Upper and Lower Limits of the Proton

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 261, No. 18, Issue of June 25, pp. 8254-8262,1986 Printed in U.S.A.

0 1986 by The American Society of Biological Chemists, Inc.

Upper andLower Limits of the Proton Stoichiometryof Cytochrome c Oxidation in Rat LiverMitoplasts* (Received for publication, March 12,1985, and inrevised form, February 24, 1986)

Baltazar ReynafarjeS, Lidia E. Costa$, and Albert L. Lehninger From the Department of Biological Chemistry, The JohnsHopkins University School of Medieine, Baltimore, Maryland 21205

The stoichiometry of vectorial H+ translocation coupled to oxidationof added ferrocytochrome c by Oz via cytochrome-c oxidase of rat liver mitoplasts was determined employing a fast-responding O2electrode. Electron flow was initiated by addition of either ferrocyWhen the rates were extrapolated tochrome c or 02. level flow, the H+/O ratios inboth cases were less than but closely approached 4;the directly observed H+/O ratios significantlyexceeded 3.0.The mechanistic H+/ 0 ratio was then more closely fixed by a kinetic approach that eliminates the necessity for measuring energy leaks andis independent of any particular model of the mechanism of energy transduction. From two sets of kinetic measurements, an overestimate and an underestimate and thus the upper andlower limits of the mechanistic H+/O ratio could be obtained. In the first set, the utilizationof respiratory energy wassystematically varied through changes in the concentrations of valinomycin or K+. From the slope of a plot of the initial ratesof H+ ejection (JH)and 0 2 uptake (Jo) obtained in such experiments, the upper limit of the H+/O ratio was in the range4.12-4.19. In the second set of measurements, the rate of respiratory energy production was varied by inhibiting electron transport. From the slope of a plot of J~uersus Jo, the lower limit of the H+/O ratio, equivalentto that at level flow, was in the range3.83-3.96.These data fix themechanistic H+/O ratio for thecytochrome oxidase reaction of mitoplasts at 4.0, thus confirming ourearlier measurements (Reynafarje, B., Alexandre, A., Davies, P., and Lehninger, A. L. (1982)Proc. Natl. Acad. Sei. U. S.A . 79, 7218-7222). Possible reasons for discrepancies in published reports on the H+/O ratio of cytochrome oxidase in various mitochondrial and reconstituted systems are discussed.

chrome-c oxidase in intact mitochondria (6-14), mitoplasts (15),inverted innermembrane vesicles (16,17), andreconstituted liposomal preparations of the purified enzyme (18-26). However, disagreement persists regarding the mechanistic stoichiometry of H+ translocation by cytochrome-c oxidase, to defined as the maximum H+/O stoichiometry for which the enzyme provides a coupling mechanism, in distinction to the observed or effective stoichiometry delivered under most in vitro or intracellular conditions. Some laboratories have reported H+/O ratios (number of Hf translocated per atom of oxygen reduced) close to 2 (11-13,18-24,26), whereas studies from this (8, 9, 15, 27) and other (10, 14, 16, 25, 28) groups have led to theconclusion that themechanistic H’/O ratio of the cytochrome oxidase reaction is significantly higher (25) and probably close to 4 (10, 14, 16, 28). Two laboratories (29, 30) have held that no H+ translocation is coupled to the cytochrome-c oxidase reaction p e r se. Recently, however, one of them (31) offered a hypothetical mechanism for a directly coupled proton-motive redox loop or redox cycle system. This disagreement demands experimental resolution since the H+ translocation stoichiometry is a central element in current research on the reaction mechanism, subunit function, and the membrane orientation of cytochrome-c oxidase, as well as on more general questions as to themolecular mechanisms of H+ translocationand the thermodynamics of respiratory chain phosphorylation. Our H+/O ratio measurements detailed in this andpreceding (15,27,32) studiesincorporate some features essentialfor determination of the mechanistic H+/O ratio of the cytochrome oxidase reaction. First, they have been carried out with a fast-responding membrane-less O2 electrode (15, 27, 33, 34), together with a glass pH electrode, whose responses were first validated against the H*/O stoichiometry of known scalar reactions (15, 27), in order to remove the criticism (1, 2, 30) that H+/O values approaching 4.0 for cytochrome oxidase result from underestimation of the rate of oxygen consumption with Clark-type electrodes. This criticism could Much direct and indirect evidence (for reviews, see Refs. 1-5) indicates that vectorial H+ translocation accompanies also be overcome by carrying out experiments in which elecelectron flow from ferrocytochrome c to oxygenvia cyto- tron flow through cytochrome oxidase was measured by dualwavelength spectrophotometry of ferrocytochrome c disap* This work supported by Grant GM05919 from the United States pearance (15) or by a technique in which a pH electrode was Public Health Service, National Institute of General Medical Sciences used to measure both electron flow and the vectorial ejection (to A. L. L.), and by Grant DAAG 29-85-G-0016 from the United of H+ (15). States Army Research Office for purchase of equipment. Some of the Second, the measurements reported in this and our precedobservations described here were briefly reported in abstract form ing (15, 27) papers were designed to overcome a more funda(Reynafaje, B., Costa, L. E., and Lehninger, A. L. (1984) Biochem. SOC. Tram.12,386-388. The costs of publication of this article were mental problem, the occurrence of energy leaks during elecdefrayed in part by the payment of page charges. This article must tron transport, such as those responsible for state 4 respiratherefore be hereby marked “aduertisement” in accordance with 18 tion. These leaks, which are believed to be due to cycling of U.S.C. Section 1734 solelyto indicate this fact. H+ and other cations, cause underestimation of the mecha4 To whom reprint requests should be addressed Dept. of Biolog- nistic stoichiometry of respiration-coupled transmembrane ical Chemistry, The Johns Hopkins Univ., School of Medicine, 725 ion movements under most experimental conditions in vitro N. Wolfe St., Baltimore, MD 21205. Fellow of the Consejo Nacional de Investigaciones Cientificas y or in vivo. Energy leaks cannot be eliminated by inhibitors and are not measurable under all circumstances; moreover, Tecnicas de la Republica Argentina.

