given time during the titrations. The measuring wavelength were 564-552 nm.
MITOCHONDRIAL
CYTOCHROME
iron, ubiquinone, and small amounts of acid-nonextractable flavin. Acid-extractable flavin was very low (less than 10% of the acid-nonextractable flavin) and phos-
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FIG. 2. A plot of the absorbance changes during oxidative [ferricyanide(O)] and reductive LNADH (a)] titration of an anaerobic preparation of cytochrome b-c, complex against the amount of oxidant or reductant added. The oxidative titration is that shown in Fig. 1. Diaphorase and 80 /.LM flavin mononucleotide were added to facilitate the equilibration with NADH. The standard NADH solution used for titration was 11.6 mM. The absorbance measurements are the differences between the absorbance at 564 nm and that at 552 nm.
520
540
bx:,
135
COMPLEX
pholipids were not measured because the preparation was suspended in an 0.05% lysolecithin-0.05% lecithin mixture. Heme c was present in a concentration of approximately 3 Fmol/g of protein, while heme b was at a level of between 5 and 6 pmol/g of protein, the ratio between the two being 1:2. These concentrations and stoichiome: try are essentially the same as those found in the highly purified preparations of complex III described by Rieske et al. (6) and Hateti et aZ. (21). The concentration of acid-nonextractable flavin (which can be taken as a measure of succinic dehydrogenase content) is about 10% of the heme c concentration. Nonheme iron is present in amounts slightly higher than the heme b content, i.e., twice the concentration of heme c. It consists of the iron-sulfur centers of complex III itself (Rieske’s ironsulfur center) plus the contribution from succinic dehydrogenase iron-sulfur protein. The latter must be less than 0.8 iron/ cytochrome c, even if all of the flavin is associated with fully active succinate dehydrogenase [eight irons per flavin (2211 as the total concentration of succinic dehy-
560 X hm)
580
600
620
FIG. 3. Difference spectra obtained for the cytochromes b of cytochrome b-c, complex during anaerobic titration with ferricyanide. The cytochrome b-c, complex was suspended at 14.8 pM heme c concentration under the conditions described in the legend to Fig. 1. The spectrum of the fully reduced anaerobic sample was measured and stored in the memory of a digital computer. The flat baseline is the reduced minus reduced difference spectrum. Spectra 1 through 11 are the difference spectra obtained after stepwise additions of ferricyanide minus the reduced spectrum stored in the computer memory. The reference wavelength was at 540 nm.
136
ERECINSKA,
WILSON
AND
MIYATA
I
I
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X (nml FIG. 4. Difference spectra obtained for cytochrome cI of the cytochrome b-c, complex during anaerobic titration with ferricyanide. Conditions are those of Fig. 3. The titration is the continuation of the one presented in Fig. 3. Spectrum 11 of Fig. 3 was stored in the computer memory; thus, the baseline of Fig. 4 is for the preparation with the b cytochromes oxidized and cytochrome c, reduced. Spectra 1 through 7 are difference spectra obtained after each addition of ferricyanide. The reference wavelength was at 540 nm.
5io
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FIG. 5. Dual wavelength difference spectra of the cytochromes of the b-c, complex obtained during anaerobic titration with NADH. Conditions are those of Figs. 1 and 2 and the technique is described in the legends to Figs. 3 and 4. The baseline is obtained with all of the cytochromes oxidized. Spectra 1-14 are difference spectra (NADH reduced-oxidized) obtained after stepwise addition of NADH.
drogenase is only 10% of that of cytochrome c,. Subtraction of the approximate value of the contribution of succinic dehydrogenase iron-sulfur centers from the overall content of the nonheme iron provides a figure equal to nearly twice the
heme c concentration. This is consistent with the presence of two irons per mole of the protein in the Rieske iron-sulfur center; this value is in exceIlent agreement with the results of Rieske et al. (6, 7). Since succinate dehydrogenase accepts 4
MITOCHONDRIAL
CYTOCHROME TABLE
COMPOSITION
b-c,
OF CYTOCHROME
Component
Preparation rnM
-~ Heme Cytochrome cI Cytochrome b Nonheme iron Ubiquinone Flavin (acid nonextractable) Protein (mg/ml) -.
COMPLEX
PREPARED
1
b-c, I BY TRITON
0.12 0.226 0.211 0.0485 0.0124 42.6
rnM
~mollg
2.82 5.31 4.95 1.14 0.29 -
equivalents per flavin (23) the total reducing equivalents accepted will be approximately 0.4 per cytochrome c,. All three preparations contain ubiquinone in amounts equal to one-third of the cytochrome c1 concentration. (This value should be compared with a figure of 15 for the same ratio found in pigeon breast mitochondria.) Since ubiquinone is a two electron donor/acceptor, on an electron basis this provides a not-insignificant figure of about 0.6 per cytochrome c,. Titrations of the Cytochrome b-c1 Complex with Ferricyanide and NADH as Followed by Measurement of the Absorbance at 552 nm Minus That at 564 nm Titrations of the cytochrome b-c, complex with ferricyanide and NADH carried out anaerobically, as described in the Methods section, are shown in Figs. 1-5. In Fig. 1, the dual wavelength spectrophotometer was set to measure the difference in absorbance between 564 and 552 nm and the titration of the reduced sample was performed through stepwise addition of small amounts of ferricyanide. After each addition of ferricyanide a change in absorbance is observed due to oxidation of cytochromes b (decrease in absorbance at 564 nm relative to 552 nm). After complete oxidation of cytochromes b the spectrophotometric trace flattens and reverses due to subsequent oxidation of cytochrome c, , revealed as the decrease in absorbance at 552 nm relative to 564 nm. The two “arms” of the trace are unequal because the absorbance change at 564 nm which accompanies oxidation of cytochromes b is greater than the absorbance change at 552 nm due to
X-100
Preparation
protein
~__
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137
COMPLEX
DEOXYCHOLATE
2 cLmol/g protein
rnM
3.08 5.38 5.73 1.06 0.23 -
0.11 0.205 0.248 0.041 0.0124 42.2
0.148 0.258 0.275 0.051 0.011 48.0
PROCEDURE
Preparation
3 pmolig
protein 2.60 4.85 5.88 0.97 0.29 -
oxidation of cytochrome c, . The equilibration is rapid as evidenced by sharp leveling of the traces after each addition of the oxidant. When oxidation of cytochrome c, is complete, the anaerobic preparation of the oxidized sample is titrated with NADH in the same stepwise manner. The trace obtained is an almost “mirror image” of that presented in Fig. 1. Equilibration with NADH is also rapid; the absorbance changes following NADH addition are complete within 1 min. The plot of the two titrations (absorbance against the number of the reducing or oxidizing equivalents used) is presented in Fig. 2. The inequality of the two sides with respect to the total absorbance change is discussed above. An interesting observation is, however, that the total number of the reducing or oxidizing equivalents used as cytochrome c, is titrated is only slightly smaller than that consumed when cytochromes b are titrated. Moreover, there are practically no equivalents used up between the end of cytochrome c, titration and the onset of cytochromes b titration. This indicates the absence of electron donors/acceptors in the oxidation-reduction potential gap between cytochrome c, and the cytochromes b in concentrations set by the limits of detectability of the present approach (see Discussion and below). Titrations of the Cytochrome b-c, Complex by Ferricyanide and NADH as Followed Measuring the Absorption Spectra from 500 to 630 nm The titrations dual wavelength
