respiratory chain mitochondria. i. electron …plant physiol. (1969) 44, 115-125 the respiratory...

Plant Physiol. (1969) 44, 115-125

The Respiratory Chain of Plant Mitochondria. I. Electron TransportBetween Succinate and Oxygen in Skunk Cabbage Mitochondria

Bayard T. Storey and James T. BahrJohnson Research Foundation, University of Pennsvlvania, Philadelphia. Pennsylvania 19104

Received September 18, 1968.

A bstract. The kinetics of oxidation of ubiquinone, flavoprotein, cytochrome c, and thecytochrome b complex in skunk cabbage (Symplocarpus foetidus) mitochondria made anaerobicwith succinate have been measured spectrophotometrically and fluorimetrically in the absenceof respiratory inhibitor and in the presence of cyanide or antimycin A. No component identi-fiab,le by these means was oxidized rapidly enough in the presence of one or the other inhibitorto qualify for the role of alternate oxidase. Cycles of oxidation and rereduction of flavoproteinand ubiquinone obtained by injecting 12 jM oxygen into the anaerobic mitochondrial suspensionwere kinetically indistinguishable in the presence of cyanide or antimycin A, implying thatthese 2 components are part of a respiratory pathwav between succinate and oxygen whichdoes not involve the cytochromes and does involve a cyanide-insensitive alternate oxidase. Thecytochrome b complex shows biphasic oxidation kinetics with half times of 0.018 sec and0.4 sec in the absence of inhibitor, which increase to 0.2 sec and 1 sec in the presence ofcyanide. In the presence of antimycin A, the oxidation of the cytochrome b complex showsan induction period of 1 sec and *a half-time of 3.5 sec. A split respiratory chain with 2terminal oxidases and a branch point between the cytochromes and flavoprotein and ubiquinoneis proposed for these mitochondria.

Many plant tissues yield mitochondria whose rateof respiration is partiallv insensitive to inhibition bycvanide (34.42). The most notable of such tissuesare the spadices of the flowers of Arum maculatum(1, 2) and the skuinik cabbage. Symplocarpus foetidus(3, 28, 299). Mitochondria isolated from these tissuesare often totally insensitive to respiratory inhibitionby cyanide or antimycin A. The cvtochromes ofskunk cabbage mitochondria have been characterizedby their spectra (4, 16, 19) which are virtuallyidentical to those observed in other plant mitochon-dria, regardless of tissue source and degree ofcvanide sensitivity (4, 37). In particular, skunkcabbage mitochondria have 3 cytochromes b whichare mostlv reduced in the aerobic steadv state in thepresence of substrate and HOQNOI or antimycin A.as is the case with mitochondria from other plants(5, 6. 16). [Mitochondria from Arum maculatumare an exception, in that cvtochrome b7 in thesemitochondria remains oxidized in the presence ofantimycin A (1)]. This observation has been in-terpreted in terms of 2 terminal oxidases in plantmitochondria, 1 a cytochrome sensitive to inhibitionby cyanide, the other an enzyme of unknown struc-ture insensitive to cyanide inhibition (4, 42). Ben-

1Abbreviations: IIOQNO: 2-Heptyl hydroxyquino-line-N-oxide; MOPS: morpholinopropane sulfonic acid;BSA: bovine serum albumin; TES: tris (hydroxy-methyl) methylaminoethyl sulfonic acid; 1799: bis(hexafluoroacetonyl) acetone.

dall, Bonner, and Plesnicar (3) have postulated thatthe oxidase of unknown structure is a non-heme ironprotein and that the alternate pathway of electrontransport through this oxidase in skunk cabbagemitochondria involves onlv pyridine nucleotide, flavo-protein, and -the oxidase.

The property of resistance to respiratory inhibi-tors makes skunk cabbage mitochondria a most usefulpreparation with which to study the oxidationkinetics of the respiratorv chain carriers bv meansof oxygen pulses into an anaerobic suspension ofmitochondria. Using this technique, Chance. Schoe-ner, and DeVault (20) have shown that the timesequence of oxidation of the cytochromes is charac-teristic of their positions in the respiratorv chain.The comprehensive kinetic studies of this tvpe whichhave been carried out on mammalian mi-tochondriaand derived submitochondrial particles are summar-ized in a recent review (25); much insight into themolecular mechanism of electron transport has beengained from them (13, 22). Such oxygenation cyclescan be carried ouit with mammalian mitochondria inthe presence of cyanide or antimycin A only if theseare at concentrations low enough that the cytochromechain is only partially inhibited (24); with plantmitochondria, it is possible to use concentrationshigh enough to ensure virtually complete inhibitionof the cytochrome chain and still obtain oxidation-reduction cycles with oxygen pulses. In this manner,the kinetic organization of the respiratory chain canbe studied in both the inhibited and uninhibited state.Further, the question of which respiratory carriers,

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identifiable by spectrophotometry or fluorimetry, are

kinetically competent in the inhibited state to functionas an alternate oxidase, or as part of an enzvmecomplex forming an alternate oxidase, can also bestudied. Chance and Hackett (16) and Chance andBonner (19) studied the kinetics of oxidation-reduc-tion cycles of the respiratory chain carriers in skunkca;bbage mitochondria-these being the only 2 in-stances of the application of this kinetic techniqueto plant mitochondria-but only in the absence ofrespiratory inhibitors. In this paper, we reportcomparative kinetics for the oxidation of respiratorycarriers in skunk cabbage mitochondria in the absenceand presence of the respiratory inhibitors. cyanide,and antimycin A.