8254

H+/ORatio of Cytochrome Oxidase they can be expected to vary in rate depending upon conditions. As one approach to overcome this problem, we have extrapolated the rates of H+ ejection and Oz uptake to zero time in order to approximate the H+/O flow ratio at level flow, the hypothetical instant atwhich electron transport and H+ translocation are initiatedagainst zero load or resistance, ix. zero AiH+, in the chemiosmotic model. At level flow, the rate of Hf backflow, whose driving force is A;H+, is zero, and the extrapolated H+/O ratio represents the closest possible approach to the mechanistic stoichiometry from rate data alone (cf. Refs. 35-37). We have also employed ferrocytochrome c as the electron donor to avoid use of nonbiological reductants such as ferrocyanide or tetramethylphenylenediamine. To allow direct access of ferrocytochrome c to the oxidase, rat liver mitoplasts were employed (15). This paper shows, first, that theH+/O ratio for cytochrome oxidase of mitoplasts, extrapolated to level flow, is close to 4 when the reaction is initiated by addition of either ferrocytochrome c or Oz,thus rendering less cogent the recent criticism (38) that initiation of the reaction by a step change in O2 concentration results in overestimation of the H+/O ratio. Second, the mechanistic H+/O stoichiometry has been more closely fixed from two sets of kinetic measurements that overestimate and underestimate the H+/O ratio and thusyield its upper and lower limits through a kinetic rationale that does not require measurement of energy leaks and that is independent of any particular model of energy transduction. This approach, which has already been applied to determination of the H+/O ratio of succinate oxidation (27), shows that the mechanistic H+/O ratio of the cytochrome oxidase reaction can be close to 4 in rat liver mitoplasts, in essential agreement with our earlier measurements (9, 15) and some other reports utilizing independent methods (14,16,28).

8255

within 0.8 s. In the second set, whichwas used in many of the experiments in which ferrocytochrome c was the initiating reagent, the 90% response time of the Oztrace was less than 0.2 s, which allowed more accurate measurement of the early kinetics of 0 2 uptake. The electrodes and recording systems were frequently checked by injection of a combined H+ and0, standard intoan anaerobic medium of the same composition as used in the stoichiometric experiments. Both H+ and 0, electrodes tend to become slower after a series of micothcondrial experiments owing to adsortpion of protein and other components. They could be rejuvenated by dipping them in 0.1 N NaOH for 1-2 min, followed by extensive washing with distilled H20. Since the range of 0,concentration changes in these experiments is relatively small, cross-talk interference between the electrodes when used with a common reference electrode is negligible and inpreceding studies (15,27)was precisely compensated by an appropriate bucking circuit. In the experiments described here, separate reference electrodes were employed. The electrodes used in thisstudy were validated as described before (15, 27) for their accuracy in recording the rate of change of H+ and 0 2 concentrations intwo enzymatic reactions in which H+ and 0, are reactants in a known stoichiometric ratio: (i) the scalar oxidation of NADH by 0 2 (40) in well-washed, uncoupled, inverted submitochondrial particles prepared from frozen-thawed rat liver mitochondria according to thereaction NADH

+ H+ + 95 0 2 e NAD+ + Hz0

and (ii) the scalar oxidation of ferrocytochrome c by cytochrome oxidase by rat liver mitoplasts in thepresence of excess protonophore (27),according to thereaction

2 cyt +'c

+ 2 H+ + ?h

0 2

+ 2 cyt c3+

+ Hz0

(2)

where cyt c2+ and c3+represent ferro- and ferricytochromes c, respectively. The electrodes of set 1 gave within 2% of the theoretical scalar H+/O ratio of 2.0 for Reaction 2 at all time points tested from the end of the dead time (-0.8 s) to over 90% of the reaction course, as shown in Ref. 27. Validation of the electrodes of set 2 is shown in Fig. lA, which shows the traces of an experiment in which ferrocytochrome c was added last to the already aerobic mitoplast suspension