also can be followed in the scanning spectrophotom-
138
ERECIr;JSKA,
WILSON
eter. The oxidative titration with ferricyanide is shown in Figs. 3 (cytochromes b) and 4 (cytochrome c,). The spectrum of the fully reduced anaerobic sample is recorded as the reference spectrum. Stepwise addition of ferricyanide results in oxidation of the components of the cytochrome b-c, complex. The first component oxidized is cytochrome bsGs, as evidenced by the appearance of the characteristic “double peak” absorbance change with a maximum at 565 nm and a shoulder at 558 nm (12). The oxidation of cytochrome bjG5 is followed by that of cytochrome bj6,. As the oxidation of cytochromes b is completed, the oxidation of cytochrome c, begins as indicated by the disappearance of the absorbance at the short wavelength side of the cytochromes b absorbance minima. At this point in the experiment (cytochromes b oxidized, cytochrome c1 mostly reduced) a new baseline was recorded (Fig. 4) and the oxidative titration continued until 100% oxidation of cytochrome c, was attained. It should be stressed here that the preparation of succinate-cytochrome c reductase used in the present work was essentially 100% reducible by the NADH, i.e., there was no additional reduction upon addition of dithionite. There was no measurable CO-sensitive component present in the preparation. The titration of the b-c, complex with NADH is shown in Fig. 5. The spectrum of the fully oxidized anaerobic preparation was taken as the reference and the spectrum was recorded after each addition of NADH and a sufficient amount of time to attain the corresponding level of reduction. At least two such oxidative and reductive titrations were carried out for each of the three preparations of the cytochrome b-c, complex. An example of the plot of the data is shown in Fig. 6. The titrations with NADH constitute the ascending arms of the curves while the titrations with ferricyanide constitute the descending ones. Different symbols refer to the three different wavelengths for which the calculations were made: 554,563, and 568 nm. It can be calculated from the known concentration of cytochrome c, (heme c) and the amount of the reducing (or oxidizing) equivalents
AND
MIYATA
consumed that there are approximately two electrons accepted and donated per cytochrome c 1 when cytochrome c, is titrated and approximately three electrons when the cytochromes b are titrated. It has been shown previously (13) that the cytochrome c1 midpoint potential (Em,,, = 0.280 -C 0.01 V) is very close to that of Rieske’s nonheme iron protein [Em7,2 = 0.275 k 0.02 V (2411, both being one electron donorslacceptors. Reduction of the b cytochromes is accompanied by reduction of ubiquinone with the half-reduction potential of 0.060 ? 0.02 V (a two-electron donor/acceptor) and the components of succinic dehydrogenase which “contaminates” the preparation of the cytochrome b-c, complex (0.4 equivalents per cytochrome c,). Since a quantitative estimate of all these components was made for each of the three reductase preparations we can make a precise comparison between the number of electrons consumed or accepted in the experimental situation and that calculated theoretically. A summary of all of the experimental data and their comparison with the information obtained by computing the values determined by analytical procedures is given in Table II. The calculations were performed on the basis of the heme c concentration in various preparations. It is seen that there are approximately 5.2 elec60
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FIG. 6. A plot of an oxidative (ferricyanide) and reductive (NADH) titration of the cytochromes of bc, complex. The experimental data were obtained from titrations similar to those shown in Fig. 5. cm), absorbance changes at 554 nm; (01, absorbance changes at 560 nm; (A), absorbance changes at 566 nm. The heme c concentration was 16.9 pM.
MITOCHONDRIAL
CYTOCHROME TABLE
ELECTRON
ACCEPTORS
Preparation number
Ieme ( (@f)
OF b-c,
COMPLEX
NADH
OF THE AND
Number
Ferricyanide
139
COMPLEX
II
RESPIRATORY FERRICYANIDE
Ieme e-k
I
b-c,
CHAIN
AND
of electron 1LIeme
C
b
0.12
0.226
I.148
0.258
0.11
0.205
THEIR
TITRATIONS
equivalents
NHI”
(rnM) SDH’
WITH
NADH
in preparation Total
e-k
t
14.4 16.8
0.0486
5.07
Average 2
17.8 17.8
48.7 46.5
14.8 14.8
35.0 32.5
0.687
4.64
Average 3
28.0 37.5
Average Average fo three prepa rations
67.5 75.0
,
i6.1
14.9
0.082
0.581
5.5 5.25 2.61
0 Heme c + heme b/3 b A two electron donor/acceptor. c Flavin (2-1 + NH1 (2~).
trons taken up or given away per heme c, as determined experimentally, while the number obtained from the addition of the concentrations of all the known components present in this preparation is approximately 5.0 per heme c. Thus the total unknown electron acceptor(s) in the preparation of the cytochrome b-c, complex must be present at a total concentration of less than 0.5 per cytochrome c, for oneelectron acceptor(s) or less than 0.25 per cytochrome c1 for two-electron acceptor(s). Titrations of “Aged” Preparations Cytochrome b-c, Complex
of the
The cytochrome b-c, complex is stable for several hours at room temperature, as judged by the spectral properties of cytochromes bsG5 and b,,,. The titrations of such a preparation (Fig. 7) with NADH and ferricyanide show, however, that fewer equivalents are required in the region where cytochrome c, is oxidized or reduced than in parallel titrations carried out on “fresh” preparations. Analysis of the data indicates that this decrease is only apparent and arises from the fact that
in the aged preparation the E, value of the spectrally invisible Rieske iron-sulfur protein is approximately 40 mV more positive than that of cytochrome c,, while in fresh preparations the E, values for the two are equal within k-20 mV. As the E, value of the iron-sulfur protein becomes more positive relative to cytochrome c,, the cytochrome c1 titration curve becomes asymmetric. In the initial phase of ferricyanide titrations only cytochrome c1 is oxidized and therefore only one equivalent per cytochrome c, is given to the ferricyanide. At more than 50% oxidation of cytochrome c 1, the slope decreases markedly and approaches the value of two because the iron-sulfur protein provides an increasing fraction of the reducing equivalents for the ferricyanide reduction. DISCUSSION
Chemical analysis of the cytochrome be, complex isolated from pigeon breast mitochondria shows that the preparation contains four one-electron carriers present in equimolar concentrations: cytochrome c,, cytochrome b,,, , cytochrome bje5, and
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ERECINSKA,
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FIG. 7. A plot of an oxidative (ferricyanide) and reductive (NADH) titration of the cytochromes of an aged preparation of cytochrome b-c, complex. The preparation was aged by diluting to 14.8 pM cytochrome c, and maintaining it for 4 h at 25°C. The titrations were carried out as described in the legends to Figs. 1 and 2 and difference spectra similar to those presented in Fig. 5 were recorded. (D), absorbance changes at 554 nm; (O), absorbance changes at 560 nm; (A), absorbance changes at 566 nm.
