The properties of the mitochondrial succinate-cytochrome c reductase

The properties of the mitochondrial succinate-cytochrome c reductase

ARCHIVES OF The RIOCHEMISTRY AND Properties BIOPHYSICS of the 161, 112-121 (1972) Mitochondrial Succinate-Cytochrome c Reductase’ DAVID ...

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ARCHIVES

OF

The

RIOCHEMISTRY

AND

Properties

BIOPHYSICS

of the

161,

112-121

(1972)

Mitochondrial

Succinate-Cytochrome

c Reductase’ DAVID

F. WILSON, AND

Johnson

MARIA ERECI%IA, JOHN MICHELE ROPPELMAN

S. LEIGH,

JR.,

Research Foundation, Department of Biophysics and Physical Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania 1910.4 Received

January

18, 1972;

accepted

April

6, 1972

The cytochromes b and 62 of pigeon heart mitochondria have half-reduction potentials (Em’s) of 1-30 mV and -30 mV at pH 7.2. The midpoint potentials of these cytochromes become more negative by 3040 mV per pH unit when the pH is made more alkaline. Detergents may be used to prepare a succinate-cytochrome c reductase free of cytochrome oxidase in which the activation of electron transport induced by oxidation of cytochrome c1 causes the half-reduction potential of cytochrome bT to become at least 175 mV more positive than in the absence of electron transport. This change is interpreted as indicating that the primary energy conservation reaction at site 2 remains fully functional in the purified reductase. Preliminary electron paramagnetic resonance spectra of the succinate-cytochrome c reductase as measured at near liquid helium temperatures are presented.

The succinate-cytochrome c reductase of the mitochondrial respiratory chain involves several oxidation-reduction components, including succinate dehydrogenase, two (l-3), and possibly three (4, 5), b cytochromes, two or more iron-sulfur proteins (7, 8) coenzyme & (9,lO) and cytochrome cl (11). An integral part of this assembly of oxidation-reduction components is the mechanism for conserving free energy for the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), also called energy conservation site 2. It has been demonstrated that cytochrome bT @I& has a half-reduction potential which is dependent on the phosphate potential (1, 2, 4, 6) and thus is considered to have a primary role in energy transduction at site 2. Moreover the halfreduction potential of cytochrome & in purified succinate-cytochrome c reductase (12,

13) has been observed to become more positive by at least 175 mV when electron transport is activated. This has been interpreted as direct evidence that the primary energy conservation event is fully functional in the purified succinate-cytochrome c reductase (12,13). Anomalous reduction of cytochrome b has been observed on addition of oxidants to antimycin A-treated anaerobic yeast cells (14), intact mitochondria (15), detergenttreated mitochondria (16), and in the b-s complex (17, 18). This reduction appears also to be a reflection of the energy dependent change in the half-reduction potential of cgtochrome 6, (12, 13). Erecinska (19) has reported a similar reduction in uncoupled mitochondria in the absenceof antimycin A at 0°C. In this communication we will present a study of the oxidation-reduction components of succinate-cytochrome c reductase in intact mitochondria and the solubilized, partially purified reductase with particular attention to their optical and electron paramagnetic resonance (epr) properties and the

i Supported by National Science Foundation Grant GB 28125 and National Institutes of Health Grant GM 12202. D. F. Wilson is the recipient of U.S. Public Health Service Career Development Award I-KO4-GM 18154. 112 Copyright All rights

@ 1972 by Academic Press, of reproduction in any form

Inc. reserved.