Experimental Procedures

Skunk cabbage (Symplocarpus foctidus) flowerswere collected from selected swampy points in Dela-ware and Montgomery Counties of Pennsylvania,Union County, New Jersey, and Tompkins County.New York. They were stored at 4°. Mitochondriawere prepared from the excised spadices by means

of the method described by Bonner (7) with minormodifications. The spadices were diced into smallpieces about 5 mm on a side and suspended in coldmedium containing 0.3 M mannitol, 10 mar EDTA,4 mM cysteine, 10 ml MOPS, and 0.1 % BSA,fraction V, !at pH 7.4. About 80 g spadix tissue per

liter of medium proved optimal. The tissue was

homogenized in a Waring Blendor with 2 periods ofsec at high speed separated by 5 sec. The grinding

head of the Blendor was replaced by a "Polvtron"head (Bronwill Scientific) which provides very

rapid and efficient disruption of the tissue. The pHremained constant during the homogenization. Thehomogenate was squeezed through 4 layers of nyloncloth and centrifuged for 20 min at 1000 g to remove

cellular debris and starch. The mitochondria were

sedimented at 10,000 g for 15 min, and then resus-

pended in wash medium of the same composition as

the grinding medium but with cvsteine omitted,100 ml of wash medium being used for each liter ofgrinding medium. Residual starch was removed bycentrifugation at 750 g for 15 min, and the finalmitochondrial preparation was isolated by sedimen-tation at 6000 g for 15 min.

The respiratory activity of each miiitochondrialp)reparation and its sensitivity to cvanide inhibitionwas determined polarigraphically with a Clark oxygenelectrode (Yellow Springs Instrunmenit Co.) in a

stirred cuvette of 3.3 ml capacity; for this deter-mination, the mitochondria wvere suspended at 0.6to 1.2 mg pro,tein/ml in a medium containing 0.3 almannitol, 10 mM TES and 5 mm phosphate adjustedto pH 7.2 with KOH. This medium was used forall experiments reported in this paper.

Mitochondrial integrity was checked by measuringthe energy-linked reduction of pyridine nucleotidebv succinate, using an Eppendorf fluorimeter (Brink-

man Instruments, Inc.) with a 313 mju + 366 m,primary filter and a 400 m,u to 3000 m,u secondaryfilter. The energy-linked reduction of fluorescentflavoprotein (23) under the same conditions wasalso measured with this fluorimeter fitted with aHeraeus ST-75 mercury arc lamp, using a 436 mnprimarv and a 500 mp.t to 3000 mMu secondary filter.

The kinietic measurements were carried out in amantually operated regenerative flow apparatus witl0.1 cm light path and in a similar, hydraulicallvoperated apparatus with 0.4 cm light path. Thelatter apparatus is similar to the one described indetail by Chance (22); the calibration of the formerone, also designed by Chance (12). has been re-ported by Storey l(40). In these experiments, theaerobic mitochondrial suspension was preincubatedfor 10 min with 130 J.M ADP and 10 jr 1799(designation for the uncoupler bis-l(hexafluoroace-tonyl) -acetone, which was kindlv provided by Dr.P. Heytler of E. I. duPont de Nemours, Inc.) todeplete the mitochondria of energy conservationcapacity. The mitochondrial suspension was madeanaerobic by addition of 3 mM succinate; 6 mMmalonate was then added to partially inhibit succinicdehydrogenase and thus maintain the flux of reducingequivalents through the respiratory chain at a verylow level (B. Chance, unpublished method). Underthese conditions, the extent of oxidation of therespiratory carriers is greater than 95 % in theaeroibic steadv state, as determined in separatecontrol experimenits. A large reservoir of reducin-equivalents is available. however, so that numerousxoxygenation cycles may be carried out on a singlesample. The flow experiment is initiated by mixingthe anaerobic mitochondrial sutspension with oxygen-saturated meditum at a volume ratio of 100/1, giving aninitial oxygen concentration of 12 ptM. Absorbancechanges corresponding'to the oxidation of ubiquiinone,cytochrom,e c, cytochrome b, and flavoprotein weremeasured as a function of time with the dual wave-length spectrophotometer (10). Fast changes weredisplayed on a storage oscilloscope and photographedto give permanent records; slow changes were dis-played on a pen recorder chart. Ubiquinone wasmonitored wiith the wavelength pair 275 to 290 myor 282 to 295 mMu (17, 39). For cytochrome c, thepair 549 to 540 my was used. Absorbance changesdue to the cytochrome b components were followedat 560 to 575 my (9), while those due to flavoproteinwere followed with 'the wavelength pair 468 to488 m,u, which minimizes interference from the cvto-chrome components. The wavelength pairs 465 to510 mL (18) and 475 to 510 m 1(23) are notkineticallv "clean" in these mitochondria and showabsorbance changes attributable to the rapidly oxi-dized cytochromes. The oxidation kinetics of fluo-rescent flavoprotein and pyridine nucleotide werealso measured in the flow apparatus by means of arotating disc fluorimeter which permits the simul-taneous recording of changes in both components(22). Pyridine nucleotide fluorescence emission

116

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STOREY AND BAHR-ELECTRON TRANSPORT IN SKUNK CABBAGE

detected at 450 mMu was excited at 366 mMu; flavo-protein fluorescence detected at 570 m;, was excitedat 436 m,. The exciting light source was a water-cooled high pressure mercury arc, and the wave-lengths indicated were selected by means of appro-priate primary and secondary filters.

Difference spectra of the mitochondrial suspen-sions were obtained with the split beam spectropho-tometer described by Chance (10). Spectra in theultraviolet region were obtained at room tempera-ture: those in the visihle region were obtained atliquid nitrogen tenlperature tusing the techniquedescribed by Estabrook (26) as modified by Bonner(4).