EXPERIMENTAL PROCEDURES

The reactions were carried out in a thermostatted closed cell (1.5 ml) with no gas phase, resembling that described in Ref. 39. The Oz electrode was inserted into the side of the cell and theglass electrode into thethreaded Teflonstopper. A narrow port in the stopper allowed insertion of the needle of a Hamilton syringe for injection of small volumes of reagents. The cell contents were stirred at 2000 rpm with a Teflon-coated magnetic bar driven by a motor underneath the cell. The positioning of the electrodes and the portfor additions, as well as the geometry and stirring rate, were empirically adjusted after many trials to minimize mixing time, as well as mechanical stirring noise, to which all 0 2 electrodes are subject. Such adjustments were monitored with test injections of small aliquotsof a combined standard of HCl (100 nmol) and O2 (50 nmol) into the standard 50 mM KCl, 200 mM sucrose medium buffered with 2.5 mM KHepes,l pH 7.1. The signals from the electrodes were suitably amplified and fed into a Soltec 330 multichannel recorder (full-scale response time, 250 ms; chart speed, 120 cm/min). The temperature of the cell in most experiments was maintained at 10 "C, except as indicated, by circulation from a regulated bath. The pH electrode employed was a Beckman Altex Futura. The oxygen electrodes were prepared according to Refs. 15, 33 and 34. The 30-gauge platinum wire was sealed into 3-mm glass tubing, with its tip flush with the seal. Generally, from five to seven layers of sintered glass were applied as described. After application of each layer, the response time of the anaerobic electrodes to an instantaneous application of a jet of air was monitored with an oscillograph. Two pairs of electrodes were used. In set 1,such as used in anearlier study (15),the response of the Oz electrode was adjusted to that of the pHelectrode by suitablechoice of the number of layers of sintered glass, so that the observed 90% response times of both electrode recording systems, under working conditions, were about equal,

(1)

W

E.

I 2 3 4 5 6 TIME (PI

12.3nmol H+ 8.3 m o l 0

i t

02

FIG. 1. Validation of the electrodes. The basic medium (1.5ml; 10 "C) was 200 mM sucrose, 50 mM KCI, 2.5 mM KHepes, pH 7.05,

2.0 p~ rotenone, 10.0 p~ FCCP, and 50 nmol of succinate. After its 0% content was reduced to about 20% of air saturation by bubbling Nz, the cellwasclosed and mitoplasts (1.0 mg of protein) were injected. The succinate was in slight excess over that required to consume the remaining 0 2 in the system. After anaerobiosis was complete as indicated by the 0, trace, 0.1 nmol/mg antimycin A was added to prevent further electron flow from the traces of remaining succinate. To thesystem was then added air-saturated basic medium at 10 "C to make a total of 42.5 nmol of oxygen in the system. One minute later,the cytochrome oxidase reaction was initiated by injecting 180 nmol of enzymatically reduced cytochrome c. A, electrode traces of changes in medium H+ and 0 2 concentration. The vertical burs on the traces set off the segments used for the kinetic analysis The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- in B. B, plots of the rates of H+ and 0 2 uptake at 0.3-s intervals zineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tet- obtained in digital form from the unsmoothed traces. The lines were raacetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenyl- fitted by regression; r = 0.99. The extrapolated zero time H+/O rate hydrazone. ratio was 2.01;theory is 2.00. cyt c", ferrocytochrome c.