SUCCINATE-CYTOCHROME

relationship of these properties to energy conservation. MATERIALS

AND METHODS

Pigeon heart mitochondria were prepared by the method of Chance and Hagihara (20). Chicken heart mitochondria prepared by the method of Low and Vallin (21) for beef heart mitochondria were st,ored frozen at 60 mg protein/ml and thawed when needed. ‘The thawed mitochondria were used after one of three treatments: A. Mitochondria at 20 mg/ml suspended in 0.1 M phosphate buffer at pH 7.4 with 0.6% cholate were fractionated by ammonium sulfate precipitation at 4°C. The fraction between 0.25 and 0.4 saturation of ammonium sulfate was collected. This fraction was resuspended in 0.1 M phosphate to give an optically clear solution containing no cytochrome c but unchanged ratios of the other cytochromes. B. Succinate-cytochrome c reductase was prepared by the method of King and Takemori (22). C. Soluble succinate-cytochrome c reductase was prepared from detergent-treated chicken heart mitochondria fractionated with ammonium sulfate, the entire procedure at 0’4’C (unpublished method of It. O&no). A suspension of froxenthawed chicken heart mitochondria (18 mg protein/ml in 0.1 M phosphat,e buffer, pH 7.4) was t.reated with 0.5y0 Triton X-106 and 0.5% deoxycholate. After thorough homogenization, the suspension was stirred for 10 min and then centrifuged for 20 min at 23,000 9. The white precipitate was discarded and the supernatant adjusted to 25yo saturation with (NHI)&S04. After 40 min the suspension was centrifuged for 15 min at 23,000 g. The pellet and/or flotate contained cytochrome oxidase. The lsuccinate cyt.ochrome c reductase was in the supernatant. The supernatant was adjusted to 45%) saturation with (NH4)801 and after 40 min the precipitate was collected by centrifugation at 23,000g for 15 min. The reductase can thus be collected essentially free of cytochrome oxidase and refractionated as necessary. For spectra.1 studies a final centrifugation at 105,OOOgfor 30 min was done and the white flotate was removed with absorbent paper. For epr studies it was found helpful to concentrate the reductase further by centrifuging at 180,OOOg for 3 hr. Although this does not fully sediment the reductase, it is concentrated in the lower portion of the tubes and the colorless upper portion may be removed. The oxidation-reduction potential titrations were carried out using the method of Dutton et al. (23-25). When samples were prepared for electron paramagnetic resonance (epr) measurements the samples were first adjusted to the desired oxidation-reduction potential, then aliquots anaerobi-

113

c REDUCTASE

tally transferred to 3 mm i.d. epr tubes (calibrated using copper EDTA solutions) and frozen by immersion in liquid nitrogen. The spectra were then measured with a Varian E3 or E4 spectrometer at liquid nitrogen temperature or near liquid helium temperature. The oxidation-reduction mediators used are indicated in the figure legends. Other reagents used were the same as previously described (4). RESULTS

The spectral properties of the cytochromesof succinate-cytochromec reclucfase.The purified succinate-cytochrome c reductase contains one c cytochrome (cl) which has an alpha maximum at 553 nm (Fig. 1) and two b cytochromes (b, and bT). The two b cytochromes have alpha maxima at 561 nm (called b& and 565 nm with a shoulder at 558 nm (called b&, respectively. These spectral properties are the same as have been reported for the b 560nm 552nm

4 565nm ‘L\’ ’ i \

i

ii

500

520

i

/ )

540‘--G%TWOvelength kmd

FIG. 1. The difference between the spectra of the reduced and oxidized cytochromes of the succinate-cytochrome c reductase. A purified succinate-cytochrome c reductase was diluted to 3.5 pM cytochrome cl with a 50 mM morpholinopropane sulfonate buffer, pH 7.2. (. . . .) measure cuvette additions; 100 ~CLM N,N,N’,N’-tetramethyl-pphenylene diamine (TMPD) and 3 mM sodium ascorbate: Reference cuvette additions; none. (-,-,-) measure cuvette additions; 100 PM TMPD, 3m~ sodium ascorbate, 6 mM succinate and 1 rnM fumarate: Reference cuvette additions; 100 PM TMPD and 3 mM sodium ascorbate. (--) measure cuvette additions; dithionite: Reference cuvette additions; 100 ~11 TMPD, 3 mM sodium ascorbate, 6 mM succinate and 1 mM fumarate: (- -) measure cuvette additions: dithionite: Reference cuvette additions; none,