Results

Integrity of the Mitochondrial Membraec. Evi-dence that cytoclhrome b functioniis on a branch path-way of electron transport in mammalian submito-chondrial particles, rather than on the main pathway,was presented some years ago by Chance (11).This finding raised the point that damage to themitochon,drial membrane can alter the organizationof the respiratorv chain. Further evidence has accu-mulated that such alteration indeed occurs, and thatboth cytochrome b and ubiquinone are affected; theoxidation rate of cytochrome b is slower, the morealtered the mitochondrial particle, while that ofubiquinone is faster (25). It was therefore neces-sary to show that the skunk cabbage mitochondriaused in this kinetic study had undramaged membranes.Bonner gives 3 properties which isolated mitochon-dria must possess if they are to be considered intact(7). These are: A) good respiratory control byADP concentration (15) and retention of this prop-erty for some hr following isolation; B) no accelera-tion by externally added NAD+ of the oxidationrate of malate, or acceleration by added cytochromec of any respiration rate; C) retention of mitochon-drial fine structure and minimal contamination bvmicrosomal fragments. Skunk cabbage mitochondria,when isolated ias described above, regularly possessthe last 2 properties, but respiratory control by ADPconcentration is usually difficult to demonstrate.The energy-linked reduction of endogenous pyridinenucleotide by succinate oxidation was regularly ob-served with these mitochondria, however, and thisproperty was retained for at least 4 hr after isolationin ice-cooled preparations. It was taken as indi-cating an intact mitochondrial membrane, not becausethe energy conservation capacity is functional, butbecause the endogenous pyridine nucleotide is evi-dently prevented from leaking out. Reduction ofpyridine nucleotide by succinate oxidation, measuredfluorimetrically, is shown in figure IA; the reductionis inhibited by preincubating the mitochondria withADP and uncoupler, as shown in figure 1B. Thecorresponding energy-linked reduction of fluorescentflavoprotein is shown in figure IC. The fluores-cence decrease is much less after preincubation with

S.CM. i0.8mg Prot/mil PN A180M,M ATP 8iM Succ

Incr. Fluor.S.CM -seK PN Reduc4,

0.8mg Prot./mIl180MuMADP=7C7TMM 1799 PNS8min Deplefion 8mM Sum

S.C.M. 8mM Succ0.5mg Prot./ml Fp180pM ATP +I \

c

\ ~\ \ \ _ Decr Fluor.

S.C.M - . 60jZ Fp Reduc.0.5mg Prot./ml 8mMSucc-180M,M ADP Fp

l0min Depletion

FIG. 1. Extent of reduction of pyridine nucleotideand flavoprotein as measured by fluorescence in thecoupled and depleted states. Experimental details aregiven in the text. In traces A and B, the fluorescenceincrease caused by pyridine nucleotide in the coupledand depleted states, respectively is recorded for 2 samplesfrom the same mitochondrial preparation. In the de-pleted sample, 24 % of the pyridine nucleotide is reducedby succinate as compared to the sample treated withATP. In traces C and D, the fluorescence decrease dueto flavoprotein for the coupled and depleted conditionis recorded. In the depleted sample, the extent of thefluorescence decrease is 18 % of that in the coupledsample.

ADP and uncoupler as is evident from figure 1D.The problem of respiratory control and energy con-servation in skunk cabbage mitochondria is consid-ered in greater detail in an accompanying paper(41). In this study, the mitochondria were treatedwith uncoupler and ADP to produce the depletedcondition which is characterized by the fluorescentflavoprotein and pyridine nucleotide responses shownin figures 1B and 1D. Under these conditions, theenergy conserving capacity of the mitochondria islost, and the process being examined during oxida-tion cycles is presumably electron transport alone.Attempts to measure carrier kinetics in the coupledstate were unsuccessful, because the mitochondriabecame uncoupled within one-half hr of being sus-pended in the reaction medium, even at 180.

Stability of the Mitochondrial Suspenision. Oneof the great advantages of the regenerative flowapparatus is that numerous measurements may becarried out with the same sample. This requiresthat the mitochondrial suspension be held for thefull period of the experiment at the desired reactiontemperature, and the stability of the preparationbecomes a problem. Breakdown of the suspensionwas signalled by coagulation of the mitochondriaand, on occasion, by a sharp decrease in the oxida-tion rate of the respiratory carriers. The problemwas aggravated by higher temperatures: at. 26°,maximum working time was 3 hr or lessi while at

117



PLANT PHYSIOLOGY

180, the suspension did not appear to have alteredafter 5 hr. The experiments with cyanide and anti-mycin A required that the rate measurement becarried out for each carrier on the same sample,first without inhibitor and then with inhibitor. Theexperiments were carried out at 180 and were com-plete in about 4 hr, thus ensuring that the kineticproperties of the mitochondria remained constantduring the period of the measurements.

Kinetics of Flavoprotein and Pyridine Niclco-tide Oxidationl. One difficulty in nmeasuring thekinetics of mitochondrial flavoprotein by absorbancechange is that of interference from cytochrome ab-sorbance changes. The use of fluorimetry offers amore specific method (21). Since the mitochondriahave been depleted with ADP and uncoupler accord-ing the conditions described in figures lB and ID,onlly the higher potential flavoproteins of low fluores-cence, presumnably FPD9 and FpS 1(23), are reducedby succinate while the highly fluorescent componentof the lower potential flavoproteins. presumably FPDiand FpT, (27), remain oxidized. The kinetics ofoxidation of flavoprotein, as measured both by ab-sorbance and by fluorimetry, are shown in figuire 2.

A-Flavoprotein And Pyridine Nucleotide FluorescenceStart Stop

Veloci y W IFor Fp, PNtTrace - ~~~~~ncr.Oxid

Fp, 436- 570mM FluoJr.