H+/O Ratio of Cytochrome Oxidase

8256

to initiate the reaction. Fig. 1B shows Guggenheim plots of changes in the rates of H+ and 0,uptake measuredat 0.3-5 intervals from the end of the dead time (-0.8 s) to over 80% ofthe reaction course.The plots extrapolate to an H+/O flow ratio of 21.3:10.6 = 2.01 at zero time, close to the theoretical 2.0, and are precisely parallel, indicating that the electrodeiscorrectlyrecording the scalarstoichiometry throughout the reaction. Mitoplasts were prepared from rat liver mitochondria accordingto Ref. 41. Most showed respiratory control ratios (FCCP)of 4-5 with succinate. Ferrocytochrome c was prepared from ferricytochrome c (Sigma, Type111) by two methods. In the first, reduction was carried out with hydrosulfite, followed by removal of low molecular weight contaminants on a Sephadex G-25 column. However, very highconcentrations of ferrocytochrome c, free of ferricytochrome c, could not alwaysbereadily obtained since some autoxidation inevitably occurred during dialysisor storage. The presence of ferricytochrome c was avoided sinceit inhibits oxidation of ferrocytochrome c; this and other adverse effectsof chemical reduction, suchas the formation of nonreactive polymers,are considered in Ref. 42. The second and more useful procedure involved enzymatic reduction of added ferricytochrome c by rat liver mitoplasts under anaerobic conditions in the presence of a small stoichiometric excessof added succinate. To 0.5 ml of 10 mM ferricytochrome c in a small test tube made anaerobic by flushing with100% argon were added5.0 pl of a rat liver mitoplast suspension (0.5 mg of mitoplast protein) and 6.0 p1 of 0.5 M potassium succinate, a 20% excess overthe ferricytochrome c; the initial pH was 7.0. The amount of succinate added was sufficient to exhaust the system of remaining dissolved 02 and then t o reduce the added ferricytochrome c completely. The tube was closed and incubated at 20 "C for at least 1h until the cytochrome c was completely reduced, as determinedspectrophotometrically in analiquot taken in the presence of cyanide to prevent its reoxidation. Since reduction of ferricytochrome c by succinate under anaerobic conditions results in acidification of the system by net release of one H+/ferricytochrome c reduced, the H+ formed was back-titrated under anaerobic conditions with a predetermined amount of KOH so that the pH of the solution matchedthe pH of the final test system. The tube was then transferred to an ice bath and kept under argon. Spectrophotometric analysis indicated that the cytochrome c was over 98-99% reduced by this procedure; no further reduction of cytochrome c couldbe produced by addition of dithionite. For the H+/O ratio determinations, 20 p1 of the ferrocytochrome c solution prepared in this way, containing about 195 nmol of ferrocytochrome c, were added to the standard H+/O test system (see figure legends), without prior separation of the mitoplasts. The 20-pl addition of the stock solution of ferrocytochrome c contains only 20 pg of mitoplast protein, i.e. less than 1%of the amount of fresh mitoplast protein inthe test system for determination of H+/O ratios. 02 uptake due to oxidation of the traces of remainingsuccinatein the mitoplast-reduced ferrocytochrome c was not detectable and in any case is prevented by the presence of antimycin A in the H+/O test system. The solubilityof oxygen in theair-saturated test medium employed (50 mM KCl, 200 mM sucrose, and 2.0 mM KHepes, pH 7.05) was determined to be 632 nmol of oxygen/ml at 10 "C by a recently developed kinetic method based onthe strict stoichiometrybetween the rates of H+and 0,uptake duringNADH oxidationby noncoupled 0 2 and H+ submitochondrial particles carriedoutwithmatched electrodes (40).

symport; moreover, N-ethylmaleimide was added to inhibit endogenous sources

H+ influx with phosphate arising from (43).

An important consideration in the experimental design is the rate and extent of H+ ejection in relation to the matrix buffering power. Since reduction of each atom of oxygen is accompanied by disappearance of 2 scalar H+ from the matrix (to form H20)plus the outward tranlocationof as many as 4 H', the matrix will lose a maximum of 6 H+/atom of oxygen consumed. The buffering power of the ratliver mitochondrial matrix is at most 50 ng ions of H+/mg of protein for the interval from pH 7 to 8 (44), which would be consumed by thereduction of about 8.3 nmol of oxygen/mg if no H+ backflow occurred. The total amount and of rate electron flow were therefore arranged to avoid exceeding the matrix buffering power over the period of the reaction utilized for the H+/O flow ratio measurements. At no time under theconditions used was there a net consumption of more than onethird of the matrixbuffering power over the range pH7-8. In principle, the energy-coupled cytochrome oxidase reaction must be initiated under conditions inwhich there is no opposing resistance in the form of Ah,+. This is afforded by initiating the reaction by adding either0, or ferrocytochrome c. In the former type of experiment, a small amountof 0, was of air-saturated injected in the form of aknownvolume standard KCl/ sucrose/Hepes medium intotheanaerobic system, which already contained all other components, including a n excess of ferrocytochrome c. Experimental details of O,-initiated electron flow measurements have already been described (15, 27) and will not be repeated here. When the reaction was initiated with ferrocytochrome c, the system was initially aerobic, with 0, always below the ferrocytochrome c added. In this case, the pH of the ferrocytochrome c stock solution was very precisely adjusted so that no scalar pH change resulted whenwas it mixedwith the rest of the system. Initiation of the energy-coupled reaction with valinomycin (or Ca") is in principle not appropriate since, in this case, ferrocytochrome c oxidation would already be under way in state 4 (ie. static head) at the time of valinomycin addition, conditions underwhich AEH+and thus the H+ leak ratewould already be maximal and could thus be expected yield to lower initial H+/O flow ratios (cf. Ref. 37). Ferrocytochrome c was present in a final concentration between 100 and 140 PM, close to the estimated concentrationof cytochrome c, about 100 p ~ in, the intermembrane spaceof intact rat liver mitochondria in 0.15 M KC1 (45). Most of the experiments in this paper were carried out at 10 "C at which the rate of H+ backflow is much lower than at 25 "C. Fig. 2 shows the tracesof a set of experiments demonstrating theeffect o f inhibitors and of omitting or replacing components of the complete system. The reaction was initiated RESULTS by addition of ferrocytochrome c to the otherwise complete Test System-It was first necessary to establish that the aerobic system. The A traces show the ratesof O2uptake and components and properties of the reaction system yield ap- vectorial H+ ejection by the complete system (K+ valinopropriate conditions for determination of the mechanisticH+/ mycin). Injection of ferrocytochrome c was accompanied by rapid disappearanceof 02,which was complete to theoriginal 0 ratio. The system describedhere, essentiallythatused base line in about3 s. H+ ejection reached a peak in about2 before (15), employs ferrocytochromec as the electron donor, rate H+ ejection. rat liver mitoplasts instead of intact mitochondria to elimi- s, at which time theH+leak rate equals the of natethepermeabilitybarriertoaddedferrocytochrome c Slow net reuptakeof H+ then ensued. The B traces show that imposed by the outer membrane, rotenone antimycin A (or when ferrocytochrome c was replaced with ferricytochrome c, myxothiazol) to prevent electron flow from site 1 and site 2 no endogenous 0, uptake and only a very slight decrease in substrates, and K+ valinomycin (or Ca2+) ascharge-com- H+ occurred. The C traces show the effect of adding ferrocypensating permeant cation, in a medium of 50 mMKC1, 200 tochrome c t o a nanaerobic system; no measurable0 2 uptake mM sucrose, and 2.5 mM Hepes buffer, pH 7.1. Phosphate, nor H+ ejection took place. The D traces show the effect of acetate, and other proton-carrying anions were withheld from omission of antimycin A; no O2 uptake nor H+ ejection prethe medium to preventpossible backflow of H+ by H+/anion ceding the addition of ferrocytochrome c occurred. After fer-