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WILSOY

cytochromes in intact pigeon heart mitochondria by Sato et al. (3, 6). In the succinate-cytochrome c reductase the cytochrome cl is readily reduced by ascorbate plus N, N, N’ ,N’- tetramethyl -p phenylenediamine (TMPD). In the presence of high concentrations of T_14PD (more than 100 PM) some b cytochrome is also reduced. Cytochrome br,cl is readilyreduced by succinate. A small amount of fumarate was also added to prevent partial reduction of the

5 250 E I w 200

‘“h -1.0

0.0 Log Ox/Red

FIG. 2. The oxidation-reduction potential dependence of the cytochrome C, in the purified succinate-cytochrome c reductase. The purified reductase was suspended at 3 PM cytochrome ci in a 50 InM morpholinopropane sulfonate buffer at pH 7.2. Theoxidation-reduction mediators used were 50 PM diamin odurene, 40 PM phenazine methosulfate and 20 PM ferricyanide (ferricyanide was also used to provide oxidizing equivalents during the oxidative titration). The titration was carried out as previously described (4,25) using the wavelength pair 552 nm minus 540 nm. (0) reductive titration; (W) oxidative titration.

ET

AL.

cytochrome bS6h. When desired cytochrome b5e5may be reduced by dithionite. The half-reduction. potentials of the cytochromes of succinate-cytochrome c reductase. When cytochrome CI is titrated as a function of t,he oxidation-reduction potential it behaves as a single component wit,h an n value of 1.0 and a half-reduction potential at pH 7.2 of +245 mV (Fig. 2). This value is slightly more positive than that of +225 mV (4) found for intact pigeon heart mitochondria and submitochondrial particles from beef heart,. The redox titration of the b cytochromes as measured at 562 nm minus 575 nm is presented in Fig. 3A. The curve is sigmoidal and can be resolved into two components as shown in Fig. 3B. The two components have 1~ values of 1 and half-reduction potentials of +65 mV and -25 mV at pH 7.2. When a number of titrations were averaged the mean values are +60 f 10 mV and -25 f 10 mV. Also included in Fig. 3A and B is a titration of the b cytochromes in the presence of excess antimycin A. The wavelength pair used to measure the cyt,ochromes was selected to avoid the antimycin A-induced shift in the spectrum of reduced cytochrome bs61 (bK) (6, 25, 27). Under these conditions there is no effect of antimycin A on the titration curve, indicating that in this preparation, as in intact mitochondria (13) and submitochondrial particles (26) the

FIG. 3. The oxidation-reduction potential dependence of the reduction of the b cytochromes of the purified succinate-cytochrome e reductase. The purified reductase was suspended at 3 PM cytochrome ci in a 50 mu morpholinopropane sulfonate buffer at pH 7.2. The oxidation-reduction mediators used were 50 PM diaminodurene, 40 PM phenazine methosulfate, 40 PM phenazine ethosulfate, 30 PM duroquinone, 10 PM pyocyanine and 30 pM 2-hydroxy naphthoquinone. The titration was carried out as previously described (1, 35) using the wavelength pair 561.5 nm minus 575 nm to measure the b cytochrome reduction. The titrations were carried out both oxidatively and reductively but in the figure the symbols are used to identify the titrations in the absence (A) and presence (0) of 2 pg/ml antimycin A. A. The titration curves are plotted assuming a single component. B. The same data plotted after mathematical resolution of the sigmoid curve in A into two components (see Ref. 1).

SUCCINATE-CYTOCHROME

antibiotic does not alter the half-reduction potential of either cytochrome. The pH dependence of the half-reduction potentials of the b cytochromes. The half-reduction potentials of the b cytochromes in intact pigeon heart mitochondria are pH dependent as shown in Fig. 4. The titration curve for the reduction of the 6 cytochromes is sigmoid and must be mathematically separated into the individua.1 components. This

L,

60

65

70

75

SO

PH

FIG. 4. The pH dependence of the half-reduction potentials of cytochromes br and bK in pigeon heart mitlochondria. The pigeon heart mitochondria were suspended in a medium containing 0.2 M sucrose and 40 rnr,r morpholinopropane sulfonate (pH 6.3-7.6) or 40 mM Tris(hydroxymethy1) aminomethane (pH 7.6-8.5) at the pH indicated on the abscissa. The potentiometric titrations were carried out anaerobically and cytochromeb (5~) and bT measured using either 561.5575 nm or 436-412 nm. The individual midpoint potentials were obtained by mathematically resolving the sigmoid titration curve into two n = 1 components as previously described (1).

