The Respiratory Chain of Plant Mitochondria

Plant Physiol. (1972) 49, 314-322

The Respiratory Chain of Plant MitochondriaXII. SOME ASPECTS OF THE ENERGY-LINKED REVERSE ELECTRON TRANSPORT FROM THE

CYTOCHROMES c TO THE CYTOCHROMES b IN MUNG BEAN MITOCHONDRIA

Received for publication August 24, 1971

BAYARD T. STOREYJohnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT

The cytochromes c of mung bean (Phaseolus aureus) mito-chondria become reduced when sulfide, a cytochrome oxidaseinhibitor free from uncoupling side effects, is added to theaerobic mitochondrial suspension in the absence of added sub-strate. The cytochromes b remain largely oxidized. Subse-quent addition of ATP results in partial oxidation of the cyto-chromes c and partial reduction of the cytochromes b due toATP-driven reverse electron transport through the second siteof energy conservation, or coupling site, of the respiratorychain. Cytochrome a is also oxidized under these conditions,but there is no concomitant reduction of the flavoprotein com-ponents, of ubiquinone, or of endogenous pyridine nucleotide.The reaction is abolished by oligomycin. The reducing equiva-lents transported from the cytochromes c and a in ATP-drivenreverse electron transport are about 2-fold greater than thosewhich appear in the cytochromes b. It is suggested that theequivalents not accounted for are present in a coupling siteenzyme at the second site of energy conservation which inter-acts with the respiratory chain carriers by means of the dithiol-disulfide couple; this couple would not show absorbancechanges with redox state over the wavelength range examined.With succinate present, reverse electron transport can bedemonstrated at both coupling sites in both the aerobic steadystate and in anaerobiosis. ATP-driven reverse electron trans-port in anaerobiosis maintains cytochrome a 30% oxidizedwhile endogenous pyridine nucleotide is 50% reduced.When mung bean mitochondria, oxidizing succinate in the

presence of sulfide through the alternate, cyanide- and sul-fide-insensitive terminal oxidase, become anaerobic, cyto-chrome b557, which has remained largely oxidized, becomesslowly reduced. The slow reduction is observed in coupled,energized mitochondria and in uncoupled mitochondria; thetime course parallels the reduction of cytochrome a, under thesame conditions. It appears that sulfide-liganded, oxidizedcytochrome a3 may be in close enough proximity to cytochromeb557 in the membrane to inhibit the reduction of the latter.

Electron transport from substrate to oxygen through thecoupled mitochondrial respiratory chain conserves the avail-able free energy in a way which can then be utilized for ATPformation from ADP and P,; the reaction is reversible, in thatinput of free energy in the form of ATP can reverse the flowof electrons through the respiratory chain carriers (7, 11, 26).Reversed electron transport can also utilize directly the high

energy intermediates generated during coupled substrate oxida-tion as was shown some years ago in mammalian mitochondria(5, 8, 12-15, 17, 28) for the energy-linked reduction of endog-enous pyridine nucleotide by succinate. This reaction has re-cently been studied in some detail in plant mitochondria (39,41), using mitochondria from etiolated mung bean (P. aurelis)seedlings, and two paths for the reduction were found. Oneinvolves the first energy conservation site of the respiratorychain and is entirely analogous to the pathway in mammalianmitochondria. The second pathway operates independently ofthe respiratory chain and olh,ains e !valents for reduction ofendogenous NAD from mlI.te derived in turn from the fu-marate formed by succin:-.te oxidation. The second pathway isinoperative in the absence of added Pi, so that it does not in-terfere with studies of the interaction of ATP with the plantrespiratory chain, which are of necessity carried out in the ab-sence of Pi to maintain a high phosphate potential' (17).The reduction of endogenous NAD by succinate in mung

bean mitochondria treated with sulfide and mCLAM2 to in-hibit the two terminal oxidases (31) and utilizing ATP as thefree energy source demonstrates the activity of the first energyconservaticn site between the respiratory chain's NADH de-hydrogenase and the ubiquinone/flavoprotein/cytochrome bregion of the chain. Succinate is required for this reaction; noreduction of NAD occurs with ATP alone, in contrast to thesituation observed in pigeon heart mitochondria (1 1, 17) whereendogenous cytochrome c can provide the reducing equivalentswhen it is in turn reduced in the presence of added sulfide. Theoxidation with added ATP of the cytochromes c reduced inanaerobiosis by endogenous substrate has also been demon-strated in mung bean mitochondria (20); this oxidation wasaccompanied by a concomitant reduction of the cytochromesb.The second energy conservation site in plant mitochondria

has been located between the b cytochromes and the c cyto-chromes by application of the crossover theorem (16. 19) tothe observed changes in redox state of these carriers duringthe state 4 to state 3 transition (4) and during the state 4 touncoupled state transition (43). This lccation is also supportedby measurement of the midpoint potentials of these cyto-chromes (20). The observed oxidation of the cytochromes cand reduction of cytochromes b on addition of ATP to anae-robic mitochondria implies that the redox state of the carriers

1 The phosphate potential is given by the free energy term RTln (ATP)/(ADP)-(P,); as used in this paper, the term high phos-phate potential means that the ratio (ATP)/(ADP) -(Pi) is large.