+

+

+

8257

H+lO Ratio of Cytochrome Oxidase

nmol 19.2 nmol 'H

L! t i I

02

C

cyt c2+

FIG. 2. Requirements of test system. The basic medium (1.5 ml; 10 "C) of 200 mM sucrose, 50 mM KCl, and 2.5 mM KHepes, p H 7.05, was flushed with N2to reduce its O2content toabout 10% of air saturation. The cell was closed and supplemented with 4.0 p~ rotenone, mitoplasts (3.0 mg of protein), and 100 nmol of succinate. After O2 was exhausted by the mitoplasts, 40 nmol/mg N-ethylmaleimide, 300 ng/mg valinomycin, 0.1 nmol/mg antimycin A, and 31.6 nmol of oxygen/system in the form of 50 pl of air-saturated basic medium a t 10 "C was added Thecytochrome oxidase reaction was then initiated by injecting 180 nmol of ferrocytochrome c to yield the traces labeled A . The B traces are of an experiment exactly as A , but with ferricytochrome c replacing ferrocytochrome c. The C truces are ferrocytochrome c added to the A system in the absence of oxygen. The D truces represent antimycin A omitted from the experiment inA. The E traces are N-ethylmaleimide omitted from A. The F truces represent the system as inA but with 15 nmol of FCCP replacing valinomycin. Note that theH+ and O2traces have different scales.

rocytochrome c addition, the rates were the same as in the presence of antimycin. The B-D experiments thus show that there isno O2uptake due to endogenous substrates. Although intact rat liver mitochondria show considerable endogenous respiration, the procedure for preparation of the mitoplasts effectively removes endogenous substrates, which derive largely from the outer membrane. Separate spectrophotometric determination of the rate of ferricytochrome c reduction by endogenous substrates in the presence of rotenone & antimycin under anaerobic conditions confirmed that it is less than 2%of the aerobic rate of ferrocytochrome c oxidation by cytochrome oxidase. The E traces show the effect of omitting N-ethylmaleimide, which did not significantly alter the rate of 0,uptake but caused a slight decrease in the net extent of H' ejection, possibly due to a slight increase in the rate of H' backflow. The only modest effect of omitting N ethylmaleimide on the rate of H' ejection, compared to the large decrease in H+ ejection rate observed when intact rat liver mitochondria were employed in the absence of this inhibitor (8), is due to the fact that endogenous Ca2+and Pi are almost completely removed during preparation of the mitoplasts, owing to the presence of EGTA in the medium, whereas intact mitochondria prepared in the absence of EGTA (8, 42) contain 30 or more nmol of Pi/mg of protein, most of which leak out during the anaerobic preincubation but is takenup again in N-ethylmaleimide-sensitivesymport with H' when electron flow is subsequently induced. The F traces show the effect of replacing valinomycin with a very . uptake took place at high concentration of FCCP (10 p ~ )O2 a higher rate together with a fast net uptake of H', i.e. the scalar H+ required for Hz0 formation. When lower concentrations of FCCP areemployed (not shown), some H' ejection occurs initially, but H' reuptake is greatly accelerated, in accordance with Ref. 46. When mitoplasts were omitted, no displacements of the H+ and 0, traces were observed on