FIG. ductase propane indicated sulfate), tions of adjusting

c REDUCTASE

115

increases the uncertainty in the measurement of each of the two cytochromes. It is evident, however, that the half-reduction potentials of each of the b cytochromes becomes more negative as the pH is made more alkaline. This pH dependence is approximately 60 mV per pH unit from pH 7.0 to pH 8.5 but appears to be less than that from pH 7.0 to pH 6.3. The limitations in the experimental technique make it impossible to decide if the small difference between the pH dependencies of the cytochromes b, and bT are real. Urban and Klingenberg (28) have reported that the half-reduction potential of the succinate reducible 6 cyt,ochrome of submitochondrial particles is pH independent on the acid side of pH 7 but has a dependence of 60 mV per pH unit on the alkaline side of pH 7. It is likely that their measurements refer primarily to cyt,ochrome bK (with some contribution from a b cytochrome with a halfreduction potential of +120 mV (4)). Th,e e$ect of the addition of oxidants for cytochrome cl to anaerobic samples of succinate-cytochrome c reductase. The anomalous behavior of mitochondrial cytochrome b in the presence of antimycin A has been noted by many workers. The addition of succinate to a preparation of the reductase leads to a reduction of the b cytochromes as measured at 565 nm minus 575 nm (Fig. 5). The oxygen consumption by the reductase is extremely slow but phenazine ethosulfate (PES) is

5. Energy conservation in the purified succinate-cytochrome c reductase. The rewas suspended at 3.5 pM cytochrome 61 in a medium containing 59 mM morpholinosulfonate at pH 7.2, placed in a sealed cuvette with an argon atmosphere and the additions made. Abbreviations used are succ (succinate), PES (phenazine ethoy (micro gram), Fum (fumarate) and Ferri (potassium ferricyanide). The addipotassium ferricyanide were made after appropriate equilibration time and after the dual wavelength spectrophotometer to the indicated wavelength pair.

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WILSON

readily reduced by the reductase and reoxidized by oxygen. The addition of 5 pnr PES is adequate to allow the oxygen in the medium to be depleted. This is aided by using the special cuvette used for potentiometric measurements. ,4 stream of ultrapure argon (lessthan 1 ppm 02) is used as the gas phase and oxygen is lost from the medium bot’h by diffusion and by oxygen consumption by the preparation. After the oxygen is exhausted, 20 pg antimycin A is added and then fumarate added to adjust the oxidation-reduction potential to a more positive value. In the experiment in Fig. 5 the potential is +65 mV as measured both by t.he succinate-fumarate ratio and by potentiometric measurement of the phenazine ethosulfate. Ferricyanide addition causesa large reduction of a b cytochrome which is reoxidized when the ferricyanide has been converted to ferrocyanide. The spectrum of t.he ferricyanide-induced absorbance change shows a maximum at 565 nm, a shoulder at 558 nm and a minimum at 553 nm (12, 13). This is the spectrum expected for a reduction of cytochrome b, and an oxidation of cytochrome cl . A similar reduction of cyt,ochrome & can be obtained by using cytochrome c, cytochrome e peroxidase and HzOz or cytochrome c, cytochrome c oxidase and oxygen. In each case the cytochrome bT reduction is essentially complete and continues until the oxidant is exhausted (12, 13). Although the actual experimental data is from the purified succinate-cytochrome c reductase (preparation C, Methods) identical results are obtained using either detergent-treated mitochondria (preparation A) or the succinate-cytochrome c reductase prepared using cholate (preparation B). Thus, the phenomenon is observed not only in intact mitochondria (15, 19) but also in several varieties of detergent-treated preparations derived from mitochondria (see also 12, 13, 16, 18). The time relationship betweenthe oxidation of cytochrome cl and the reduction of cytochromeb, and the magnitude of the shift in the cytochrome b, half-reduction potential. When the ferricyanide is added to the succinatecptochrome c reductase the kinetics of cytochrome cl oxidation as measured at 552 nm minus 575 nm is very similar to the reduction