' Abbreviations: Em7.2: oxidation/reduction midpoint potentialmeasured at pH 7.2; mCLAM; m-chlorobenzhydroxamic acid;1799: bis (hexafluoroacetonyl acetone).

314

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RESPIRATORY CHAIN OF MITOCHONDRIA. XII

at the second coupling site can equilibrate with the phosphatepotential with relatively little interference from the first cou-pling site. This reaction could thus provide an additional probefor studying the second coupling site and, hopefully, clarifythe role of the three cytochromes b. This paper reports a studyof the reverse electron transport reaction between the c and bcytochromes with ATP as the source of energy.

MATERIALS AND METHODS

Mitochondria were prepared from the hypocotyls of 5- or6-day-old etiolated seedlings of mung bean (Phaseolus aureus),using substantially the method described by Bonner (3) andIkuma and Bonner (24), with the modifications of Storey andBahr (42). The mitochondria were assayed for respiratory con-trol in a medium containing 0.3 M mannitol, 10 mM TES, 5ms P1 and brought to pH 7.2 with KOH. This medium isdesignated TP; the same medium without phosphate is desig-nated T. All experiments in this paper were carried out inmedium T, unless otherwise specifically noted. Oxygen con-sumption by the mitochondrial suspension with succinate ormalate as substrate was measured in a closed cuvette with aClark electrode (Yellow Springs Instrument Co.) as describedby Estabrook (23). Mitochondrial protein content was deter-mined by a modified Lowry method (29).

Adenine nucleotides and NADH were obtained from Boeh-ringer Mannheim Corp.; succinic acid from Aldrich Co.; so-dium sulfide, potassium cyanide, and mannitol from J. T.Baker Chemical Co. These were used without further purifi-cation. The uncoupler 1799 was generously supplied by Dr.Peter Heytler of E. I. du Pont de Nemours Co., and a sampleof mCLAM was generously provided by Dr. Gregory Schon-baum of the University of Alberta.Absorbance changes corresponding to the reduction or oxi-

dation of the respiratory chain carriers were recorded using adual wavelength spectrophotometer (6) with a compensationcircuit to reduce noise from light source fluctuations (18). Thefollowing wavelength pairs were used for the various carriers:468 to 490 nm for flavoprotein; 282 to 295 nm for ubiquinone;549 to 540 nm for cM7; 556 to 540 nm for b.; 560 to 540 nmfor b.7; and 565 or 566 to 540 nm for b.. The subscripts givethe reduced minus oxidized difference absorbance maxima forthe cytochromes in spectra obtained at 77 K (32).

Fluorescence changes corresponding to the oxidation andreduction of low potential, fluorescent flavoprotein Fp,, (36,40) were recorded by an Eppendorf fluorimeter equipped withan Heraeus (Hanau) mercury arc No. ST-75 as described inearlier papers (21, 40, 42). For this work, the primary filterfor the excitation light was an interference filter with maximaltransmittance at 463 nm with 10-nm band width; the trans-mittance of the secondary filter for the emitted light lay be-tween 490 and 3000 nm.

Difference spectra of the mitochondrial suspensions wereobtained at liquid nitrogen temperature with the split beamspectrophotometer described by Chance (6), using the tech-nique developed by Estabrook (22) as subsequently modifiedby Bonner (1).

RESULTS

The cytochromes c and cytochrome a in aerobic mung beanmitochondria become partially reduced upon treatment withcyanide (37) or sulfide, the reducing equivalents coming fromendogenous substrate. There is very little reduction of thecytochromes b or of flavoprotein under these conditions.Coupled mitochondria with cytochromes c partially reducedand with cytochromes b essentially all oxidized are a conve-

Cytochrome c 549-540nmM.B.M. 43mg Protein/mlt ImM mCLAM 240

AAbs= IxIO~2

10.25mMNozS

0.75mM-5ml- c Red.NADH Succ A

4 1799

---I Incr Abs.549nm

60sec

B I-LAAbs= I1x10-2

t j~~~O~M 0.7mM 5mMc

1.2m~M 1799__NADH Sc

0.25mMATII

-1 -~~~H

60sec

C -I

AAbs= I x10-2

t

0.25mM

IO,MO.75mmM_I 1.2mM 1799 N DHScI I~~~______I

Oligomycin0.5,Lg/mg Prot

*60 sec

c Red.