PN, 366-"450ma . T2

12Be M 02

B - Flavoprotein Absorbance 468-488m,4Stort Stop

Flow FpdVelocity - - Oxid. I

TrAce

IncrrAbs.468mp. AO~~~~~~.D.= O.C

L)009

t 0.2 sec12,LM °2

FIG. 2. Oxidation rate of flavoprotein and pyridinenucleotide in skunk cabbage mitochondria at 220, theformer measured both by fluorescence and absorbance.The mitchondrial suspension contains 4 mg protein/ml;the light path is 0.1 cm. Succinate and malonate are

present at 3 mm and 6 mM, respectively. Fluorescencechanges corresponding to flavoprotein and pyridine nu-

cleotide were measured simultaneously on freshly pre-pared, depleted mitochondria, followed by measurementof flavoprotein absorbance on the same sample in thesame flow apparatus. Pyridine nucleotide fluorescence wasexcited with light at 366 mu; flavoprotein fluorescencewas excited with light at 436 m/L.

There is no deflection of the trace during the flowin either record; when the flow stops, both thefluorescence (fig 2A) and ajbsorbance (fig 2B)changes proceed with a half-time of 180 msec, avalue quite comparable to that observed with mam-malian and avian heart mitochondria (14). Thekinetics of pyridine nucleotide oxidation are meas-ured simultaneously with those of fluorescent flavo-protein on the record in figure 2A; this reactionproceeds with a half-time of 260 msec. The fullcycle of oxidation and reduction. whose initial partis recorded in figure 2. is shown in figure 3. Theflavoprotein fluorescence and absorbanice changes arein synchrony, with the time to half-reduction-t,., off (31)-being about 80 sec in each case.Reduction is complete for flavoprotein. but not forpyri,dine nucleotide. This component apipears toundergo biphasic r-eduction, but the second slow

A-FLAVOPROTEIN AND PYRIDINE NUCLEOTIDEFLUORESCENCE

366- 450m/.L PNOxid

12,1M 02 - 60sec

-. -.-. j t+FpV<; _ 436-570Om, Oxid

-Vi -60sec

FluorDecr

t

FluorIncr

B- FLAVOPROTEIN ABSORBANCE 468-488muet-ZOD=00011T"'ncr+

~ 6Osec Abs Fp12,*LM02 468mrn Oxid.

FIG. 3. Oxidation and reduction cycle of pyridinenucleotide and flavoprotein in depleted skunk cabbagemitochondria recorded simultaneously wtih the tracespresented in figure 2, but on the slower time scale indi-cated. A) Simultaneous recording of flavoprotein andpyridine nucleotide fluorescence. Oxidation of pyridinenucleotide is shown by a rapid downward deflection ofthe trace in the upper record; that of flavoprotein by arapid upward deflection in the lower record. This isfollowed by a slow reduction of each component, thatof pyridine nucleotide apparently being biphasic andnot attaining its initial degree of reduction. A half-time(t12'off) of about 80 sec is estimated for the reductionof flavoprotein. B) Oxidation of flavoprotein is shownby the sharp upward deflection of the trace correspond-ing to increased absorbance at 468 mg. This is fol-lowed by a slow reduction with a half-time (t1/2 off)estimated to be about 80 sec in agreement with the valueobserved for fluorescent flavoprotein.

118




UBIQUINONE 275-290mrnL

Start1 rStopFlow

VelocityTrace

Final- . . . .

t 05sec-i el2,hM02 oUQd

Start Stop

LB veIO~'Flow.