ET AL.

of cytochrome bT as measured at 565 nm minus 575 (Fig. 5). Because cytochrome bK remains essentially unchanged under these conditions, the actual time relationship can be measuredvery sensitively at a wavelength pair (such as 557 nm minus 575 nm) at which both cytochromcs b, and cl contribute but with opposite signs. The observation that the initial change is in the direction of cytochrome cl oxidation suggests that the oxidation of cytochrome cl precedes the reduction of cytochrome & . After the ferricyanide is exhausted the rereduction of cytochrome cl precedes the reoxidation of cytochrome bT . This is the expected behavior if the reduction of the cytochrome bT requires the activation of electron transport from succinate to cytochrome c1. Measurements at the wavelength pairs which are more specific for cytochrome b, (565-575 nm) show that when the Eh of the succinate-fumarate couple is +65 mV, cytochrome bT is approximately 97 % oxidized prior to the addition of oxidant and becomes at least 95% reduced when the oxidant is present (corresponding to a half-reduction potential of at least + 145 mV). Thus, during an energy conservation cycle the half-reduction potential of cytochrome bT exhibits a shift of at least 175 mV. Another estimate of the shift in half-reduction potential of cytochrome b, may be made from the observation that the rereduction of cytoehrome cl precedes the reoxidation of cytochrome & . This suggests t.hat during the energy conserving cycle the half-reduction potential of the cytochrome b, may become somewhat (lo-20 mV) more positive than that of cytochrome CI . That is, perhaps as high as +255 mV. The reduction of cytochrome b on addition of an oxidant for cytochrome cl as observedin the absenceof antimycin A. Erecinska (19) has reported that the reduction of cytochrome & on addition of an oxidant for cytochrome cl can be observed in uncoupled mitochondria if the temperature of the suspension is lowered to near 0°C. This provides additional evidence that the phenomenon is a function of the energy conservation mechanism at site 2 and is not peculiar to the presence of antimycin A. When the temperature of a sample of succinate-cytochrome c reduc-