Rieske’s iron-sulfur protein. In addition, ubiquinone and succinic dehydrogenase are found in concentrations 0.3 and 0.1 that of cytochrome c,, respectively. Anaerobic titrations of this preparation with a reductant (NADH) and an oxidant (ferricyanide) demonstrate that the order of reduction or oxidation of the components is consistent with the known values of their half-reduction potentials. In the potential span where cytochrome c1 is titrated (i.e., between an E, value of -0.32 and -0.2 V) two reducing equivalents are taken up or given away, which indicates that Rieske’s iron-sulfur protein with a half-reduction potential very close to that of cytochrome c, undergoes parallel oxidation and reduction such that at each point it is reduced to the same extent as cytochrome c,. In the region where cytochromes b are oxidized and reduced (the potential region between an Eh of 0.160 and -0.08 V) there are approximately three oxidizing or reducing equivalents involved, two being accounted for by the two b cytochromes present in equal concentrations while the third is shared between ubiquinone (0.6 equivalents since two equivalents are required per ubiquinone) and succinic dehydrogenase flavin and iron sulfur centers (0.4 equivalents). Succinic dehydrogenase is a “contaminant” of the b-c, complex while
ubiquinone may or may not be an intrinsic part of the complex. Its presence in substoichiometric quantities in preparations which retain full durohydroquinone cytochrome c reductase activity leads us to conclude that it is highly unlikely that ubiquinone is an obligatory redox component at site 2 (see, however, 25). It should be further pointed out that because ubiquinone donates (and accepts) two electrons and is reduced at intermediate levels of reduction of the b cytochromes, its effect on their titration curves is readily observed, which permits the conclusion that its Em7.2 value is similar to that observed in intact mitochondria [Em7.2 of ubiquinone = 0.045 + 0.02 V (26)]. It has been suggested repeatedly during the last 10 years that site 2 contains yet other components in addition to those discussed above. Two of them, component X of Rieske (9) and Y of Eisenbach and Gutman (10) were characterized with respect to the values of their half-reduction potentials. The numerical values given were 0.15 V for component X and 0.25 V for component Y. A precise analysis of the titration curves reported in this work could confirm or exclude the existence of such additional components. Two points are relevant: First, the titration of cytochrome c, requires 2.0 ? 0.1 equivalents/
MITOCHONDRIAL
CYTOCHROME
cytochrome c1 and the slope of the titration curve is a straight line. This means that except for cytochrome c, and the Rieske iron-sulfur protein there are no other redox components present with E, values 20.06 V of that of cytochrome c, in concentrations greater than 0.1 that of cytochrome c,. Second the possible existence of redox components with E, values between 0.2 and 0.06 V can be determined from careful comparison of the experimental titration curves and those calculated for the known components (Fig. 8). The three curves shown in Fig. 8 represent the calculated plots of absorbance changes at 552 nm (extreme left) and at 564 nm (the middle and the right curve) against the number of electrons accepted or donated per unit concentration (cytochrome c, content in this case). The circle symbols refer to the curves constructed by taking into account the known components of cytochrome b-c, complex [cytochrome c, (E,,, = 0.25 V), Rieske iron-sulfur protein (E, = 0.25 V), cytochrome b,,, (E, = 0.09 V), ubiquinone (E, = 0.06 V), cytochrome b,,, (E, = 0.00 V), and the succinate dehydrogenase flavin (E, = -0.04 V)] in their experimentally measured stoichiometries of 1:1:1:0.3:1:0.1. The triangles describe the curves which, in addition to the information given above, assume the presence of a hypothetical component with a midpoint potential of 0.15 V and an n value of 1 in a stoichiometry equal to that of cytochrome c,. The plots allow very precise measurements of the number of equivalents required to go from 75% reduction of cytochrome c, to 75% reduction of cytochromes b. The value is 2.5 equivalents per cytochrome c, in the absence of the hypothetical component (the difference between the points B and A) and 3.5 in the presence of the additional component (the difference between points C and A). Moreover, the calculated values must be increased to 2.7 and 3.7 equivalents per cytochrome c, to account for the iron-sulfur centers of succinate dehydrogenase which were not considered in calculating the theoretical curves. Analysis of the experimental titration curves gives 2.63 * 0.51 equivalents per cytochrome c, for titrations with
b-c,
141
COMPLEX
lb
2’0
3:o
4:o
50
6’0
e-/cyt c, FIG. 8. Theoretical curves of the absorbance changes at 552 nm (left-hand curve) and 564 nm (middle and right-hand curves) against the number of oxidizing or reducing equivalents. The circles refer to the curves constructed by taking into account the known components of the cytochrome b-c, complex: cytochrome c,, Rieske’s iron-sulfur center, cytochrome bz6,, ubiquinone, cytochrome b,,,, and the succinate dehydrogenase flavin in their experimentally measured stoichiometries of 1:1:1:0.3:1:0.1. The triangles refer to the curves which, in addition, assume the presence of a hypothetical component with a midpoint potential of 0.15 V and an n value of 1 in a stoichiometry equal to that of cytochrome cf. The arrows at points A, B, and C indicate 75% reduction of cytochrome c, (A) or cytochromes b (B and C) under both sets of conditions. The two b cytochromes are assumed to contribute equally to the absorbance change at 564 nm.
NADH and 2.61 -t 0.27 equivalents for titrations with ferricyanide. The number of reducing equivalents accepted or donated by the cytochrome b-c, complex determined experimentally is equal to the value expected from the stoichiometry of the known redox components. This is true for all potential regions of the titrations and thus the data are incompatible with the existence of any unknown redox components at site 2 (i.e., with E, values between 0.32 and -0.1 V). It also follows from the results presented above that in functionally fully competent cytochrome b-c 1 complex, the iron-sulfur center 5 (27) is present in quantities less than 10% that of cytochrome c, and cannot be considered as an obligatory member of the redox components at site 2. This is consistent with its suggested role in a flavoprotein responsible for reoxidation of the electron transferring flavoprotein of the fatty acyl coenzyme A dehydrogenating system (28). In addition to the components discussed
142
ERECINSKA,
WILSON
above the mitochondrial respiratory chain has been periodically reported to contain more than two cytochrome b species (see, for example, 29). The results presented in this paper show that the sum of all of the b cytochromes present in the b-c, region of the respiratory chain is twice the cytochrome c1 concentration. Moreover, they accept and donate reducing equivalents in a manner consistent with the presence of only “two species in a 1:l molar ratio. In summary, the results presented in this paper indicate that energy transduction site 2, like site 3, consists of four oneelectron transfer components: two b cytochromes, cytochrome c, , and Rieske’s ironsulfur center. Two of the components are on the low oxidation-reduction potential side of the site (the two b cytochromes) and two are on the high potential side (Rieske’s iron-sulfur center and cytochrome c,) and the transfer of electrons between the two is coupled to the synthesis of ATP. Moreover, it has been demonstrated that the chemical properties of two redox components at each site [cytochromes b,,, and c1 at site 2 (30-32) and cytochromes a and a3 at site 3 (33-35)l are dependent on the phosphorylation state of the mitochondria which has led to the proposal (36, 37) that these particular redox components are directly involved in the energy transduction process. Finally, the similarities in composition of the energy transduction sites 2 and 3 and in the reactivities of their components support the concept that the general principles which underlie the basic mechanism of energy transduction are the same at all three phosphorylation sites and apply equally to site 1. REFERENCES 1. VAN GELDER, B. F., AND BEINERT, H. (1969) Biochim. Biophys. Acta 189, 1-24. 2. RIESKE, J. S., BALJM, H., STONER, C. D., AND LIPTON, S. H. (1967) J. Biol. Chem. 242, 48544866. 3. SILMAN, H. I., RIESKE, J. S., LIPTON, S. H., AND BAUM, H. (1967) J. Biol. Chem. 242, 48664875. 4. BAUM, H., SILMAN, H. I., RIESKE, J. S., AND LIPTON, S. H. (1967) J. Biol. Chem. 242, 48764887. 5. RIESKE, J. S., LIPTON, S. H., BAUM, H., AND SILMAN, H. I. (1967) J. Biol. Chem. 242, 48884896.