Velocity..Trc_race...{s,... *1OA°D=OOO43

Final - lncrt

~~~~Abs1t -1 F~-5sec 275mgi

12/1M 02Start-1 vStop

(C Flow-Velocity.... t

Trace ; =t10D=0.0017+O3mM KCN T

Final - :it e5sec

12MM 02

OXYGEN CYCLES ± KCN

FIG. 4. Oxidation and reductioni cycles of ubiquinoneas measured at 275 to 290 m,u in the presence and ab-sence of cyanide at 18°. Ubiquinone content of thesemitochondria is 1.6 nmol/mg protein. Mitochondrialprotein concentratioll is 2.8 mg/ml and the light path is0.1 cm in these experiments; succinate and malonate arepresent at 3 mm and 6 mm respectively. A) Oxidationof ubiquinone in the absence of inhibitor; the half-timefor oxidation is 300 msec. The apparent decrease inabsorbance at 275 mr during the flow is caused by dilu-tion of the suspension; upon completion, the trace returnsto this position as shown by the arrow marked "final",rather than to the original position. Oxidation is es-

sentially complete in the steady state. B) Completecycle of oxidation and reduction of ubiquinone in theabsence of inhibitor on a time scale 10-fold slower thanin A. The absorbance change caused by dilution isclearly evident. C) Complete cycle of oxidation andreduction of ubiquinone in the same mitochondrial sus-

pension as in A and B, but in the presence of 0.3 mmKCN, recorded on the same time scale as B. The half-time for oxidation is 2 sec, and the degree of oxidationin the steady state is 48 %.

phase does not go to completion. The fluorescencerecords of figure 2 and figure 3 were obtained witha fresh mitochondrial suspension, the absorbancemleasurenment being made directly after the fluores-cence one. If the fluorescence measurements are

made after a large number of oxygen cycles, thepyridine nucleotide fluorescence is then virtuallyundetectable. The flavoprotein fluorescence is stillreadily measured under these conditions and gives

the same result obtained by means of the spectro-photometric measurement at 468 to 488 mju.

The difference extinction coefficient AE' for re-duced minus oxidized flavoprotein at 468 to 488 muwas estimated to be 4 mM-1 cm-' from the spectrumof isolated succinic dehydrogenase presented by King(36), using 10 mm-1 cm-1 at the extinction coeffi-cient for the wavelength pair 475 to 510 m/1 (27).The concentration of flavoprotein reducible bv suc-cinate in the depleted condition is calculated to be0.5 to 0.7 nmol/mg protein from the absorbancechange observed. This is about onie-third to one-fourth of the flavoprotein reducible by dithionite inskunk cabbage mitochondria reported by Lance andBonner (37), who used the wavelength pair 460 to510 mM for their calculationl.

Oxidation Kinetics in the Absence and Presenceof Cyanide. These experiments, and those reportedbelow with antimycin A, were carried out withmitochondria pretreated with ADP and uncoupler todeplete their energy conservation capacity as de-scribed above. The cyanide concentration was0.3 mM in this series of experiments, which, fromthe results of Ikuma and Bonner (33) on cyanideinhibition of respiration of mung bean mitochondria.should provide essentiallv complete inhibition ofcvtochrome oxidase. The time course of a cycle of

AZQ Flavoprotein 468-488mM (CStarts; f Stop Flow StortI F Stop

;4,-V y-._______Troce60D.=0000 8b+.3mM___'0D8O00008

>_ZtKCN*-O~.2sec t Fp 2o-2sec

12oM 02 lOxid. 12yM 02Incr.

12,uM 02 Abs.12< M 02I _ *468m

OD.0.00'001+3M1IVTiiL0D,OO0I

60sec 6Osec

B3 Oxygen Cycles ± KCN 0D

FIG. 5. Oxidation and reduction cycles of flavo-protein, as measured with the wavelength pair 468 mjuto 488 m,u, in the absence and presence of cyanide at 180.In records A and B, the mitochondrial suspension con-tains 3.0 mg protein/ml; in C and D, it contains 2.7 mgprotein/ml. Otherwise, the reaction conditions are iden-tical. The light path is 0.1 cm; the response time is 50msec in A and 220 msec in C. A) Oxidation in theabsence of cyanide. No reaction occurs during the flow.When the flow stops, oxidation proceeds with a half-time of 180 msec, as indicated by an upward deflectionof the trace. A slight disturbance during the flow re-sults in a small downward deflection of the trace. Oxi-dation is essentially complete in the steady state. B)Trace obtained on a recorder with the time scale indi-cated, showing the time course of reduction of this com-ponent in the absence of cyanide. C) Oxidation in thepresence of cyanide, recorded on a time scale 10-foldslower than that of A. The half-time for oxidation is1 sec; the extent of oxidation is 75 % in the steady state.D) Trace obtained on a recorder with the time scaleindicated, showing the time course of flavoprotein re-duction in the presence of cyanide.

119



Table I. H1-f Times of Oxidation, t1. on, and Cycle Times to Half-reduction, t 1 off, of the Respiratory, Carriers ofSkunk Cabbage Mitochzondria, as Determined bAy 02 Pnilse Experiments

Fp

18010002001000

Inhibitor

0.3 m m KCN-

antimycin A0.7 ug/mg prot

0.3 inM KCNo

antimycin A40.7 Ag/mg prot

1-Half-time of oxidation. -t1/ 2 °" msecQ b

3002000400

2000

18: 400'200; 1000'15: 400'35002

Cycle timiie to half-reduction. -t / 2 ,ff, SeCFp Q b30 6 2050 18 5230 6 1850 18 270 ]

3.13.83.23.4

c

i.60.151.616

Oxidationi is biphasic: half-times giveni are estimates for the 2 phases.2 Oxidation did not start unitil about 1 sec after imiixing the mitochondrial suspenisioil with oxygeniated medium.

Maximum extent of oxidationi during the cycle is 75 % for Fp, 48 % for Q, 76 for b, and 30 % for c in thepresence of 0.3 mM KCN.

4 Maximum extent of oxidation during the cycle is 85 % for Fp, 53 % for Q, 20 % for b, and 100 % for c in thepresence of 0.7 ,g antimycin A/mg protein.

oxidation and reduction is shown in figure 4 forubiquinone, figure 5 for flavoprotein, figure 6 forcytochrome b, and figure 7 for cytochrome c, in theabsence and presence of 0.3 mM cyanide. Theserecords were all obtained with the same mitochon-drial suspension and thus are directly comparableallowing for the decrease in mitochondrial proteindue to dilution as the experiment proceeded. Thehalf-times for oxidation and re-reduction. t,1, O and

t]/ off (31). are summarized in table I.

In the absence of inhibitor. the half-time forubiquinone oxidation-t,/12 ,n,,,iS 300 msec. a valuevery similar to that obtained with mammalian andavian heart mitochondria under the same conditions(14). The time to half-reduction, t1l/ off, is 6 sec.

In the presence of cyanide. the oxidation rate isdecreased by a factor of 6, the half-time being 2 sec.