SUCCINATE-CYTOCHROME

c REDUCTASE

117

Fig. 7. Addition of 350 PM ferricyanide to an anaerobic suspensionof the reductase causes 35pM Ferria very rapid oxidation of cytochrome cl which is then followed by a reduction of Qio&~~ [email protected]+]@ii,A cytochrome b, . The reduction of cytochrome bT is maximal in approximately 70 msec and I-L1 then is followed by slow reoxidation. -f f [email protected],oclThe electron paramagnetic resonance (epr) p (FIG. 6. The reduction of cytochrome by by the properties of the componentsof the succinateaddition of ferricyanide in the absence of anticytochrome c reductase. Purified succinatemycin A. Purified succinate-cytochrome c reductase was suspended at 1.3 PM cytochrome CI in a cytochrome c reductase is convenient for epr measurements because heme concentra0.05 M Tris-maleate buffer at pH 6.5. The preparations can be used well above those readily tion was placed in the cuvette as described in the attained using submitochondrial particles. legend of Fig. 2, a dilute suspension of yeast cells added and the temperature cooled to 5°C. After In addition, cytochrome oxidase with its anaerobiosis: succinate (4 mM) and fumarate (4 strong epr absorption has been removed. mna) were added to adjust the oxidation-reduction EPR spectra of the purified succinate-cytopotential and then ferricyanide added as indicated. chrome c reductase measured for samples The absorbance scale for the tracings at 566-540 frozen when aerobic or anaerobic at various nm and 560440 nm is given on the left while that oxidation-reduction potentials are shown in for 553-540 nm is given on the right. Fig. 8. The most prominent feature of the spectrum is a strong 4.3g signal which is tase is lowered to 5°C a similar result is obpresent in the aerobic sample. In addition, tained (Fig. 6:). high-spin heme signal at 6g and low-spin The addition of ferricyanide causes a reduction of cytochrome b, as measured at heme signals at near 3g and 2.39 are present. The iron-sulfur epr spectra are well-known 566-540 nm, an oxidation of cytochrome by (8, 29, 30) and this region of the spectra is as measureda,t 560-540 nm and an oxidation not presented here. of cytochrome cl as measuredat 553-540 nm. The signal amplitudes of the various peaks The kinetics of the absorbance change at are plotted as a function of the oxidation566-540 nm is quite complex. Reduction of reduction potential of the sample prior to cytochrome & (lo-15% of total) proceeds freezing in Fig. 9. The 4.39 signal is maximal rapidly, followed by oxidation of 6, to a in the aerobic sample and decreasesin amsteady state (the negative absorbancechange which results from cytochrome bK oxidation is equal to the positive absorbance change which results from cytochrome & reduction). After approximately 30 set cytochrome bT is reoxidized (concomitant with cytochrome cl reduction) and then cytochrome bK is reduced again as the absorbance returns to its initial value. At this wavelength pair the oxidation of cytochrome cl contributes a slight (apprloximately 0.001 A) negative FIG. 7. The rate of reduction of cytochrome bT absorbance change. The rate oj’ the reactions involved in the re- when ferricyanide is added. Purified succinateduction of the b cytochrome. Erecinska et al. cytochrome c reductase was diluted to 1 GM cyto(15) have demonstrated that the reduction chrome CI in a 0.02 M phosphate buffer at pH 7.0. The suspension was made anaerobic by adding 10 of cytochromle b, which occurs when oxidant mg (wet weight) of 8. cerevisiae yeast cells/ml and is added to antimycin A-treated mitochon- 10 mu glucose. After anaerobiosis succinate (1.5 dria is very rapid at 25°C (7 msechalf-time). mM), fumarat,e (15 mM) and 0.1 rg antimycin A/mg A similar experiment using purified succi- reductase protein were added and the experiment nate-cytochrome c reductase is shown in run in a stopped-flow apparatus. 566.54Onm

560-540nm

35pM Ferri

553-540nm

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WILSON

plitude as t,he potential is made more negative corresponding to n = 1.0 and a halfreduction potential of 70 mV. The 1.9g signal of an iron-sulfur protein increases as the potential is made more negative. The n value is 1.0 and the half-reduction potential is 25 mV (seealso 24). The amplitude of the highspin heme signal at 6g increaseson anaerobiosis,remains constant to an oxidation-reduction potential of approximately 10 mV, and decreases with more negative values as expected for a component with an n value of 1.0 and a half-reduction potential of -60 mV. The amplitude of the low-spin heme signal as a function of oxidation-reduction potential is rather complex: the amplitude is small but both the 2.3g and 3g signalsappear to belong to the same heme component.

Aerobtc

t286mV

+175mV

; .

+35mV

: -102nl”

ET AL.

iL 4eroblc

300

200

100

0

-100

E,,CmVl

FIG. 9. The oxidation-reduction potential dependence of some of the epr signals of the purified succinate-cytochrome G reductase. The experimental conditions were the same as for Fig. 8.