AND
MIYATA
6. RIESKE, J. S., HANSEN, R. E., AND ZAUGG, W. S. (1964) J. Biol. Chem. 239, 3017-3022. 7. RIESKE, J. S., LAUGG, W. S., AND HANSEN, R. E. (1964) J. Biol. Chem. 239, 3023-3030. 8. ORME-JOHNSON, N. R., HANSEN, R. E., AND BEINERT, H. (1974) J. Biol. Chem. 249, 19281939. 9. RIESKE, J. S. (1971) Arch. Biochem. Biophys. 145, 179-193. 10. EISENBACH, M., AND GUTMAN, M. (1974) FEBS Lett. 46, 368-371; (1975) Eur. J. Biochem. 52, 107-116; 59, 223-230. 11. WIKSTR~M, M. K. F., AND BERDEN, J. A. (1972) Biochim. Biophys. Actu 283, 403-420. 12. ERECIASKA, M., OSHINO, R., OSHINO, N., AND CHANCE, B. (1973) Arch. Biochem. Biophys. 157, 431-445. 13. LEIGH, J. S., JR., AND ERECII+KA, M. (1975) Biochim. Biophys. Acta 387, 95-106. 14. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. (1949) J. Biol. Chem. 177, 751-766. 15. BASFORD, R. E., TISDALE, H. D., GLENN, J. L., AND GREEN, D. E. (1957) Biochim. Biophys. Acta 24, 107-115. 16. BRUMBY, P. E., AND MASSEY, V. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. X, pp. 463-471, Academic Press, New York. 17. WILSON, D. F., AND KING, T. (1964) J. Biol. Chem. 239, 2683-2690. 18. KRGGER, A., AND KLINGENBERG, M. (1965) Biothem. 2. 344, 317-336. 19. CHANCE, B., AND GRAHAM, N. (1971) Rev. Sci. Instr. 42, 941-945. 20. DUTTON, P. L. (1971) Biochim. Biophys. Actu 226, 63-80. 21. HATEFI, Y., HAAVIK, A. G., AND GRIFFITHS, D. E. (1962) J. Biol. Chem. 237, 1681-1685. 33 KING, T. E. (1963) J. Biol. Chem. 238,4037-4051. --. 23. OHNISHI, T., SALERNO, J. C., WINTER, D. B., LIM, J., Yu, C. A., Yu, L., AND KING, T. E. (1976) J. Biol. Chem. 251, 2094-2104. 24. WILSON, D. F., AND LEIGH, J. S., JR. (1972). Arch. Biochem. Biophys. 150, 154-163. 25. MITCHELL, P. (1975) FEBS Lett. 56, l-6. 26. URBAN, P. F., AND KLINGENBERG, M. (1969)Eur. J. Biochem. 9, 519-525. 27. OHNISHI, T., WILSON, D. F., ASAKURA, T., AND CHANCE, B. (1972) Biochem. Biophys. Res. Commun. 46, 1631-1638. 28. BEINERT, H. AND RUZICKA, F. J. (1975) in Electron Transfer Chains and Oxidative Phosphorylation (Quagliariello, E., Papa, S., Palmieri, F., Slater, E. C., and Siliprandi, N., eds.), pp. 37-42, North-Holland, Amsterdam. 29. WIKSTRGM, M. K. F. (1973) Biochim. Biophys. Acta 301, 155-193. 30. WILSON, D. F., AND DUTTON, P. L. (1970) B&him. Biophys. Res. Commun. 39,59-64. 31. CHANCE, B., WILSON, D. F., DUTTON, P. L., AND
MITOCHONDRIAL
M. (1970) Proc. Nat. Acad. Sci. 66, 1175-1182. DUTTON, P. L. AND LINDSAY, J. G. (1973) in Mechanisms in Bioenergetics (Azzone, G. F., Ernster, L., Papa, S., Quagliariello, E., and Siliprandi, N., eds.), pp. 535-544, Academic Press, New York. WILSON, D. F., AND DUTTON, P. L. (1970) Arch. B&hem. Biophys. 136, 583-584. EREC~NSKA,
USA
32.
33.
CYTOCHROME
b-c,
COMPLEX
143
34. HINKLE, P., AND MITCHELL, P. (1970) Bioenergetits 1, 45-60. 35. LINDSAY, J. G., AND WILSON, D. F. (1972) Biochemistry 11, 4613-4621. 36. WILSON, D. F., DUTTON, P. L., ERECIASKA, M., LINDSAY, J. G., AND SATO, N. (1972)Account.s Chem. Res. 5, 234-241. 37. WILSON, D. F., ERECIASKA, M., AND DUTTON, P. L. (1974) Reu. Biophys. Bioengrg. 3, 203-230.
OF
BIOCHEMISTRY
Mitochondrial
AND
BIOPHYSICS
177,
Cytochrome Components
MARIA
ERECINSKA,
Department
of Biochemistry
133-143
(19’76)
b-c, Complex: Its Oxidation-Reduction and Their Stoichiometryl DAVID
F. WILSON,
and Biophysics, Philadelphia, Received
University Pennsylvania April
AND
YURIKO
of Pennsylvania
MIYATA Medical
School,
19, 1976
A cytochrome bx, complex was isolated from pigeon breast muscle mitochondria and purified to a content of 3 nmol of cytochrome c, per milligram of protein. Anaerobic suspensions of the preparation were titrated with reducing equivalents (NADH) and oxidizing equivalents (ferricyanide). The oxidation-reduction components of the complex were measured by the number of reducing equivalents accepted or donated per cytochrome cI and compared with the stoichiometries of the known redox components as measured by independent methods. The preparation accepts or donates 5.2 i 0.3 equivalents per cytochrome c I, while the measured content of cytochrome cl, cytochrome b,,, , cytochrome b,,,, Rieske iron-sulfur protein, ubiquinone, and succinate dehydrogenase accounts for 5.0 i 0.2 equivalents per cytochrome c,. It is concluded that there are no unknown redox components in the cytochrome bx, complex. The cytochrome bx, complex (energy transduction site 2) appears to be a structural unit containing equal amounts of cytochrome c,, cytochrome b,,,, cytochrome b,,,, and the Rieske iron-sulfur protein.
One of the major objectives in the study of mitochondrial oxidative phosphorylation is identification of the redox components of the respiratory chain, in particular those associated with each of the three phosphorylation sites and the description of their thermodynamic properties. Transduction of energy at site 3 occurs in cytochrome c oxidase and has been shown to involve four one-electron (n. = 1) redox components (1) while the reactions at site 2 (b-c, region of the respiratory chain) and site 1 (NADH dehydrogenase) are, on the other hand, less well characterized. Rieske and co-workers (2-5) established that after isolation, the cytochrome b-c, region of the respiratory chain (complex III) contains cytochromes b and c, (in a 2:l molar ratio), ncnheme iron, ubiquinone, and the antimycin A binding site. The results from titrations with ascorbate, however, raised the possibility that the complex contained GM
’ Supported 12202.
by U.S.