addition of ferrocytochrome e. Results virtually identical to those in Fig. 2 are observed when the reactions are initiated by addition of O2to anaerobic systems, rather thanby addition of ferrocytochrome e. These experiments thus show that the test system used provides appropriate conditions for determination of the vectorial H+/O ratio of electron flow from ferrocytochrome c to 02. Protocol for Determination of the Vectorial H+/O RatioFig. 3 shows the complete protocol of a typical measurement of the vectorial H+/O ratio, in which the cytochrome oxidase reaction was initiated by addition of ferrocytochrome e. Each step isshown in thetraces anddescribed in thelegend, which includes details of the electrode calibrations. After all reagent additions and the preincubation period, the reaction was initiated by injection of ferrocytochrome e. O2 uptake began almost immediately and was accompanied by net H' ejection, which reached a maximum before completion of O2 consumption, showing that H+ back-leakage proceeded at an increasing rate that soon equaled andthen exceeded the declining rate of H+ ejection as oxidation of the ferrocytochrome c neared completion. New reuptake of the ejected H' continued to and thenbeyond the original H+ base line, to reach astableend value. The overall net loss of medium H+ via membrane leaks from theinstant of O2 injection to the stable end value, which corresponds to the scalar uptake of H' during cytochrome oxidase activity (Reaction 2), was 62.8 nmol; the total 0 2 consumption (0,added plus dissolved O2 in the antimycin stock solution) over the entire reaction period was 33.5 nmol. The final scalar H+/O ratio was therefore 62.833.5 = 1-90,within 5% of the value 2.0 required by Reaction 2. After O2uptake was complete, the O2 trace remained at the original base line for long periods, demonstrating that no back-diffusion of O2 occurred.

0,

1.6 mol

FIG. 3. Complete protocol of a measurement of the vectorial H+/O ratio. The basic medium (1.5 ml; 10 "C) contained 200 mM sucrose, 50 mMKC1, 2.0 p M rotenone, and 2.5 mM KHepes, pH 7.05. After its O2 content was reduced to about 10% of air saturation a t 10 "C by bubbling NO, the cell was closedand thefollowing additions were made in the sequence numbered on the traces. After each addition, the system wasallowed to reach equilibrium with the medium. 1, mitoplasts (2.61 mg of protein); 2, potassium succinate (about 50 nmol) predetermined to be slightly more than enough to exhaust the remaining O2 of the medium; 3, N-ethylmaleimide (40 nmol/mg); 4, valinomycin (300 ng/mg); 5, antimycin A (0.1 nmol/ mg); 6,air-saturated medium at 10 "C to make a total of 33.5 nmol of oxygen in the system; 7, 180 nmol of ferrocytochrome c; 8,first, 100 nmol of standard 0.1 N HC1 for calibration of the H' electrode, which was followed by two small additions of 0,to oxidize the remaining ferrocytochrome c in the system. The last addition of 02 was 50.0 pl of air-saturated medium a t 10 "C, containing 31.6 nmol of oxygen, for calibration. The chart speed was changed at specific points as indicated. The vertical burs on the traces set off the segments used for kinetic analysis. The dashed trace represents O2 uptake assuming that itoccurs a t a single exponential rate throughout its course.

8258

H+/O Ratio of Oxidase Cytochrome

Again, it will be noted that endogenous 0, and H+changes are essentially nil, confirming Fig.2, as indicated by the completely flat traces immediately following addition of O2 (Fig. 3, point 6) and just before addition of the calibrating HCl (Fig. 3, point 8). Processing of Rate Data-Although processing of the rate data to yield the H+/O flow ratio at level flow was briefly described earlier for the case of experiments inwhich electron flow was initiated by injection of O2 (15, 27), some details require emphasis. As already indicated, in thepresence of an optimal concentration of valinomycin, i.e. sufficient to almost completely discharge the membrane potential generated by extrusion of H+, the mechanistic H+/O ratio is most closely approached from the flow ratio only at the hypothetical instant of level flow, at which opposing force ( A ~ H +is) zero. Since the dead time, i.e. the time required for mixing, electrode response, and recorder response, is finite, even the earliest experimentally observed rate of H+ ejection and Oz uptake is already beyond the instant of level flow. To obtain the H+/O flow ratio at level flow, the observed rates of H+ ejection and O2 uptake taken at many successive time intervals duringthe reaction were extrapolated back to apparent zero time, the instant at which level flow presumably prevails. Because of the finite time required for mixing, the true zero time cannot be precisely fixed. However, from a combination of measurements of the 0, electrode response time per se obtainedfrom oscilloscope observations (15) and the response time of the O2 trace obtained from the recording system, a close approximation of the virtual or apparent zero time was empirically fixed as the time at which the O2 recorder trace begins its response to injection of ferrocytochrome c. To obtain the H+/ 0 ratio at apparent zero time, i.e. level flow,rate datafor both H+ ejection and O2 uptake were taken from the end of the dead time of the H+electrode to thetime at which H+ejection approaches its peak; this period is set off by short vertical lines on the H+ trace (Fig. 3). Since the final concentration of ejected H+ is not known owing to theincreasing rate of H+ backflow as ApH increases and since there is a slight degree of uncertainty regarding the truezero time owing to thefinite time required for mixing, mathematical considerations led to use of the procedure of Guggenheim (cf. Ref. 47)for situations in which the initial or final concentration of a reactant or product are notprecisely known. Many points were taken by computer from the unsmoothed traces and digitized, the reaction rates were determined at the midpoints of successive 0.1-s intervals, and various functions of the rate data were plotted uersus time. Under the conditions employed, the rates of both H+ ejection and 0, uptake at corresponding time intervals were found to correspond very closely to monotonic exponentials, as shown by the Guggenheim plots (Fig. 4) of the rates of H+ ejection and Oz, which are linear to r 3 0.99. Back-extrapolation of the plots to apparent zero time yielded level flow rates of86.6 nmol of H+/s or 1990 nmol of H+ ejected per min/mg and 21.8 nmol of oxygen/s or 501 nmol of oxygen uptake/min/mg. The H+/O ratio at level flowwas therefore 1990:501 = 3.97. The earliest H+/O ratio obtained from the directly measured part of the reaction was 3.2. From many experiments of this kind, with ferrocytochrome c added last, the extrapolated level flow H+/O ratios lay in the range 3.50-3.97, with the highest directly observed H+/O ratios in the range 3.0-3.3. Earlier experiments carried out with reactions initiatedwith Oz gave almost identical results, with level flow H+/O ratios of 3.60-3.96 (15). Determination of the Upper and Lower Limits of the H+/O Ratio: Rationale-Measurements of the H+/O flow ratio extrapolated to approximate level flow, such as those described