Both signals increasein amplitude with more negative potentials in the region from 120 mV to 20 mV and then decreasewith a halfreduction potential of near -70 mV. A signal which may be associated with a low-spin heme is observed at 3.49 but is somewhat masked by the 4.3g signal under these measuring conditions. The 3.4g signal is greatly decreasedat +175 mV and absent by f3.5 mV. A complete oxidation-reduction potential dependence of this signal has not been reliably measured. In the reductase preparation, as in the isolated cytochrome oxidase (31), if a reductive titration is completed and then aliquots taken in an oxidative titration, the measured epr spectra differ somewhat from those of the reductive titration. Unfortunately this lack of complete reversibility meansthat any identification of the epr signalswith a particular component of the respiratory chain must be considered very tentative. DISCUSSION

FIG. 8. The epr spectra of the purified succinate-cytochrome c reduct’ase. The purified succinate-cytochrome c reductase was suspended at approximately 18 PM cytochrome c1 in a 0.1 M phosphate buffer, pH 7.0. The oxidation-reduction mediators used were 30 PM diaminodurene, 24 PM phenazine methosulfate, 24 PM phenazine ethosulfate, 36 PM duroquinone, 30 pM 2-hydroxy naphthoquinone and 10 PM pyooyanine. The sample was made anaerobic by addition of NADH. The oxidation-reduction potential was made more negative with additions of NADH solution and more positive with potassium ferricyanide. Aliquots were anaerobically transferred to epr sample tubes and frozen as previously described (24).

The portion of the respiratory chain which is concerned with the transport of reducing equivalents from succinate to cytochrome c contains many components. These include succinate dehydrogenase with its associated iron-sulfur proteins, at least two b cytochromes (bK and bT), coenzyme Q, cytochrome cl , a high-potential iron-sulfur protein as well as other unidentified compoponents. The 4.3g epr signal belongs to a component which has a half-reduction potential of near +70 mV and could be considered at least a prospective member of the respiratory chain but this is not likely.

SUCCINATE-CYTOCHROME

During the preparation of this manuscript Orme-Johnson et ~2. (32) reported an epr study of the heme components of the cytochrome b-cl region. They observed a seriesof components with signals from near 3.3g to 3.89 which were attributed to the individual cytochromes. In our preparation and at sample temperatures somewhat below those of Orme-Johnson and co-workers (13’ K vs. approximately 8” K) we are unable to resolve the complete oxidation-reduction potential dependence of the signals in the region from 3.4g to 3.8g. Certainly the greater part of the unresolved signal at 3.49 disappears in the potential region expected to reduce cytochrome cl , in agreement with the previous reports (32). In addition, we observe highspin heme signals at 6g and low-spin heme signalsat 3g and 2.3g [not observed by OrmeJohnson et al. (32)] which disappear in the potential region expected for cytochrome bT . Resolution and identification of the individual components will require experiments utilizing both oxidation-reduction potential profiles and complete temperature control in the region from 4.2” K to 13” K. These experiments are in progress. It is interesting to note the increase in the signal intensity of the low-spin heme (2.3g, 3g) which occurs in the potential region of 100-O mV. This transition is observed both in reductive and oxidative titrations and thus is presumably the result of interactions between this heme and a component with a half-reduction potential of +20-50 mV (cytochrome b, ?). Azzi and Santato (33) have reported that the half-reduction potential of cytochrome & becomesmlorepositive with increasing pH. This is apparently due to the failure of the authors to distinguish between an effect causedby substrates providing a more negative oxidationreduction potential and that caused by cgtochrome & acquiring a more positive half-reduction potential. The cytochrome bT h.alf-reduction potential actually becomes more negative (not more positive) as the suspending medium is made more alkaline (seeFig. 4 and Ref. 28). The oxidation-reductioln potential provided by the substrate utilized by the authors (33) (as measured potentiometrically with suitable mediators) becomesmore negative, resulting in an increassedreduction of the cytorhrome