Public
Health
Service
yet another electron acceptor in amounts stoichiometric with cytochrome c1 (6, 7). Similar suggestions were made by OrmeJohnson et al. (8) on the basis of titrations of isolated ubiquinone-cytochrome c reductase with dithionite and NADH and by several other authors (9-11) on the basis of indirect evidence. Cytochrome b-c, complex prepared using a Triton X-lOO-deoxycholate method (12, 13) retains full durohydroquinone cytochrome c reductase activity and the unique spectral properties of the two b cytochromes (b,,, and b,,,). This preparation contains high concentrations of the cytochromes and of the Rieske iron-sulfur protein, but is deficient in ubiquinone and in succinate dehydrogenase. In the present paper we will present evidence that the purified cytochrome b-c, complex contains only cytochrome bsG5, cytochrome bj6,, cytochrome c 1, and the Rieske iron-sulfur protein in stoichiometric amounts. Thus site 2, like site 3, is an organized complex
Grant 133
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
134
ERECINSKA,
containing four one-electron nents in equal amounts. MATERIALS
AND
WILSON
redox compo-
METHODS
The cytochrome b-c, complex was isolated from pigeon breast muscle mitochondria by the procedure published previously for the purification of a succinate-cytochrome c reductase (12, 13). During the repeated fractionation with ammonium sulfate (in the presence of Triton X-100 and deoxycholate) employed for the isolation, a continuing loss of succinate dehydrogenase was observed. In the preparation utilized in this study the total succinate dehydrogenase (active and inactive) was less than 10% of the cytochrome c, content. Therefore, the cytochrome bx, complex appears to be a more appropriate name for this preparation. Analytical procedures. Protein was determined by the biuret method (14) using crystalline bovine serum albumin as a standard. Cytochrome b and c, contents were determined after conversion of their heme moieties into the respective pyridine hemochromogens as described by Basford et al. (15). The millimolar extinction coefficients used for calculations were 34.1 (at 557 nm) for pyridine hemochromogen b and 19.1 (552 red-540 ox) for pyridine hemochromogen c Nonheme iron was determined by the orthophenanthroline method according to Brumby and Massey (161 using iron nitrate prepared from iron wire as a standard. Acid nonextractable flavin was assayed fluorometrically according to the method of Wilson and King (17). Coenzyme Q was estimated by the procedure of Kroger and Klingenberg (18). Spectral studies and titrations. Spectral measurements were carried out using a Johnson Foundation scanning dual wavelength spectrophotometer. This instrument is provided with a digital wavelength drive on one of the two monochromators while the other is being held constant at a reference wavelength. The absorption spectrum of either the fully oxidized or fully reduced sample (or at any intermediate level of reduction) is measured and stored in the memory of a digital computer. The stored spectrum is then subtracted from any other subsequently measured and the trace which is recorded represents the difference between the two. The operation of this system is described in detail in (12, 19). Titrations were carried out in an anaerobic cuvette designed by Dutton (201 equipped with two micropipette titrators, one containing a standard solution of lo-20 mM ferricyanide, the other containing a standard solution of 5-12 mM NADH. Rapid interaction between NADH and the components of the cytochrome b-c, complex was ensured by the presence of diaphorase (20 units A/ml) and 80 FM flavin mononucleotide. At the end of each titration the concentra-
AND
MIYATA
tions of the standard solutions were estimated spectrophotometrically. The ferricyanide was measured at 420 nm using a millimolar extinction coefficient of 1.0 and the NADH was measured spectrophotometrically at 340 nm in the presence of pyruvate before and after the addition of lactate dehydrogenase (A emM = 6.23). The titrations were carried out either by measuring the absorbance changes at the wavelength pair 564-552 nm (Fig. 1) or by scanning the entire spectral region between 500 and 630 nm (Figs. 3-5). In each case, sufficient time was required to allow for the equilibration between the reductant (or the oxidant) and the respiratory chain components. Materials. Sodium deoxycholate, Triton X-100, NADH, flavin mononucleotide, and diaphorase (type II from Cl. kluyueri) were obtained from Sigma Chemical Company (St. Louis, MO.). RESULTS
Stoichiometry of the Electron Carriers in the Cytochrome b-c, Complex from Pigeon Breast Mitochondria Isolated Using Triton X-100 Deoxycholate Procedure The composition of three different preparations of the cytochrome b-c, complex isolated using a combination of nonionic (Triton X-100) and ionic (deoxycholate) detergents is shown in Table I. The preparations contain heme c, heme b, nonheme
FIG. 1. Spectrophotometric recording of the titration of an anaerobic reduced preparation of the cytochrome b-c, complex with ferricyanide. The cytochrome bx, complex was suspended at 15.4 /AM heme c in 50 mM phosphate buffer, pH 7.2 and preequilibrated with ultrapure argon gas to remove most of the dissolved oxygen. Sufficient dithionite solution was added to reduce the complex and remove the remaining oxygen. The reoxidation was carried out by stepwise addition of ferricyanide (the concentrations are given in the figure). Any excess of dithionite was destroyed by the initial addition of ferricyanide. Thereafter, at least three oxidative (with ferricyanide) and reductive (with NADH; Fig. 5) titrations were carried out on each preparation. Thus there is no dithionite remaining in the incubation mixture at any given time during the titrations. The measuring wavelength were 564-552 nm.
MITOCHONDRIAL
CYTOCHROME
iron, ubiquinone, and small amounts of acid-nonextractable flavin. Acid-extractable flavin was very low (less than 10% of the acid-nonextractable flavin) and phos-
e 0
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20 e-/cut C)
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FIG. 2. A plot of the absorbance changes during oxidative [ferricyanide(O)] and reductive LNADH (a)] titration of an anaerobic preparation of cytochrome b-c, complex against the amount of oxidant or reductant added. The oxidative titration is that shown in Fig. 1. Diaphorase and 80 /.LM flavin mononucleotide were added to facilitate the equilibration with NADH. The standard NADH solution used for titration was 11.6 mM. The absorbance measurements are the differences between the absorbance at 564 nm and that at 552 nm.
520
540
bx:,
135
COMPLEX
pholipids were not measured because the preparation was suspended in an 0.05% lysolecithin-0.05% lecithin mixture. Heme c was present in a concentration of approximately 3 Fmol/g of protein, while heme b was at a level of between 5 and 6 pmol/g of protein, the ratio between the two being 1:2. These concentrations and stoichiome: try are essentially the same as those found in the highly purified preparations of complex III described by Rieske et al. (6) and Hateti et aZ. (21). The concentration of acid-nonextractable flavin (which can be taken as a measure of succinic dehydrogenase content) is about 10% of the heme c concentration. Nonheme iron is present in amounts slightly higher than the heme b content, i.e., twice the concentration of heme c. It consists of the iron-sulfur centers of complex III itself (Rieske’s ironsulfur center) plus the contribution from succinic dehydrogenase iron-sulfur protein. The latter must be less than 0.8 iron/ cytochrome c, even if all of the flavin is associated with fully active succinate dehydrogenase [eight irons per flavin (2211 as the total concentration of succinic dehy-
560 X hm)
580
600
620
FIG. 3. Difference spectra obtained for the cytochromes b of cytochrome b-c, complex during anaerobic titration with ferricyanide. The cytochrome b-c, complex was suspended at 14.8 pM heme c concentration under the conditions described in the legend to Fig. 1. The spectrum of the fully reduced anaerobic sample was measured and stored in the memory of a digital computer. The flat baseline is the reduced minus reduced difference spectrum. Spectra 1 through 11 are the difference spectra obtained after stepwise additions of ferricyanide minus the reduced spectrum stored in the computer memory. The reference wavelength was at 540 nm.
136
ERECINSKA,
WILSON
AND
MIYATA
I
I
I
I
I
I
520
540
560
580
600
620
X (nml FIG. 4. Difference spectra obtained for cytochrome cI of the cytochrome b-c, complex during anaerobic titration with ferricyanide. Conditions are those of Fig. 3. The titration is the continuation of the one presented in Fig. 3. Spectrum 11 of Fig. 3 was stored in the computer memory; thus, the baseline of Fig. 4 is for the preparation with the b cytochromes oxidized and cytochrome c, reduced. Spectra 1 through 7 are difference spectra obtained after each addition of ferricyanide. The reference wavelength was at 540 nm.
5io
540
560
560
590
660
itnrn)
FIG. 5. Dual wavelength difference spectra of the cytochromes of the b-c, complex obtained during anaerobic titration with NADH. Conditions are those of Figs. 1 and 2 and the technique is described in the legends to Figs. 3 and 4. The baseline is obtained with all of the cytochromes oxidized. Spectra 1-14 are difference spectra (NADH reduced-oxidized) obtained after stepwise addition of NADH.
drogenase is only 10% of that of cytochrome c,. Subtraction of the approximate value of the contribution of succinic dehydrogenase iron-sulfur centers from the overall content of the nonheme iron provides a figure equal to nearly twice the
heme c concentration. This is consistent with the presence of two irons per mole of the protein in the Rieske iron-sulfur center; this value is in exceIlent agreement with the results of Rieske et al. (6, 7). Since succinate dehydrogenase accepts 4
MITOCHONDRIAL
CYTOCHROME TABLE
COMPOSITION
b-c,
OF CYTOCHROME
Component
Preparation rnM
-~ Heme Cytochrome cI Cytochrome b Nonheme iron Ubiquinone Flavin (acid nonextractable) Protein (mg/ml) -.