and t72 off iS increased to 1S sec. Flav,oprotein inthese mitochondria is affected similarly.

Cytochrome b, as might be expected of a complexmade up of 3 separate components, does not showsimple kinetics. There is an initial fast oxidationwith a half-time of 18 msec followed bv a slowerone with a half-time of 400 msec. This biphasicoxidation also occurs in the presence of cyanide. butnow the half-times have increased to 200 msec and1 sec. Cyanide also inhibits the reduction of cyto-chrome b to the extent that the reduction remains

incomplete after a cycle, as is evident from com-

parison of figtures 5C and SF. This incompletereduction was also noted by Chance anld Hackett(16).

Cytochrome c is oxidized wvith a half-time of3.1 msec in the absence of inhibitor, a valuie closeto that measured for this respiratory carrier in ratliver mitochondria (22). There is a further slowoxidation which accounts for about 10 % of theahbsorbance change; a similar slo-w oxidation phase

was noted by Chance and Bonner (19) in skunkcabbage mitochondria reduced by endogenous sub-strate only. The reduction part of the cvcle alsoshows a slow phase comprising 10 % of the ab-sorbance change. Cytochrome c shows a t1/2 off of

1.6 sec under these conditions. In the presence ofcyanide, there is oxidation of cytochrome c with a

half-time of 3.8 msec, which is nearly the half-timein the uninhibited system. The maximum extent ofoxidation is only 30 %, however. Reduction occurs

in 2 phases after a steady state lasting about 100msec. The first phase corresponds to a t,/2 off of

about 150 msec. The absorbance change due to theslow phase is again about 10 % of the total changedue to cvtochrome c in the absence of inhibitor.

The kinetic behavior of cvtochrome c upon

oxygenation of the anaerobic mitochondrial suspen-

sion in the presence of cyanide is most readilyexplained by a binding site for cyanide on reducedcytochrome oxidase which is different from the siteresponsible for inhibition of the reduction of oxi-dized cytochrome a3 by reduced cytochrome a, andwhich does not interfere with the oxidation ofcvtochrome a3 by oxygen. Yonetani has recentlysummarized the argument for 2 such sites (43)deriving both from his own work. and from that ofKeilin and Hartree (35) and Chance (9). Thepattern of cytochrome c oxidation shown in figure6C would then reflect the following sequenee; oxida-tion of reduced cytochrome a, by oxygen followedby oxidation of reduced cvtochrome a in less than0.5 msec (22) ; reduced cvtochrome c is then inturn oxidized with its normal half-time. Oncecytochrome a, is oxidized, however, the cyanidepresent binds to the inhibitory site, and the flow ofelectrons through the oxidase to oxvgen is effec-tively cut off. The effect is that of an oxygen pulsemuch lower in concentratioln than the one actuallyused.

Mitoclhondrialpreparation

28-054

28-017

28-054

28-017

120 PLANT PHYSIOLOGY




Cytochrome b 560-575m)4

F Starts- fStops Starts Stops Flow Velocity

Velocity I Cyt. b Oxid Trace

Trace

12, M 20 -1 * 0.2sec Flow03+-*2e

03tatsjStops Trae E Starts- Stops Velocity

0a0b. X Decr Abs. KCN

000159 I-'.I IOf008

t - 2I-02secM02. 02sec

00022 KCN02-- -t-- Flow1

Aoygen Cycles ± KCN

FIG. 6. Oxidation and reduction cycle of cytochromeb,as measured at 560 m,uc to 575 m,± in the absence and

presence of cyanide at 180. In records A, B, and C,the mitochondrial suspension contains 3.1 mg protein/mi;in records D, E, and F, it contains 2.7 mg protein/mi.Otherwise, the reaction conditions are identical to thosein tigure 4. The light path is 0.1 cm, and the responsetime 50 msec in all experiments. A) Oxidation of cyto-chrome b in the absence of inhibitor. Some oxidationoccurs during the flow as shown by upward deflectionof the trace to a plateau, followed by a further, rapidupward deflection on oessation of flow. This reactionts in turn followed by a slower one as shown by a con-tinuous rise in the trace. The flowr velocity is 109 cm!sec and the calculated first order rate constant for therapid oxidation is 38 sec'1, corresponding to a half-timeof 18 msec. B) The biphasic nature of the oxidation isevident from this record with a slower time scale. Thehalf time of the slow oxidation is estimated to be 400msec. Oxidation is essentially Complete in the steadystate which lasts about 1 sec reduction then begins asshown by the slow downward deflection of the traee. C)Trace obtained simultaneously with B on a recorder withthe time scale indicated, showing the slow, biphasic re-duction of cytochrome b. Response time of the recorderis 1 sec. D) Oxidation of eytochrome b in the pres-ence of 0.3 mMs KCN recorded on a fast time scaleat twice the sensitivity of the records A and B. Thereis no deflection during the flow as in A; on cessationof the flow, oxidation proceeds with a half-time of 200msec. E) A seond, slower oxidation occurring in thepresence of cyanide is evident from the record obtainedwith a time scale 5-fold slower than that for D; thehalf-time is about 1 see. The sensitivity is the sameas for record D and twiee that for records A and B;oxidation proceeds to 76 % of the extent observed inthe absence of cyanide. The arrow- marked "final" in-dicates the final position of the trace after completionof the cycle. F) Trace obtained simultaneously with Eon a recorder with the time scale indicated. The slowpart of the oxidation is evident in this trace, as is theincomplete reduction of this eomponent at the end of thecycle.

CYTOCHROME c 549-540m

Start s;Stop

Flow--_VelocityTrace

t t e02sec12MM 02 Cxytd C

LEB

Start 1 Stop

Velocity----- :Trace >t

- \AOD=OOOI5

t -+ e-2sec Decr.12?M 02 AbsI

549m,uStart- Stop

FlowVelocity

(~ Trace

.A0~~LoD=000O8+0.3mMKCN.-

.)

12,MM 02OXYGEN CYCLES ± KCN