c REDUCTASE

119

b, . This phenomenon has no relationship to the energy dependent half-reduction potential change described by Wilson and Dutton (1, 2, 34, 35). Slater and co-workers (36-38) have published a series of papers in which they describe an energy dependent “red shift” in the spectrum of cytochrome b. They utilize the wavelength pair 560 nm minus 566 nm in their measurements and stat,e that “this wavelength pair is isosbestic for ferri- and ferro-cytochrome b, so that the addition of succinate, NADH or NazSz04, after ascorbate and cyanide, has no effect on A A 566560 nm” (36). They attribute absorbance changes observed using this wavelength pair to shifts in the absorption maximum of the reduced cytochrome b. It is evident from Fig. 1 as well as from the work of Sato et al. (5, 6) and Dutton et al. (25, 39) t.hat this is not the ca,seand the “red shift” is actually due to the oxidation and reduction of at least two b cytochromes with different absorption maxima (for a recent review seeRef. 40). The function of the mitochondrial respiratory chain is to transfer electrons from one oxidation-reduction potential level to a more positive oxidation-reduction potential level with retention of the available free energy in a form suitable to drive the synthesis of adenosine triphosphate. For example, the electrons from succinate are first donated to a group of oxidation-reduction components which undergo a rapid electron exchange to attain roughly equal potentials (cytochrome b coenzyme Q, etc.). Electrons are then tknsferred from this low potential pool to an acceptor which is part of a higher potential pool (cytochromes cl , a, c, copper, etc.). Certainly in coupled mitochondria, and almost certainly in uncoupled mitochondria, this electron transfer must occur through the component responsible for energy transduction at the energy conservation site. Thus the only difference between coupled and uncoupled respiration is the relative rate of transfer of the available free energy from the energy-transducing site to an energy-requiring reaction of interest as compared to its transfer to a reaction in which it is dissipated as heat. Within the context of the chemical coupling hypothesis it would be expect#edthere-

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fore that purified fragments of the respiratory chain, such as the succinate-cytochrome c reductase and cytochrome oxidase, retain their ability to synthesize the primary “highenergy” intermediate. They respond as “uncoupled” preparations, however, because someof the ancillary enzymes (such as ATP synthetase) are removed or damaged. The dependence of the half-reduction potential of cytochromes 6, and a3 of intact mitochondria and phosphorylating submitochondrial particles on the phosphate potential (1, 2, 34, 35) has made available a technique for demonstrating the existence of the primary “high-energy” intermediate in the absence of the additional enzymes required in the conventional assaysfor energy conservation (ATP synthesis, energy-linked transhydrogenase, etc.). Experiments have established that with purified succinate-cytochrome c reductase in the presence of antimycin A, the activation of respiration causesthe half-reduction potential of cytochrome b, to become at least 175 mV and possibly 280 mV more positive. A shift in the half-reduction potent.ial of 175 mV is attained in intact mitochondria by a phosphate potential equivalent to a free energy of ATP hydrolysis greater than - 12 kcal while a shift of 280 mV is the maximum attainable by ATP addition. The presenceof antimycin A is not required for the respiration dependent effect and similar experimental results are obtained by lowering the temperature of the sample to 5°C or below (seealso 19). The transfer of energy away from the primary

.Succinate ATP

ET

AL.

high energy intermediate at site 2 has a higher energy of activation than does the oxidation-reduction reactions which generate it. Lowering the temperature thus inhibits an energy transfer reaction as does antimycin A and both result in an increase in the steady state concentration of the primary high energy intermediate. A schematic representation of the mitochondrial energy transfer pathways is shown in Fig. 10. -REFERENCES 1. WILSON, 2. 3. 4. 5. 6. 7.

D. F., AND DUTTON, P. L. (1970) Biochem. Biophys. Res. Commun. 39,59. CHANCE, B., WILSON, D. F., DUTTON, P. L., AND ERECINSKA, M. (1970) Proc. Nat. Acad. Sci. U.S.A. 66, 1175. SATO, N., WILSON, D. F., AND CHANCE, B. (1971) Fed. Eur. Biochem. Sot. Let. 16, 209. DUTTON, P. L., WILSON, D. F. AND LEE, C. P. (1970) Biochemistry 9, 5077. WIKSTROM, M. K. F. (1971) Biochim. Biophys. Acta 263, 332. SATO, N., WILSON, D. F. AND CHANCE, B. (1971) Biochim. Biophys. Acta 263, 88. RIESKE, J. S., HANSEN, R. E. AND ZAUGG, W. S. (1964) J. Biol. Chem. 239,3017.

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