COMPLEX
PREPARED
1
b-c, I BY TRITON
0.12 0.226 0.211 0.0485 0.0124 42.6
rnM
~mollg
2.82 5.31 4.95 1.14 0.29 -
equivalents per flavin (23) the total reducing equivalents accepted will be approximately 0.4 per cytochrome c,. All three preparations contain ubiquinone in amounts equal to one-third of the cytochrome c1 concentration. (This value should be compared with a figure of 15 for the same ratio found in pigeon breast mitochondria.) Since ubiquinone is a two electron donor/acceptor, on an electron basis this provides a not-insignificant figure of about 0.6 per cytochrome c,. Titrations of the Cytochrome b-c1 Complex with Ferricyanide and NADH as Followed by Measurement of the Absorbance at 552 nm Minus That at 564 nm Titrations of the cytochrome b-c, complex with ferricyanide and NADH carried out anaerobically, as described in the Methods section, are shown in Figs. 1-5. In Fig. 1, the dual wavelength spectrophotometer was set to measure the difference in absorbance between 564 and 552 nm and the titration of the reduced sample was performed through stepwise addition of small amounts of ferricyanide. After each addition of ferricyanide a change in absorbance is observed due to oxidation of cytochromes b (decrease in absorbance at 564 nm relative to 552 nm). After complete oxidation of cytochromes b the spectrophotometric trace flattens and reverses due to subsequent oxidation of cytochrome c, , revealed as the decrease in absorbance at 552 nm relative to 564 nm. The two “arms” of the trace are unequal because the absorbance change at 564 nm which accompanies oxidation of cytochromes b is greater than the absorbance change at 552 nm due to
X-100
Preparation
protein
~__
-
137
COMPLEX
DEOXYCHOLATE
2 cLmol/g protein
rnM
3.08 5.38 5.73 1.06 0.23 -
0.11 0.205 0.248 0.041 0.0124 42.2
0.148 0.258 0.275 0.051 0.011 48.0
PROCEDURE
Preparation
3 pmolig
protein 2.60 4.85 5.88 0.97 0.29 -
oxidation of cytochrome c, . The equilibration is rapid as evidenced by sharp leveling of the traces after each addition of the oxidant. When oxidation of cytochrome c, is complete, the anaerobic preparation of the oxidized sample is titrated with NADH in the same stepwise manner. The trace obtained is an almost “mirror image” of that presented in Fig. 1. Equilibration with NADH is also rapid; the absorbance changes following NADH addition are complete within 1 min. The plot of the two titrations (absorbance against the number of the reducing or oxidizing equivalents used) is presented in Fig. 2. The inequality of the two sides with respect to the total absorbance change is discussed above. An interesting observation is, however, that the total number of the reducing or oxidizing equivalents used as cytochrome c, is titrated is only slightly smaller than that consumed when cytochromes b are titrated. Moreover, there are practically no equivalents used up between the end of cytochrome c, titration and the onset of cytochromes b titration. This indicates the absence of electron donors/acceptors in the oxidation-reduction potential gap between cytochrome c, and the cytochromes b in concentrations set by the limits of detectability of the present approach (see Discussion and below). Titrations of the Cytochrome b-c, Complex by Ferricyanide and NADH as Followed Measuring the Absorption Spectra from 500 to 630 nm The titrations dual wavelength
also can be followed in the scanning spectrophotom-
138
ERECIr;JSKA,
WILSON
eter. The oxidative titration with ferricyanide is shown in Figs. 3 (cytochromes b) and 4 (cytochrome c,). The spectrum of the fully reduced anaerobic sample is recorded as the reference spectrum. Stepwise addition of ferricyanide results in oxidation of the components of the cytochrome b-c, complex. The first component oxidized is cytochrome bsGs, as evidenced by the appearance of the characteristic “double peak” absorbance change with a maximum at 565 nm and a shoulder at 558 nm (12). The oxidation of cytochrome bjG5 is followed by that of cytochrome bj6,. As the oxidation of cytochromes b is completed, the oxidation of cytochrome c, begins as indicated by the disappearance of the absorbance at the short wavelength side of the cytochromes b absorbance minima. At this point in the experiment (cytochromes b oxidized, cytochrome c1 mostly reduced) a new baseline was recorded (Fig. 4) and the oxidative titration continued until 100% oxidation of cytochrome c, was attained. It should be stressed here that the preparation of succinate-cytochrome c reductase used in the present work was essentially 100% reducible by the NADH, i.e., there was no additional reduction upon addition of dithionite. There was no measurable CO-sensitive component present in the preparation. The titration of the b-c, complex with NADH is shown in Fig. 5. The spectrum of the fully oxidized anaerobic preparation was taken as the reference and the spectrum was recorded after each addition of NADH and a sufficient amount of time to attain the corresponding level of reduction. At least two such oxidative and reductive titrations were carried out for each of the three preparations of the cytochrome b-c, complex. An example of the plot of the data is shown in Fig. 6. The titrations with NADH constitute the ascending arms of the curves while the titrations with ferricyanide constitute the descending ones. Different symbols refer to the three different wavelengths for which the calculations were made: 554,563, and 568 nm. It can be calculated from the known concentration of cytochrome c, (heme c) and the amount of the reducing (or oxidizing) equivalents
AND
MIYATA
consumed that there are approximately two electrons accepted and donated per cytochrome c 1 when cytochrome c, is titrated and approximately three electrons when the cytochromes b are titrated. It has been shown previously (13) that the cytochrome c1 midpoint potential (Em,,, = 0.280 -C 0.01 V) is very close to that of Rieske’s nonheme iron protein [Em7,2 = 0.275 k 0.02 V (2411, both being one electron donorslacceptors. Reduction of the b cytochromes is accompanied by reduction of ubiquinone with the half-reduction potential of 0.060 ? 0.02 V (a two-electron donor/acceptor) and the components of succinic dehydrogenase which “contaminates” the preparation of the cytochrome b-c, complex (0.4 equivalents per cytochrome c,). Since a quantitative estimate of all these components was made for each of the three reductase preparations we can make a precise comparison between the number of electrons consumed or accepted in the experimental situation and that calculated theoretically. A summary of all of the experimental data and their comparison with the information obtained by computing the values determined by analytical procedures is given in Table II. The calculations were performed on the basis of the heme c concentration in various preparations. It is seen that there are approximately 5.2 elec60
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.
.
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FIG. 6. A plot of an oxidative (ferricyanide) and reductive (NADH) titration of the cytochromes of bc, complex. The experimental data were obtained from titrations similar to those shown in Fig. 5. cm), absorbance changes at 554 nm; (01, absorbance changes at 560 nm; (A), absorbance changes at 566 nm. The heme c concentration was 16.9 pM.