Incr Abs.549nm

c Red

Incr. Abs.549nm

FIG. 1. Changes in redox state of cytochrome cu7 in mungbean mitochondria as monitored at 549 to 540 nm with the dualwavelength spectrophotometer. A: Reduction of CU7 by endogenoussubstrate on addition of sulfide, followed by oxidation upon addi-tion of ATP due to energy-linked reversed electron transport. Ad-dition of the uncoupler 1799 abolishes the energy-linked reactionand the cytochrome becomes reduced. Addition of NADH and suc-cinate gives full reduction of C547, and serves to quantitate the totalamount in the mitochondrial suspension. B: The same reactionconditions are employed as in A, but cyanide is substituted for sul-fide. C: The same reaction conditions as in A, but oligomycin isadded prior to ATP.

nient system for examining reverse electron transport, since itis not necessary to work under anaerobic conditions as wasdone in the experiments previously reported (20). Oxidationof cytochrome C,M7 by reverse electron transport utilizing freeenergy from ATP is shown in Figure 1A. Addition of sulfide tomung bean mitochondria suspended in medium free of P, andtreated with mCLAM to block the alternate, cyanide-insensi-tive oxidase (31) results in 50% reduction of the cytochromec. Addition of ATP causes some 60% oxidation of the cyto-chrome, and this effect is reversed by addition of the uncou-pler 1799. Reduction is complete on the addition of NADH

Plant Physiol. Vol. 49, 1972 315

A

r



Plant Physiol. Vol. 49, 1972

and succinate. The extent of reduction attained in the presenceof ATP and 1799 but absence of substrate is 76%, comparedto the 60% observed in energy-depleted mitochondria (37).The use of cyanide (Fig. 1B) gives results similar to those ob-tained with sulfide (Fig. 1A), but the extent of cytochromeoxidation on addition of ATP is less, and the reduction onaddition of uncoupler is more rapid and shows a slight over-shoot. These differences are consistent with the known un-coupling effects of cyanide (25). For this reason, sulfide, knownto have no uncoupling effects on pigeon heart mitochondria(17) and apparently also free of such effects on mung beanmitochondria, was used as the inhibitor for cytochrome oxi-dase throughout this study.The oxidation of cytochrome c-7 by added ATP is in-

hibited by oligomycin, as shown in Figure 1C. In fact, isola-tion of the respiratory chain from intramitochondrial ATP byoligomycin results in a further reduction of the cytochrome inthe presence of sulfide; addition of ATP and 1799 have littlefurther effect.The reduction of the cytochromes b accompanying the oxi-

dation of the cytochromes c appears in the four experimentalrecords of Figure 2, which were all obtained with the samemitochondrial preparation. Reduction of cytochrome Ce7 onaddition of sulfide and its oxidation on addition of ATP areshown in Figure 2A, with the use of the appropriate wave-length pair 549 to 540 nm. Cytochrome cz,9 behaves identicallyunder these experimental conditions. The same experiment isrepeated in Figure 2B with the wavelength pair 556 to 540nm) which records the redox changes of b5,3, but which alsorecords in part the redox changes of cM9: the slight increasein reduction on addition of ATP corresponds to b5. reductionwhich leads to an increase in absorbance at 556 nm and toc,49 oxidation which leads to a decrease. The total absorbancechange at this wavelength pair attributable to b5. is about twotimes that attributable to cf,9 (20); the resulting absorbancechange observed is a small net increase in absorbance at 556nm and virtually no change on addition of uncoupler. Thereis little interference from cytochrome cs49 with the absorbancechanges recorded at 560 to 540 nm, the wavelength pair

suitable for monitoring the redox state of cytochrome b557. Adefinite reduction of this cytochrome is evident on ATP addi-tion (Fig. 2C), which is reversed by uncoupler. The wave-length pair 566 to 540 records changes in the redox state ofcytochrome b562 with some interference from b5-5. The experi-ment using this wavelength pair is shown in Figure 2D. Addi-tion of ATP does give some reduction of b52, but the slowphase seen with b,57 (Fig. 2C) is missing. Uncoupler causesrapid reoxidation, and subsequent succinate addition causeslittle further reduction of this cytochrome (Fig. 2D).

Addition of succinate to the mung bean mitochondria afteruncoupler results in essentially complete reduction of thecytochromes c and b . (Fig. 2, A and B). But the reduction ofb557 by succinate is somewhat slower and incomplete (Fig. 2C)when interference from b.. with the absorbance changes at 560to 540 nm is taken into account. There is very little reductionof b562 by succinate under these conditions (Fig. 2D). Theseresults are in good agreement with those obtained usingenergy-depleted mung bean mitochondria with cyanide as in-hibitor of cytochrome oxidase and exogenous NADH as thesource of reducing equivalents (37).A difference spectrum obtained at room temperature show-

ing the effect of adding sulfide to mung bean mitochondriasuspended in medium T is shown in Figure 3A. The two cyto-chromes c and cytochrome a become reduced, yielding dif-ference absorbance maxima at 550 and 600 nm. The presenceof sulfide shifts the latter maximum 1 to 2 nm to shorterwavelengths; as pointed out by Chance et al. (9), all plantmitochondria examined so far have the reduced-minus-oxi-dized maximum for cytochrome a at 601 to 603 nm ratherthan at 605 nm as observed with mammalian mitochondria.There is a difference absorbance maximum at 482 nm whichis partially attributable to the cytochromes, but must alsocontain some contribution from some other component, possi-bly an iron-sulfur protein. The minimum is not at the properposition for flavoprotein (10). Addition of ATP to the sulfide-containing suspension reduces the amplitude of the maximaattributable to cytochromes c and a, corresponding to oxida-tion, and a new absorbance band at 562 nm appears, corre-