+I

\

0.5 1.0 1.5 2.0 2.5 TIME

(SI

FIG. 4. Processing of the data from the H+ ejection portion of Fig. 3.The l i e in line A shows changes in the rate of H+ ejection (AH+/At) measured a t 0.1-s intervals uersus time. Its slope is given by k in the Guggenheim equation ln(AH+/At)t = ln(AH+/At)o + k . t,

where (AH+/At)ois the initial rate of H+ ejection (86.6 nmol of H+/ s) and k is the slope of the line. Line B shows changes in the rate of O2 uptake measured also at 0.1-s intervals uersus time. The line is given bythe same exponential h(AO/At), = In(AO/At),, k . t, where (AO/At)o is equal to 21.8 nmol of oxygen/s and Iz has a different value than the rate constant for H+ ejection. (Note that the scale in y is not identical, and consequently, lines A and B are not parallel.) The initial H+/O ratio at approximate level flow is therefore 3.97. The zero time does not necessarily represent the moment the full reaction begins but the moment the recorder shows the initial increase in O2 concentration (see "Discussion"). Line C shows the decrease in the observed H+/O ratio as the reaction proceeds, calculated from lines A and B .

-

above and in Ref. 15, in theory yield the lower limit of the mechanistic H+/O ratio since the coupling coefficient q at level flow can be expected to have a value approaching but slightly less than 1.0 (37). Level flow H+/O ratios therefore set the lower limit to the mechanistic H+/O ratio, However, the mechanistic H+/O ratio can be more precisely fixed by a further refinement that allows determination of its upper limit from kinetic measurements, such as we have applied to determination of the upper limit of the H+/O ratioof succinate oxidation (27). In brief, the observed rate of H+ ejection (JH) under any given set of conditions is given by the expression

JH = d

o

- Jr.

(3)

in which Jo is theobserved rate of oxygen consumption, n is the number of H+ ejected per oxygenreduced, i.e. the mechanistic H+/O ratio, and JL is the rate of the H+ leak. When Equation 3 is divided by Jo,the expression JH "

Jo

- n - - JL Jo

is obtained, which is simple a statement of the obvious fact that the observed H+/O ejection ratio JH/Jounderestimates the mechanistic H+/O ratio (n) by the factor JL/Jo,i.e. the fraction of the total H+ ejected that undergoes back-leakage at a given rate of Oz uptake. Equation 3 can be used to determine the upper and lower limits of n from the effects on JH and Jo, and thuson JL,of agents that can alterthe rateof formation or the rate of utilization of respiratory energy, i.e. the high energy intermediate state,which in thechemiosmotic model is AiH+.For example, if the valinomycin concentration