FIG. 7. Oxidation and reduction cycles of cytochroniiec as measured at 549 to 540 mnu in the absence andpresence of cyanide at 180. In experiments A and B,the mitochondrial suspension contains 3.3 mg protein/ml;in experiment C, it is 2.6 mg protein/ml; otherwise thereaction conditions are identical to those in figure 4.The light path is 0.1 cm; the response time in all 3experiments is 50 msec. A) Oxidation of cytochrome con short time scale. Addition of oxygenated mediumresults in oxidation during flow; there is a rapid up-ward deflection of the trace to a plateau during theflow followed by a further rapid deflection when theflow stops. Cytochrome c is essentially completely oxi-dized under these conditions. The calculated first orderrate constant for oxidation is 226 sec-1, corresponding tohalf-time of 3.1 msec. There is then a slow upward de-flection, comprising about 10 % of the total absorbancechain, to the oxidized steady state. The reduction whichcompletes the cycle is signaled by a downward deflec-tion of the trace. B) Repeat of the experiment shownin A, but with 10-fold slower time scale. Biphasic re-duction of this component is evident; 90 % is rapidlyreduced, while the remaining 10 % is slowly reduced,resulting in the "tail" seen in the record. C) Oxidationof cytochrome c in the presence of 0.3 mm KCN, re-corded at twice the sensitivity of A and B. During theflow, there is an upward deflection of the trace to aplateau followed by a second upward deflection to anapparent steady state when the flow stops. The calcu-lated rate constant is 182 sec-1, corresponding to a half-time of 3.8 msec. This state lasts for 0.1 sec and isfollowed by rapid biphasic reduction. About 30 % ofthis component is oxidized under these conditions.

121

-r ~-uesec



PLANT PHYSIOLOGY

Kinetics in the Absence anid Presence of Anti-mycin A. As in the experiment with cyanide,these kinetics were all obtained with the same

mitochondrial suspension and hence are directlycomparable. The half-times obtained in this series

of experiments are also listed in table I. The oxi-dation-reduction cycle for ubiquinone in the presence

of antimycin A is nearly indistinguishable from thecycle carried out in the presence of cyanide (fig4C). Essentially the same extent of oxidation isattained; 53 % wi,th antimycin A as compared to48 % with cyanide. The cycle for flavoprotein inthe presence of antimycin A is also very similar tothat in the presence of cyanide. as is evident fromthe half-times in table The extent of oxidation

Cytochrome b 560-575m54 Flow

VelocityStarts -Stops Flow Velocity Starts -Stops Trace

000l5 Protein

-'fiMa 1 102sec 125M -10.2sec

Flow Velocity Velocity

Starts1 Stops ToStTrace

{B F 2 Antimycin A

*07yog/mgOg D

A00D1Ab A~AO.D

00015' ,TDecrA0008T

12,4M°20 -I [4-sec 125M 0 Ssec0s

25lM .2 Ati.ycinAntimFy--

secAbsO0001

Oxygen Cycles Antimycin A

FIG. 8. Oxidation and reduction cycles of cytochromeb, as measured at 560 to 575 m,u, in the absence andpresence of Antimycin A at 180. In records A, B, andC, the mitochondrial suspension contains 3.9 mg protein/ml; in records D, E, and F the protein content has drop-ped to 3.4 mg/ml due to dilution in the course of theexperiment. The light path is 0.1 cm and the response

time of the detector is 50 msec in records A through Dand 500 msec in records E and F. Oxidation of cyto-chrome b in the absence of inhibitor is shown in A andB; some oxidation occurs during the flow, and the oxi-dation is biphasic, as in figure 6A and 6B. The half-time for oxidation of the fast component is 15 msec,

corresponding to a first order rate constant of 46 sec-1;the half-time for the second component is 400 msec. Acycle of oxidation and reduction is shown in C on a

slow time scale; biphasic reduction similar to that infigure 6C is observed. D) Oxidation of cytochrome bin the same mitochondrial suspension in the presence

of Antimycin A recorded on a fast time scale. Nodeflection of the trace is observed. E) Oxidation as inD recorded on a slower time scale. There is a lag ofabout 1 sec after the flow has stopped before any reactionoccurs. Oxidation then proceeds with a half-time of 3.5sec; only 20 % of the 560 to 575 m,u component is oxi-

dized in the steady state. F) Cycle of oxidation andreduction; the cycle time, taken from the point of half-

oxidation to half-reduction is about 2 min.

in the steadv state was 85 %; with cyanide theextent of oxidation was 75 %. The time course ofan oxidation-reduction cycle in the absence andpresence of antimycin A is shown in figure 8 forthe cytochrome b complex. In the presence ofantimycin A there is essentially no oxidation in thefirst sec; a slow oxidation with a half-time of3.5 sec then follows. That oxidation can be ob-served at all appears to be the resuilt of the veryslow rate of reduction under these conditions, thecycling time being 270 sec. The oxidation of cvto-chrome c proceeds with the same lialf-tinme in theabsence and presence of antimycin A, 3.2 mnsec inthe first instance and 3.5 msec in the latter- one.The slow oxidation and reduction seen at 549 to540 mjL, which is observed in figure 7A is abolishedby antimycin A. The inhibitor also increases thecycling time for cvtochrome c by nearly a factorof 10.

Discussion

Two points emerge directly from the resultsreported in this paper. First, the cytochrome bcomplex must be peripheral to electron transportthrough the alternate oxidase, since its rate ofoxidation in the presence of antimycin A is far tooslow for it to play a kinetically competent role inthat process. In fact,, in the presence of cyanide,its first oxidation half-time of 200 msec is still slowbv mammalian standards for a cytochrome closelyassocia.ted with a terminal oxidase (22). Second,the oxidation rates and cycle times for flavoproteinand ubiquinone are essentially the same in thepresence of antimycin A or cyanide, whereas theoxidation rates and cvcle times for cytochrome cand the cytochrome b complex are very differentwith the 2 different inhibitors. Either inhibitorappears to isolate from the cytochromes a respira-tory pathway lying between succinic dehydrogenaseand oxygen and interacting with flavoprotein andubiquinone. The oxidation rate of flavoprotein istoo slow in the presence of either inhibitor to qjualifyit as a terminal oxidase or as closely associatedwith one. There is, in fact, no component identi-fiable by differential spectroscopic methods whichqualifies for the role of alternate oxidase on kineticgrounds. The proposal by Bendall, Bonner, andPlesnicar that a non-heme iron protein functions asan alternate oxidase in these mitochondria (3) isin accord with this observation.

In the absence of inhibitor the half-timie ofoxidation, t1,/ on, for flavoprotein is less than thatfor ubiquinone, whereas the t1/2 off for flavoproteinis greater than that for ubiquinone. The same re-lationship between the 2 half-times for these com-ponents holds in the presence of either cvaniide orantimycin A. For an analysis of the position ofthese components in the respiratory chain of skunkcabbage mitochondria, we have applied a set ofordering theorems developed by Higgins (30. 31)

122