M.B.M.-2.Omg Protein/ml.+0.7mM mCLAM 250C

A C547: 549-540nmlOmM T

j ~Succ

= ATP 1_799

0.36mM - -4-

N2S_ L Cyto.-4 -.---c:------------------ t Abs 4x10e-3

=+60 s -5

b553 556 - 540nm 0

__ .__ fIncrIOmM AbsSucc vs

HImM 14FM 1-- 540nmATP _1799, 4

0.36mM_

60/ ' ~LAAbs= 4xI0-3

- 0 C s I :ec

b557: 560-540nmI T~~~lmtk1 ,>

=L,=,~~~-Sc

bi6 I6-4n1.m 14, M

1.1mM 14E,M tiAbs=2xjO-30.36mM -ATP 1799 --No2S.. -m

sec

FIG. 2. Comparison of redox changes in cytochromes C547, b553, b557, and b56G on initiation of reversed electron transport by addition of ATPto sulfide-treated mung bean mitochondria. The same mitochondrial suspension was used in all four experiments. Note that the records C and Dwere obtained at twice the recording sensitivity as those of A and B.

B

316 STOREY

c




sponding to reduction of the cytochromes b (Fig. 3A). Thereis some change in the shape of the absorbance minimum at481 to 482 nm, but little change in amplitude. The same dif-ference spectrum obtained at -196 C is shown in Figure 3B.All three cytochromes b are partially reduced, with b.,2 barelydetectable, as expected from the experimental records of Fig-ure 2 obtained with the dual wavelength spectrophotometer.One peculiarity of the reversed electron transport from the

cytochromes c to the cytochromes b is that rather less b, andboo. are reduced than would be expected from the oxidation ofC.7 and c59 on the basis of absorbance changes. This is evidentfrom the records of Figure 2. The absorbance change observedon adding uncoupler to the ATP-treated mitochondria-whichcorresponds to the full difference of coupled minus uncoupled-is 4.3 X 10-' at 549 to 540 nm (Fig. 2A), while an estimatethe sum of the absorbance changes attributable to the b cyto-chromes in the other three records is about 2.8 X 10-. It iseven more evident from Figure 3A, where some 0.018 ab-sorbance units of the cytochromes c at 550 nm and of cyto-chrome a at 600 nm disappear on addition of ATP, to be re-placed by 0.0052 unit of the cytochromes b at 562 nm. It isdifficult to quantitate these changes because the extinctioncoefficients of the various cytochromes at their difference ab-sorbance maxima are not known with certainty (27). If oneassumes that all the extinction coefficients are approximatelythe same, as was done in a previous kinetic study with quitereasonable results (32), one finds that about half the equiva-lents lost by the cytochromes c and cytochrome a on additionof ATP appear in the cytochromes b.An attempt to search for the remainder of these equivalents

in ubiquinone or flavoprotein is shown in the experiments ofFigure 4. Because of the high intrinsic absorbance of ATP inthe ultraviolet region of the spectrum where ubiquinone ismeasured, the order of addition of sulfide and ATP was re-versed in the experiments of Figure 4 compared to those ofFigure 2, the ATP being added first in the former. Additionof sulfide to a suspension of mitochondria in medium T con-taining ATP (Fig. 4A) gives a transient absorbance changewith the wavelength pair 282 to 295 nm; subsequent additionof uncoupler has no effect. Addition of succinate causes anabsorbance decrease at 282 nm, corresponding to completeubiquinone reduction, as determined from separate controlexperiments. The response of cytochrome cM4 in the samemitochondrial preparation to the same series of additions isalso shown in Figure 4A, in the record directly below that ofthe ubiquinone responses. In the presence of ATP, addition ofsulfide produces relatively little reduction of the cytochrome:the reduction occurs on addition of uncoupler, showing thatenergy-linked reversed electron transport is maintaining thecytochrome oxidized. This order of addition does not changethe response of the mitochondria to the combination ofsulfide + ATP; only the initial phase of reduction seen inFigure 2A on addition of sulfide is missing.

The flavoprotein responses to ATP in the presence of sulfideare shown in Figure 4B together with the cytochrome c,,4, re-sponse obtained from the same mitochondrial preparationunder the same experimental conditions. It is evident fromthe higher degree of C547 oxidation maintained in the presenceof ATP + sulfide than that maintained in the presence ofthese reagents plus uncoupler that reversed electron transportfrom the cytochromes c to the cytochromes b is occurring.It is also evident from the absorbance and fluorescencechanges diagnostic of the redox state of the flavoproteins thatelectron transport into or out of these components cannot bedetected with any certainty. There is virtually no change inflavoprotein fluorescence. The increase in absorbance at 468

24°C

520

1Azs=00L1

T,547

-1960 C

450 500 550 600 650X (nm)