MITOCHONDRIAL
CYTOCHROME TABLE
ELECTRON
ACCEPTORS
Preparation number
Ieme ( (@f)
OF b-c,
COMPLEX
NADH
OF THE AND
Number
Ferricyanide
139
COMPLEX
II
RESPIRATORY FERRICYANIDE
Ieme e-k
I
b-c,
CHAIN
AND
of electron 1LIeme
C
b
0.12
0.226
I.148
0.258
0.11
0.205
THEIR
TITRATIONS
equivalents
NHI”
(rnM) SDH’
WITH
NADH
in preparation Total
e-k
t
14.4 16.8
0.0486
5.07
Average 2
17.8 17.8
48.7 46.5
14.8 14.8
35.0 32.5
0.687
4.64
Average 3
28.0 37.5
Average Average fo three prepa rations
67.5 75.0
,
i6.1
14.9
0.082
0.581
5.5 5.25 2.61
0 Heme c + heme b/3 b A two electron donor/acceptor. c Flavin (2-1 + NH1 (2~).
trons taken up or given away per heme c, as determined experimentally, while the number obtained from the addition of the concentrations of all the known components present in this preparation is approximately 5.0 per heme c. Thus the total unknown electron acceptor(s) in the preparation of the cytochrome b-c, complex must be present at a total concentration of less than 0.5 per cytochrome c, for oneelectron acceptor(s) or less than 0.25 per cytochrome c1 for two-electron acceptor(s). Titrations of “Aged” Preparations Cytochrome b-c, Complex
of the
The cytochrome b-c, complex is stable for several hours at room temperature, as judged by the spectral properties of cytochromes bsG5 and b,,,. The titrations of such a preparation (Fig. 7) with NADH and ferricyanide show, however, that fewer equivalents are required in the region where cytochrome c, is oxidized or reduced than in parallel titrations carried out on “fresh” preparations. Analysis of the data indicates that this decrease is only apparent and arises from the fact that
in the aged preparation the E, value of the spectrally invisible Rieske iron-sulfur protein is approximately 40 mV more positive than that of cytochrome c,, while in fresh preparations the E, values for the two are equal within k-20 mV. As the E, value of the iron-sulfur protein becomes more positive relative to cytochrome c,, the cytochrome c1 titration curve becomes asymmetric. In the initial phase of ferricyanide titrations only cytochrome c1 is oxidized and therefore only one equivalent per cytochrome c, is given to the ferricyanide. At more than 50% oxidation of cytochrome c 1, the slope decreases markedly and approaches the value of two because the iron-sulfur protein provides an increasing fraction of the reducing equivalents for the ferricyanide reduction. DISCUSSION
Chemical analysis of the cytochrome be, complex isolated from pigeon breast mitochondria shows that the preparation contains four one-electron carriers present in equimolar concentrations: cytochrome c,, cytochrome b,,, , cytochrome bje5, and
140
ERECINSKA,
0
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10 pM
r IO
20
WILSON
AND
MIYATA
$ 30
10
20
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30 e-/Q!
$74 40
50
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40
30 e’Cy+
40
50
60
70
Ferrt 20 c,
10
0
FIG. 7. A plot of an oxidative (ferricyanide) and reductive (NADH) titration of the cytochromes of an aged preparation of cytochrome b-c, complex. The preparation was aged by diluting to 14.8 pM cytochrome c, and maintaining it for 4 h at 25°C. The titrations were carried out as described in the legends to Figs. 1 and 2 and difference spectra similar to those presented in Fig. 5 were recorded. (D), absorbance changes at 554 nm; (O), absorbance changes at 560 nm; (A), absorbance changes at 566 nm.
Rieske’s iron-sulfur protein. In addition, ubiquinone and succinic dehydrogenase are found in concentrations 0.3 and 0.1 that of cytochrome c,, respectively. Anaerobic titrations of this preparation with a reductant (NADH) and an oxidant (ferricyanide) demonstrate that the order of reduction or oxidation of the components is consistent with the known values of their half-reduction potentials. In the potential span where cytochrome c1 is titrated (i.e., between an E, value of -0.32 and -0.2 V) two reducing equivalents are taken up or given away, which indicates that Rieske’s iron-sulfur protein with a half-reduction potential very close to that of cytochrome c, undergoes parallel oxidation and reduction such that at each point it is reduced to the same extent as cytochrome c,. In the region where cytochromes b are oxidized and reduced (the potential region between an Eh of 0.160 and -0.08 V) there are approximately three oxidizing or reducing equivalents involved, two being accounted for by the two b cytochromes present in equal concentrations while the third is shared between ubiquinone (0.6 equivalents since two equivalents are required per ubiquinone) and succinic dehydrogenase flavin and iron sulfur centers (0.4 equivalents). Succinic dehydrogenase is a “contaminant” of the b-c, complex while
ubiquinone may or may not be an intrinsic part of the complex. Its presence in substoichiometric quantities in preparations which retain full durohydroquinone cytochrome c reductase activity leads us to conclude that it is highly unlikely that ubiquinone is an obligatory redox component at site 2 (see, however, 25). It should be further pointed out that because ubiquinone donates (and accepts) two electrons and is reduced at intermediate levels of reduction of the b cytochromes, its effect on their titration curves is readily observed, which permits the conclusion that its Em7.2 value is similar to that observed in intact mitochondria [Em7.2 of ubiquinone = 0.045 + 0.02 V (26)]. It has been suggested repeatedly during the last 10 years that site 2 contains yet other components in addition to those discussed above. Two of them, component X of Rieske (9) and Y of Eisenbach and Gutman (10) were characterized with respect to the values of their half-reduction potentials. The numerical values given were 0.15 V for component X and 0.25 V for component Y. A precise analysis of the titration curves reported in this work could confirm or exclude the existence of such additional components. Two points are relevant: First, the titration of cytochrome c, requires 2.0 ? 0.1 equivalents/
MITOCHONDRIAL
CYTOCHROME
cytochrome c1 and the slope of the titration curve is a straight line. This means that except for cytochrome c, and the Rieske iron-sulfur protein there are no other redox components present with E, values 20.06 V of that of cytochrome c, in concentrations greater than 0.1 that of cytochrome c,. Second the possible existence of redox components with E, values between 0.2 and 0.06 V can be determined from careful comparison of the experimental titration curves and those calculated for the known components (Fig. 8). The three curves shown in Fig. 8 represent the calculated plots of absorbance changes at 552 nm (extreme left) and at 564 nm (the middle and the right curve) against the number of electrons accepted or donated per unit concentration (cytochrome c, content in this case). The circle symbols refer to the curves constructed by taking into account the known components of cytochrome b-c, complex [cytochrome c, (E,,, = 0.25 V), Rieske iron-sulfur protein (E, = 0.25 V), cytochrome b,,, (E, = 0.09 V), ubiquinone (E, = 0.06 V), cytochrome b,,, (E, = 0.00 V), and the succinate dehydrogenase flavin (E, = -0.04 V)] in their experimentally measured stoichiometries of 1:1:1:0.3:1:0.1. The triangles describe the curves which, in addition to the information given above, assume the presence of a hypothetical component with a midpoint potential of 0.15 V and an n value of 1 in a stoichiometry equal to that of cytochrome c,. The plots allow very precise measurements of the number of equivalents required to go from 75% reduction of cytochrome c, to 75% reduction of cytochromes b. The value is 2.5 equivalents per cytochrome c, in the absence of the hypothetical component (the difference between the points B and A) and 3.5 in the presence of the additional component (the difference between points C and A). Moreover, the calculated values must be increased to 2.7 and 3.7 equivalents per cytochrome c, to account for the iron-sulfur centers of succinate dehydrogenase which were not considered in calculating the theoretical curves. Analysis of the experimental titration curves gives 2.63 * 0.51 equivalents per cytochrome c, for titrations with
b-c,
141
COMPLEX
lb
2’0
3:o
4:o
50
6’0
e-/cyt c, FIG. 8. Theoretical curves of the absorbance changes at 552 nm (left-hand curve) and 564 nm (middle and right-hand curves) against the number of oxidizing or reducing equivalents. The circles refer to the curves constructed by taking into account the known components of the cytochrome b-c, complex: cytochrome c,, Rieske’s iron-sulfur center, cytochrome bz6,, ubiquinone, cytochrome b,,,, and the succinate dehydrogenase flavin in their experimentally measured stoichiometries of 1:1:1:0.3:1:0.1. The triangles refer to the curves which, in addition, assume the presence of a hypothetical component with a midpoint potential of 0.15 V and an n value of 1 in a stoichiometry equal to that of cytochrome cf. The arrows at points A, B, and C indicate 75% reduction of cytochrome c, (A) or cytochromes b (B and C) under both sets of conditions. The two b cytochromes are assumed to contribute equally to the absorbance change at 564 nm.