who has analyzed in detail the problem of multi-enzyme systems in which the enzymes are arrangedin a linear array. He was able to show that thetl2 off for each enzyme in the system were orderedfor a wide range of enzyme and substrate concen-trations and of reaction rate constants. In themulti-enzvme sequence A-> B-> C-> S, where S issubstrate, the direction of the ordering is t1/2 of f

(C)<tl/20ff (B)< tlf.2 off (A), and the samedirection of ordering holds for t1/2 on, In terms ofour experiments with oxygen pulses, the t/2on andthe tl12 of f for each carrier in a linear respiratorychain shotuld he smaller the nearer the carrier is tothe oxidase. This ordering theorem is not com-

pletely general, but in practice, extreme conditions,not even approached by the system under study,must be chosen to produce a disordering. In addi-tion, he showed that only the enzvme reacting withthe suibstrate shows no induction period; enzymes

further removed from the substrate show inductionperiods. In practice, these induction periods are

difficult to observe. At no point during this studvcould we observe an induction period for the oxida-tion of flavoprotein, but induction lperiods of 50 to100 msec were observed for ubiquinone in a numberof the mitochondrial preparations. An example isshown in figure 9. If we assume that flavoproteinand tubiqulinone are part of a linear sequence of

Ubiquinone 275-290mML

Succ

- - -| - - Malonate

Fp: -Q

_/

Anti. A-- - --CN-a3

62

02~~°2

FIG. 10. Proposed scheme of electron transport fromiisuccinate to oxygen in uncoupled skunk cabbage mito-chondria. The symbol Y is used for the branch pointand the symbol X is used for the alternate oxidase.Inhibition sites are shown by dashed lines.

Flow VelocityTrace

O.D.0=0.-007

121j,M 02 0.2secFIG. 9. Oxidation of ubiquinone in the absence of

inhibitor, recorded at an oscilloscope sweep speed of 200msec/cm. Reaction conditions are the same as thosedescribed in figure 4; the mitochondrial suspension con-

tains 2.7 mg protein/ml. Two experiments are shownon the same record; the letters a and b serve to cor-

relate the absorbance change with the appropriate flowvelocity trace. There is a slight downward deflectionof the trace during the flow- due to dilution: The tracereturns to this new positioil after the cycle as shownby the arrows marked "final." After the flow hasstopped, the traces remain level for about 80 msec, afterwhich they show an upward deflection corresponding tothe oxidation of ubiquinone.

respiratorv carriers, a contradiction results. On thebasis of the tl/2 off sequence, ubiquinone should beplaced between flavoprotein and oxygen. On thebasis of the tl/2 o sequence and the observed induc-tion period for quinone oxidation, flavoprotein shouldbe placed between ubiquinone and oxygen. It isevident that these 2 carriers cannot both be part ofa linear respiratory chain transporting electrons fromsuccinate to oxygen.

The analog computer was used to examine a

number of non-linear arrangements of the respira-tory carriers of skunk cabbage mitochondria in orderto ascertain which ones were compatible with thekinetic data and the ordering theorems. The sim-plest of the compatible arrangements is shown infigure 10. There are 2 paths to oxygen, one

through cytochrome c and cytochrome oxidase, theother through the cyanide insensitive alternate oxi-dase. Since the kinetics of uibiquinone and flavo-protein oxidation are the same in the presence ofantimvcin A as in the presence of cyaniide. thebranch point for these 2 pathwavs, designated Y. isplaced before the site of antimvcin A inhibition.The nature of Y is unknown, but it may well be a

inon-heme iron protein-as p6stulated for the alternateoxidase (3)).

Starts1 -Stops

a_

Finalb I1

b"_ a, -5

_ Fv1w_U__ b1Kl_ThJ~b_IT

123

>



PLANT PHYSIOLOGY

Placement of uibiquinone on a side pathway con-

necting to flavoprotein removes i.t from the stricturesof the ordering theo.rems mentioned above. Thekinetic behavior of a redox enzyme or coenzyme so

placed in a respiratory chain varies with the valuesof the rate constants controlling the redox reactionswith its partner on the "main sequence" of electrontransport. We have examined a simple non-linearsequence of this type with the analog computer.For a wide range of rate constants. the t1/2 n of thecomponent on the side pathway may be equal to or

greater than that observed for its main sequence

partner, while its tl2 off may be equal to or lessthan that of its partner. If the rate constants are

made sufficiently large, the component on the sidepath-way will be in rapid equilibrium with its mainsequence partner and the 2 will be kine.tically indis-tinguishable. This case applies to the componentwe have designated flavoprotein, which most prob-ably consists of 2 flavoproteins, Fps and FPD1, (23),here behaving as a single kinetic entity.

The fast component of the cytochrome b kineticshas been placed in figure 10 according to the re-

quirements of the ordering theorems. The tl./ off

values in the presence of cyanide or antimycin Ashow that this cytochrome b cannot be part of a

linear sequence be.tween succinate and the alternateoxidase; neither can it be part of a linear sequencebetween succinate and cytochrome c. The role ofthe other memrbers of the cytochrome b complexrequires further study.

The proposed respiratory pathway shown infigure 10 was derived from kinetic studies withuncoupled, depleted skunk cabbage mitochondria, andthus applies only to that particular condition. Thequestion of what modifications to such a scheme are

required to account for the results observed withcoupled skunk cabbage mitochondria are discussedin the accompanying paper (41).

Acknowledgment

The authors are indebted to Dr. Britton Chance forhis expert guidance in the intricate art of measuringrapid reactions and for the use of the specialized equip-ment required for these measurements. Dr. Walter D.Bonner and Dr. Derek S. Bendall have provided manyhours of thoughtful discussion which launched the authorson this study. Dr. Joseph Higgins has been most helpfulin analyzing the consequences of multi-enzyme interac-tions both with blackboard and analog computer. Skill-ful and enthusiastic technical assistance was rendered byMrs. Dorothy Rivers. This research was supported byAEC contract AT (30-1) -3724 and USPHS GM-12202and was carried out during the tenure of USPHS CareerDevelopment Award K3-GM-7311 to the senior author.

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respiratory chain mitochondria. i. electron …plant physiol. (1969) 44, 115-125 the respiratory...

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