FIG. 3. A: Difference spectra of mung bean mitochondria ob-tained at room temperature. The dashed line is the spectrum ob-tained on adding sulfide to the aerobic mitochondrial suspension;the solid line is the spectrum obtained on subsequent addition ofATP. The aerobic mitochondrial suspension contained 12 mg pro-tein/ml plus 1 mM mCLAM; this was the reference sample. Sul-fide was added to the measuring cuvette at 0.5 mm and the differ-ence spectrum obtained. ATP was then subsequently added to themeasuring cuvette, and the second spectrum was obtained. B:Difference spectrum at liquid nitrogen temperature correspondingto the solid line spectrum in A. The aerobic mitochondrial sus-pension contained 20 mg protein/ml and 1 mM mCLAM; thisserved as reference. The measuring cuvette contained this suspen-sion plus 0.5 mM sulfide and 1 mM ATP. The optical path lengthwas 1.0 cm in A and 0.2 cm in B.

nm on addition of sulfide to the mitochondrial suspension isdue to the 482-nm component observed in Figure 3A. Thesame change is also observed on addition of cyanide. Fromprevious work (36, 37), the absorbance decrease at 468 nm onaddition of succinate can be ascribed primarily to Fpha. Thisset of experiments shows that electron transport from thecytochromes c to the cytochromes b does not involve theflavoproteins or ubiquinone. Those equivalents from the cyto-chromes c which are not found in the cytochromes b must bein some component with an absorbance spectrum that doesnot change detectably on change of redox state.

If the order of succinate addition is changed such that thissubstrate is added directly to sulfide-treated mitochondria, theeffect on the cytochromes b is that shown in Figure 5. Cyto-chrome b,-;, is rapidly reduced (Fig. SA). The subsequent addi-tion of ATP produces a very small increase in reduction whichis uncoupler sensitive. By comparison with Figure SB, thischange can be attributed in large part to cytochrome b...which shows the same time course for the change, but a much





M.B.M. 2.4mg Protein/ml0.8mM mCLAM + 0.7mM ATP

lB

Ubiquinone 282-295nm

UQRed.

Cytochrome C547 549-540nm

L\ Abs =4x 1-

OAMNo2S 14MM 1799

MM, S=<IC Red

A Abs = 2

T

M.B.M. 1.5mg Protein/ml0.8mM mCLAM

Flovoprotein

FpRed.

FpRed.

Cyt.cRed.

Cytochrome C547 549-540nm

.- 4.LM I799

0.9mANT 2S

FIG. 4. A: Effect of reverse electron transport from the cytochromes c on the redox state of ubiquinone. The upper record shows the effecton ubiquinone of adding: first sulfide, then the uncoupler 1799 to the mitochondrial suspension containing ATP added 2 min prior to the startof the experiment. The downward deflection observed on addition of succinate corresponds to ubiquinone reduction; the extent of the deflectionis equal to the total change from oxidized to reduced for ubiquinone as determined separately. Note that these experiments were carried out inMedium T which contains no Pi, so that interference from pyridine nucleotide reduction is avoided. The parallel experiment for cytochromeC547 with the same mitochondrial suspension is shown in the lower record. B. Effect of reverse electron transport from the cytochromes c on theredox state of flavoprotein. The upper record shows the effect on flavoprotein of adding sulfide, followed by ATP, 1799, and succinate in thatorder. The reaction conditions are identical to that in A. The parallel experiment for cytochrome C,3-7 with the same mitochondrial suspension isshown in the lower record.

larger absorbance change at 560 to 540 nm than at 556 to540 nm. Conversely, the rapid reduction seen at 560 to 540nm (Fig. SB) on the addition of succinate is associated withthe same time course but a smaller absorbance change thanthat seen at 556 to 540 nm; the rapid reaction can be at-tributed in large part to cytochrome b5... There is a very

small, slower absorbance change observed at 565 to 540 nm

on addition of succinate to sulfide-treated mitochondria (Fig.SC). At this w-avelength pair, both b- 2 and b257 are recorded,and this absorbance change and time course, by comparisonwith those shown in Figure SB, can be attributed to b,,; thereis no reduction of b,2 under these conditions. This is the re-

sult to be expected on the basis of the different midpoint po-

tentials of the cytochromes b (20). Comparison of the rela-tively large and quantitatively similar absorbance change inFigures SB and 5C on addition of ATP shows that both b,,57and b7 2 are reduced by reversed electron transport with suc-

cinate present. This reduction is sensitive to uncoupler, as

shown by the reoxidation observed on addition of 1799.The records of Figure 5 were obtained with a mitochondrial

suspension of high enough protein concentration that anaero-

biosis was achieved in a relatively short time on addition ofNADH. even with both mCLAM and sulfide present to in-hibit both terminal oxidases. At the far right side of each ofthese three records, after an extended steady state, a slow in-crease in absorbance relative to 540 nm is observed. Previouswork (37) with energy-depleted mung bean mitochondria hasdemonstrated that this change is due primarily to the slowreduction of cytochome b57, in anaerobiosis in the presence