NADH and 2.61 -t 0.27 equivalents for titrations with ferricyanide. The number of reducing equivalents accepted or donated by the cytochrome b-c, complex determined experimentally is equal to the value expected from the stoichiometry of the known redox components. This is true for all potential regions of the titrations and thus the data are incompatible with the existence of any unknown redox components at site 2 (i.e., with E, values between 0.32 and -0.1 V). It also follows from the results presented above that in functionally fully competent cytochrome b-c 1 complex, the iron-sulfur center 5 (27) is present in quantities less than 10% that of cytochrome c, and cannot be considered as an obligatory member of the redox components at site 2. This is consistent with its suggested role in a flavoprotein responsible for reoxidation of the electron transferring flavoprotein of the fatty acyl coenzyme A dehydrogenating system (28). In addition to the components discussed
142
ERECINSKA,
WILSON
above the mitochondrial respiratory chain has been periodically reported to contain more than two cytochrome b species (see, for example, 29). The results presented in this paper show that the sum of all of the b cytochromes present in the b-c, region of the respiratory chain is twice the cytochrome c1 concentration. Moreover, they accept and donate reducing equivalents in a manner consistent with the presence of only “two species in a 1:l molar ratio. In summary, the results presented in this paper indicate that energy transduction site 2, like site 3, consists of four oneelectron transfer components: two b cytochromes, cytochrome c, , and Rieske’s ironsulfur center. Two of the components are on the low oxidation-reduction potential side of the site (the two b cytochromes) and two are on the high potential side (Rieske’s iron-sulfur center and cytochrome c,) and the transfer of electrons between the two is coupled to the synthesis of ATP. Moreover, it has been demonstrated that the chemical properties of two redox components at each site [cytochromes b,,, and c1 at site 2 (30-32) and cytochromes a and a3 at site 3 (33-35)l are dependent on the phosphorylation state of the mitochondria which has led to the proposal (36, 37) that these particular redox components are directly involved in the energy transduction process. Finally, the similarities in composition of the energy transduction sites 2 and 3 and in the reactivities of their components support the concept that the general principles which underlie the basic mechanism of energy transduction are the same at all three phosphorylation sites and apply equally to site 1. REFERENCES 1. VAN GELDER, B. F., AND BEINERT, H. (1969) Biochim. Biophys. Acta 189, 1-24. 2. RIESKE, J. S., BALJM, H., STONER, C. D., AND LIPTON, S. H. (1967) J. Biol. Chem. 242, 48544866. 3. SILMAN, H. I., RIESKE, J. S., LIPTON, S. H., AND BAUM, H. (1967) J. Biol. Chem. 242, 48664875. 4. BAUM, H., SILMAN, H. I., RIESKE, J. S., AND LIPTON, S. H. (1967) J. Biol. Chem. 242, 48764887. 5. RIESKE, J. S., LIPTON, S. H., BAUM, H., AND SILMAN, H. I. (1967) J. Biol. Chem. 242, 48884896.
AND
MIYATA
6. RIESKE, J. S., HANSEN, R. E., AND ZAUGG, W. S. (1964) J. Biol. Chem. 239, 3017-3022. 7. RIESKE, J. S., LAUGG, W. S., AND HANSEN, R. E. (1964) J. Biol. Chem. 239, 3023-3030. 8. ORME-JOHNSON, N. R., HANSEN, R. E., AND BEINERT, H. (1974) J. Biol. Chem. 249, 19281939. 9. RIESKE, J. S. (1971) Arch. Biochem. Biophys. 145, 179-193. 10. EISENBACH, M., AND GUTMAN, M. (1974) FEBS Lett. 46, 368-371; (1975) Eur. J. Biochem. 52, 107-116; 59, 223-230. 11. WIKSTR~M, M. K. F., AND BERDEN, J. A. (1972) Biochim. Biophys. Actu 283, 403-420. 12. ERECIASKA, M., OSHINO, R., OSHINO, N., AND CHANCE, B. (1973) Arch. Biochem. Biophys. 157, 431-445. 13. LEIGH, J. S., JR., AND ERECII+KA, M. (1975) Biochim. Biophys. Acta 387, 95-106. 14. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. (1949) J. Biol. Chem. 177, 751-766. 15. BASFORD, R. E., TISDALE, H. D., GLENN, J. L., AND GREEN, D. E. (1957) Biochim. Biophys. Acta 24, 107-115. 16. BRUMBY, P. E., AND MASSEY, V. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. X, pp. 463-471, Academic Press, New York. 17. WILSON, D. F., AND KING, T. (1964) J. Biol. Chem. 239, 2683-2690. 18. KRGGER, A., AND KLINGENBERG, M. (1965) Biothem. 2. 344, 317-336. 19. CHANCE, B., AND GRAHAM, N. (1971) Rev. Sci. Instr. 42, 941-945. 20. DUTTON, P. L. (1971) Biochim. Biophys. Actu 226, 63-80. 21. HATEFI, Y., HAAVIK, A. G., AND GRIFFITHS, D. E. (1962) J. Biol. Chem. 237, 1681-1685. 33 KING, T. E. (1963) J. Biol. Chem. 238,4037-4051. --. 23. OHNISHI, T., SALERNO, J. C., WINTER, D. B., LIM, J., Yu, C. A., Yu, L., AND KING, T. E. (1976) J. Biol. Chem. 251, 2094-2104. 24. WILSON, D. F., AND LEIGH, J. S., JR. (1972). Arch. Biochem. Biophys. 150, 154-163. 25. MITCHELL, P. (1975) FEBS Lett. 56, l-6. 26. URBAN, P. F., AND KLINGENBERG, M. (1969)Eur. J. Biochem. 9, 519-525. 27. OHNISHI, T., WILSON, D. F., ASAKURA, T., AND CHANCE, B. (1972) Biochem. Biophys. Res. Commun. 46, 1631-1638. 28. BEINERT, H. AND RUZICKA, F. J. (1975) in Electron Transfer Chains and Oxidative Phosphorylation (Quagliariello, E., Papa, S., Palmieri, F., Slater, E. C., and Siliprandi, N., eds.), pp. 37-42, North-Holland, Amsterdam. 29. WIKSTRGM, M. K. F. (1973) Biochim. Biophys. Acta 301, 155-193. 30. WILSON, D. F., AND DUTTON, P. L. (1970) B&him. Biophys. Res. Commun. 39,59-64. 31. CHANCE, B., WILSON, D. F., DUTTON, P. L., AND
MITOCHONDRIAL
M. (1970) Proc. Nat. Acad. Sci. 66, 1175-1182. DUTTON, P. L. AND LINDSAY, J. G. (1973) in Mechanisms in Bioenergetics (Azzone, G. F., Ernster, L., Papa, S., Quagliariello, E., and Siliprandi, N., eds.), pp. 535-544, Academic Press, New York. WILSON, D. F., AND DUTTON, P. L. (1970) Arch. B&hem. Biophys. 136, 583-584. EREC~NSKA,
USA
32.
33.
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b-c,
COMPLEX
143
34. HINKLE, P., AND MITCHELL, P. (1970) Bioenergetits 1, 45-60. 35. LINDSAY, J. G., AND WILSON, D. F. (1972) Biochemistry 11, 4613-4621. 36. WILSON, D. F., DUTTON, P. L., ERECIASKA, M., LINDSAY, J. G., AND SATO, N. (1972)Account.s Chem. Res. 5, 234-241. 37. WILSON, D. F., ERECIASKA, M., AND DUTTON, P. L. (1974) Reu. Biophys. Bioengrg. 3, 203-230.