of either cyanide or sulfide. This slow reduction occurs in thepresence of high substrate concentrations and thus representsa different phenomenon from the slow reduction of cyto-chrome b,,7 observed in oxygen pulse experiments which arecarried out with succinate as substrate at high concentrationsof malonate (32). This behavior is somewhat puzzling, sincethere is no evidence for a specific inhibition by cyanide orsulfide of the reduction of this cytochrome; its midpoint po-tential Em,.2 = +42 mv is close enough to that of cytochromeb.. with Em,7.2 = +75 mv (20) that no qualitative difference inbehavior would be expected. Since the previous experimentshad been carried out with energy-depleted mung bean mito-chondria, it was necessary to examine the reduction rate of b....in coupled mitochondria in the presence of sulfide to ascertainthe effect of energization. The records from this set of experi-ments are shown in Figure 6. Addition of sulfide to the mito-chondrial suspension results in partial reduction of cytochromea and cytochrome c517 (Fig. 6, A and D), with little effect oncytochromes b. and b-,2 (Fig. 6, B and C). Addition of ATPcauses oxidation of cytochromes a and c,7 with concomitantreduction of b,,,7 and b7.- demonstrating that the mitochondriaare indeed coupled and capable of reversed electron transportat the second site of energy conservation. Addition of suc-cinate causes reduction of all components (with a slight over-shoot) to their redox levels characteristic of the coupled en-ergized aerobic steady state with inhibited cytochrome oxidase.Oxygen is consumed via the alternate, sulfide-insensitive oxi-dase pathway, and the suspension becomes anaerobic. Cyto-chrome a, then becomes slowly reduced, as shown in Figure

318 STOREY




M.BM. 6.0mg Protein/ml + 0.67mM mCLAM240C

aX Cyt. b553 556-540nm

13mM ATP 0, I-D

b553 Red

I< t m : hAbs=0.004 < ncr Abs

-I 556nm

b567 Red

Incr. Abs.560nmn

¢ Cyt. b562 565-540nm

bw Red

Incr Abs.565rm

FIG. 5. Reverse electron transport to cytochrome b553 (A), cytochrome b557 (B), and b562 (C) driven by ATP in the presence of added succinate.

6A; the process requires about 4 min, in general agreementwith previous results obtained with cyanide as inhibitor (35).Comparison of the records of Figures 6B and 6A shows thatreduction in anaerobiosis of cytochrome bM7 is also slow andfollows nearly the same time course as the reduction of cyto-chrome a3. The same is true of b.2 (Fig. 6C), but the smallerabsorbance change indicates only partial reduction when in-terference at 566 to 540 nm from bM, is taken into account.Cytochrome C547 attains nearly its full degree of reduction inthe aerobic steady state (Fig. 6D), and little further reductionoccurs on anaerobiosis. These results with coupled mung beanmitochondria, held at high phosphate potential by added ATPin the absence of P, and supplied plentifully with substrate,demonstrate that the slow rate of reduction of cytochromeb,,- in the presence of sulfide or cyanide is not due to an en-ergy-linked process: it is also observed in energized, coupledmitochondria.The set of experiments parallel to those of Figure 6, with

uncoupler added prior to succinate, are presented in Figure7. The same mitochondrial preparation was used in both setsof experiments. Comparison of the records of Figures 7A and7B again shows that b, is reduced in anaerobiosis with nearlythe same time course as cytochrome a3. With uncoupler pres-ent, less b^,, is reduced in the aerobic steady state with suc-cinate (Fig. 7B) than in the energized coupled state (Fig. 6B),the difference being attributable to reversed electron trans-port driven by ATP in the latter case. With uncoupler pres-ent, there is at best only a partial reduction of cytochromeb., as shown by the smaller upward deflection of the trace

seen in Figure 7C compared to Figure 6C. Cytochrome C547 iscompletely reduced by succinate in the uncoupled aerobicsteady state (Fig. 7D); no further reduction is observed inanaerobiosis.Comparison of Figures 6A and 6D with Figures 7A and

7D allows one to assess the extent to which ATP-driven re-verse electron transport operates from the cytochromes c andcytochrome a with succinate present. The operation of thisreversed electron transport in both the aerobic steady stateand in anaerobiosis is particularly well illustrated by com-parison of the records of Figures 6A and 7A. The magnitudeof the slow absorbance change corresponding to cytochromea3 reduction in anaerobiosis is the same in both cases, as ex-pected, but the over-all change observed with the energizedmitochondria is less. Cytochrome a remains partially oxidizedin the presence of ATP in anaerobiosis as well as in theaerobic steady state; the extent of oxidation is about 30% ascalculated from these records. The same is true of cytochromeC57 (compare Figs. 6D and 7D), but the extent of oxidation inthe energized state is less than that of cytochrome a, as wouldbe expected from the more positive midpoint potential of C,7(20). It has been shown (41) that, under the conditions of theexperiment of Figure 6, endogenous pyridine nucleotide isabout 15% reduced in the aerobic steady state and becomes50% reduced in anaerobiosis. Endogenous pyridine nucleotide,therefore, acts as the ultimate acceptor for reducing equiva-lents in reversed electron transport driven by ATP, but cyto-chromes bM- and b2 also maintain a more reduced steadystate (Fig. 5). With the first and second sites of energy con-





M. B.M - 1.9mg Protein/ml - 25°Ca a3 445 - 455nm

2min --A~Abs=l XIO2- ~~~~- - h~~~Abs 4 x 10-7 3_- T

__5b557 560- 540nrn2mn'__

B _ .-Ab=x0

__ b562 566-540nm__ 2mi n' AAs4x

.C _.-- t:I. -- -

IE1M ATP036mM .---OmM Succinate tNo2S __IAs Abs 4 x10-3

_- -_- 1. c54- 549-540nm

-2minm

Cyt. Red.

conditions.) Further, the state of 50% reduction is obtainedwith ATP and succinate when mCLAM is also present inthe sulfide-blocked system (41). This is further evidence thatonly the first energy conservation is operative in plant mito-chondria treated with cyanide or sulfide which oxidize sub-strate through the alternate oxidase.

DISCUSSION

The demonstration of ATP-driven reverse electron trans-port between the cytochromes c and the cytochromes b ofmung bean mitochondria, added to the evidence from thecrossover experiments (4) and potential measurements (20)cited earlier, confirms the location of the second energy con-servation site between these respiratory chain carriers. Thepattern of the reverse electron transport helps to resolve some

of the fine structure of this site. The high potential carriers in-volved are cytochromes C.,, C;j49, and a, which all respond in

like manner to energization with ATP and de-energizationwith uncoupler. Cytochrome a is also the low potential car-

rier of the third coupling site. This is in accord with theirmeasured midpoint potentials: Em7.2 =+235 mv for the twoc cytochromes and +190 mv for cytochrome a. CytochromeC.47 is the c cytochrome extractable with salt, while c.54 re-

mains bound to the mitochondrial membrane (2, 27). Cyto-

M.B.M - 1.9mg Protein/mI - 25°C

incr. Abs.Rel. To

540nm Or455nm

FIG. 6. Reverse electron transport from cytochromes a (A) andcU7 (D) to cytochromes bw (B) and bw2 (C) driven by ATP in thepresence of sulfide but absence of mCLAM, followed by succinateaddition to give the aerobic steady state of succinate oxidation whichis, in turn, followed by transition to anaerobiosis, with the mitochon-dria in the coupled, energized state. Oxygen consumption is throughthe alternate, sulfide-insensitive oxidase. The order of addition issulfide, ATP, and succinate as indicated in the record of D.

servation participating under these experimental conditions,it is possible to keep cytochrome a (Em7.2 =+ 190 mv) par-tially oxidized and endogenous pyridine nucleotide (EB,11.2 =-320 mv) partially reduced with ATP as the source of freeenergy.

In the experiments of Figures 1 and 2, the reversed elec-tron transport reactions were carried out in the presence ofmCLANM to block the cyanide- or sulfide-insensitive alternateoxidase. In the experiments of Figures 6 and 7, no mCLAMwas present and exactly the same results were obtained. Thereducing equivalents transported from the cytochromes c tothe cytochromes b do not have access to the alternate oxidase,in accord with the observation that they do not have accessto the flavoproteins associated with the cytochromes b. Thesame is true for cytochromes b even when succinate is presentin addition to ATP, as shown in Figure 5. With succinatepresent, however, reducing equivalents are transported toendogenous NAD by reverse electron transport, and these dohave access to the alternate oxidase, since the endogenous NADis but 15% reduced in the aerobic steady state, but 50% re-duced in anaerobiosis, as mentioned above. (The transition tothe more reduced state occurs far more rapidly for the NADthan does the same transition for cytochrome b.. under these

003 445-455nm

_- - -

-Abs=IxIO2

b557 560-540nm

_-__-Ab =

__t _ AAbs 4xlO 3

__

---_K

-

b562 566-540nm

c_~~~

1-_, 4. --AAbs = 4 x 10-3 -

;

Cyt.Red.t

Incr. AbsRel To540nm

Or455nm

C547 549 -540nm

-/ -t~-lOmM -- tAbs=4xlO-3---- - Succo

-+ lmM-___-- T -ATP -

0.36mM 14E1M 60secNo2S 1799

FIG. 7. Parallel experiment to that of Figure 6, but with addi-tion of the uncoupler 1799 after addition of ATP but prior tosuccinate. The order of addition and time scale for all four recordsis indicated in the record of D. The same mitochondrial preparationwas used under the same experimental condition as in Fig. 6.

320 STOREY




chrome c,4, corresponds, therefore, to mammalian cytochromec, while CMS corresponds to mammalian c1. Wohlrab (44) hasdemonstrated that cytochrome c is required for the cytochromea pool to equilibrate rapidly with itself and with cytochromec1; by analogy, c,H7 is assigned this role in plant mitochondria.This, in turn, places cytochrome CHS as the high potential car-rier acting directly at the second energy conservation site.Cytochrome Cs7 maintains this cytochrome and cytochrome a,the low potential carrier of the third site, essentially in equilib-rium.The low potential part of the second energy conservation or

coupling site comprises the cytochromes b. The pattern ofATP-driven reverse electron transport indicates that all threecytochromes can accept electrons from the high potentialcarriers. but that bw, as expected from its low midpoint po-tential (Em7.2 = -77 mv) (20), is reduced to a very limitedextent. The results of the experiments reported here, added tothe kinetic measurements of the oxidation of the b cyto-chromes (32), are compatible with the placement of bothb53 and b,7 as the low potential carriers acting directly at thesecond energy conservation site. It is suggested tentativelythat reducing equivalents from NAD-linked substrates passthrough the first coupling site and reach the second sitethrough cytochrome by,2 to b,,, while reducing equivalentsfrom succinate or exogenous NADH reach cytochrome b5through ubiquinone and Fp,. (37). Both cytochromes b, andboo can be oxidized via the coupling site by the cytochromesc; the extent with which they actually interact with each otheris still problematical.When reverse electron transport is induced by ATP in

mung bean mitochondria treated with sulfide but without addedsubstrate (Figs. 1-3), more reducing equivalents are takenfrom the cytochromes c547, C.9, and a, all acting as a singlepool, than appear in the cytochromes b by a factor of 2 orso. As pointed out above, these equivalents do not appear ineither flavoprotein or ubiquinone. If energy conservation is tooccur between the cytochromes b and cytochromes c, theremust be some sort of coupling site enzyme which can trans-form the free energy of the oxidation/reduction reactions me-diating electron transport into the free energy of an acid an-

hydride eventually convertible to ATP. This problem has beenanalyzed in detail (33, 38), and the chemical hypothesis de-rived from this analysis predicts that each coupling site en-zyme should partake in the electron transport process by meansof the dithiol-disulfide redox couple. In order to function, thecoupling site enzyme must exist as two species, one of lowenergy and one of high energy. The hypothesis predicts thatthe low energy species has the lower midpoint potential andundergoes redox reactions with low potential carriers of thecoupling site; the high energy species has the higher midpointpotential and interacts with the high potential carriers. It isthe oxidized disulfide form of the high energy species whichconserves the free energy and makes it available for energy-linked reactions and ATP synthesis; it is also this oxidizedform which, at the second site, reacts with reduced cytochromec to become reduced to the dithiol, thereby oxidizing twomolecules of the cytochrome. The reduced forms of the twospecies are freely interconvertible, thereby mediating reverse

electron flow. In summary, the hypothesis predicts that ener-gization of the coupling site enzyme, as has been done withATP in these experiments, will produce the oxidized energizedform which is the direct oxidant for cytochrome c. The ener-gized state is characterized, therefore, by oxidation of thecytochromes c and reduction of both the cytochromes b andthe coupling site enzyme. The latter would not show any ab-sorbance change over the wavelength range currently available

for mitochondrial studies. The amount of coupling site en-zyme at the second site can be estimated from the cytochromecontent (27) and the apparently lost equivalents to be about0.1 to 0.2 nmole/mg protein. While these experiments scarcelyprove the existence of such a coupling site enzyme in mungbean mitochondria, they do suggest evidence for it.

The slow reduction of cytochromes b, and b5. which isobserved in the presence of cyanide or sulfide appears to cor-relate with the redox state of cytochrome a3 in both thecoupled energized (Fig. 6) and uncoupled states (Fig. 7). Inthe coupled energized state, the situation is somewhat com-plicated by ATP interaction at both the first and secondcoupling sites, which maintains pyridine nucleotide partiallyreduced and cytochrome a partially oxidized; part of the re-duction of the cytochromes b is due to this reverse electrontransport. In the uncoupled state, only about 30% of cyto-chrome b, is reduced in the aerobic steady state, and the restbecomes reduced in the slow, somewhat biphasic reaction seenin the record of Figure 7B. The apparent ability of cytochromeb, to "sense" the redox state of cyanide- or sulfide-ligandedcytochrome a3 suggests that these two carriers are in closeproximity in the membrane and that oxidized cytochrome a3,with sulfide or cyanide bound to the inhibitory site of thatcarrier, can somehow inhibit the reduction of b,*. The idea ofa close interaction between cytochrome a3 and the cyto-chromes b of the respiratory chain is an explicit part of thechemical hypothesis for energy conservation in the mito-chondrial membrane (33, 38). The hypothesis points out thata fourth site of energy conservation may operate betweencytochrome a3 and oxygen; experimental support for this ideahas been put forward by Muraoko and Slater (30). This siteis postulated to be thermodynamically coupled to the secondand third coupling sites by utilizing the free energy of thereaction to bind the water molecules generated at the couplingsites during the energy-conserving reaction accompanyingelectron transport. (Ref. 38 gives full details of this mecha-nism.) Such a formulation requires close physical proximityof cytochrome a3 to the second coupling site, as well as to thethird coupling site of which it is a part. To the extent that theforegoing suggestion of inhibition of b.sn reduction by sulfide-liganded, oxidized a3 has any validity, this observation may beinterpreted as evidence for such proximity.

Acknowledgments-The author wishes to thank 'Mrs. Dorothy Rivers for skill,patience, and ingenuity in the preparation of mitochondria and other technicalassistance. He is indebted to Dr. John R. Williamson for the use of an Eppendorffluorimeter and to 'Mr. Norman Graham for technical expertise in setting up thecompensated dual wavelength spectrophotometer. This work was supported byUnited States Public Health Service Grant GM-12202 and National ScienceFoundation Grant NSF-GR-23063 and was carried out during the tenure ofUnited States Pubhc Health Service Career Development Award K3-GMI-7311.

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The Respiratory Chain of Plant Mitochondria