H+/O Ratio of Cytochrome Oxidase is increased from zero to some maximal level in a series of test systems, the rateof utilization of respiratory energy (ie. AjiH+)to drive net K+ uptake will increase. In this case, both J Hand Jo will increase as valinomycin concentration is increased. However, the leak rate J L is not likely to remain constant since it will depend on the magnitude of the driving force for H+ backflow, namely A i i ~ +Since . Ab,+ will become smaller as it is increasingly utilized to drive K' uptake, JI,is therefore likely to decrease as valinomycin concentration is increased. From the algebraic form of Equation 3, the slope of a plot of JH versus JO from a series of tests at increasing valinomycinwould be expected to be greater thanthe mechanistic stoichiometry n and would thus set its upper limit. However, if the rate of 0, uptake and H+ ejection during ferrocytochromec oxidation is experimentally varied by adding increasing concentrations of a respiratory inhibitor, such as cyanide, to a system already supplemented with optimal K+ + valinomycin, tb,e rate of energy production would be systematically decreased. The rates of both electron flow and H' ejection will decrease with increasing cyanide concentration. However, in thiscase, A,&+ will tend to remain constant down to relatively low rates of O2 uptake but in any case is likely to decrease rather than increase. The leak rate JLwould therefore remain constant or decrease under these circumstances. The slope of a plot of JH versus Jo from such a set of measurements with a respiratory inhibitor would be close to the H+/O flow ratio at level flow and would thus constitute the lower limit of the mechanistic stoichiometry n. The effects of graded additions of 1)valinomycin and of 2) cyanide in the presence of valinomycin on the observed rates of 0,uptake and H' ejection thus will yield plots of JH versus Jo whose slopes are respectively above and below the mechanistic HC/ 0 ratio. By this rationale, the upper and lower limits of the mechanistic H+/O ratio can be determined without actually eliminating H' backflow or measuring it, as was shown in a preceding study (27). A detailed kinetic and nonequilibrium thermodynamic treatment of the rationale will be presented for publication elsewhere.' Upper Limit of the Mechanistic H+/O Ratio-The basic test system employed was that describedforFig. 3, with the reaction initiated by addition of a known amount of 02.The K+ concentration was held constant at 50 mM, but the valinomycin concentration was varied from 0 to 1.0 pg (0.2 nmol/ mg of protein), thusvarying the rates of both H' ejection and 0 2 uptake and presumably also the H' leak rate. Conversion of the rate datafrom the raw traces into digital form, preparation of Guggenheim plots, and line fitting were carried out by computer as described above (cf. Refs. 15 and 27). At each valinomycin concentration, the primary plots of log rate uersus time for both oxygen uptake and H+ ejection were linear from the end of the mixing and electrode dead time (0.5-0.8 s) to about 60-70% reduction of the added 02.For uniformity of treatment and tominimize errors attending use of single observations, the extrapolated zero time rates of H+ ejection and 0 2 uptake at each level of valinomycin were obtained from Guggenheimplots. as described above,and the resulting rates were plotted against each other. Fig. 5 shows the effect of varying the valinomycin concentration on the zero time rates of H+ ejection (JH) plotted versus the zero time rate of 0' uptake (Jo).Two series of experiments were performed with two different preparations of mitoplasts; in one set, antimycin A was omitted. All the zero time JHand Jo values obtained, six from each series,are plotted on the same coordinates. The plot, fitted by regression A. Beavis and A. L. Lehninger, manuscript in preparation.

1

8259

,

I

40

80 120 Jo , nmol O / min.rng

160

FIG. 5. Effect ofvalinomycin on the ratesof H+ejectionand O2 uptake: the upper limit of the H+/O ratio. The data were obtained from 12 experiments onthe same preparationof mitoplasts, six in the absence and six in the presence of antimycin A, according to thefollowingprotocol. The testmedium (1.35 ml; 10 "C) contained 200 mM sucrose, 50 mM KCI, and 2.0 mM KHepes, pH 7.05. The O2 content of the medium was first reduced to about 20%of air saturation at 10 "C by bubbling wet Nz.The cell was then closed, and additions of 4.0 PM rotenone, 200 nmol of ferricytochrome e, 3.4 mg of mitoplasts, and 270 nmol of succinate were made through the small port. After the system became completely anaerobic, as indicated by the Oz electrode trace, N-ethylmaleimide (40 nmol/mg) was added, and the system was incubated anaerobically to allown the ferricytochrome c to become fully reduced at the expense of the succinate, under monitoring with the H+ electrode, which required -3 min. Valinomycin was added in concentrations up to 400 ngfmg. In one series, antimycin A (0.1 nmol/mg) was added; in the other, antimycin A was omitted. After the traces stabilized under the anaerobic conditions, 63.5 nmol of oxygen wereinjected, and therates of 02 uptake and H+ ejection were recorded. After the remaining ferrocytochrome c was the .Oz completely oxidized by a series of small injections of 02, electrode was calibrated by adding small dlquots of the air-saturated medium (10 "C) at the end of the experiment. The pHelectrode was calibrated with 100-nmol pulses of standard HC1 before and after each experiment. Both sets of data were plotted on the same coordinates to show the close agreement.

analysis to r = 0.98, has a slope of 4.12, which thus represents the upper limit of the mechanistic H+/O ratio. Omission of antimycin A did not affect the essential linearity or slope of the plot, confirmingthe experiments in Figs. 2 and 3 showing that no significant endogenous electron transport or H+ ejection occurred under the conditions used. Lower Limit of the Mechanistic H+/O Ratio-To confirm the lower limit of the H+/O ratio for cytochrome oxidase as given by the extrapolated H+/O flow ratio at level flow as determined earlier (15) and inFig. 4, the rates of H+ ejection and 0, uptake were systematically varied in another series of experiments in which valinomycinwas present at a constant, near-maximal concentration by adding freshly prepared neutral cyanide at concentrations from 0 to 3 phl. Fig. 6 shows the plots Of JHversus JOfor two sets of experiments performed with twodifferent mitoplast preparations; again, in one series of experiments, antimycin A was omitted. The JH versus Jo plot for series A, obtained from six experiments, has a slope of 3.86 and that for series B, from six experiments with antimycin omitted, has a slope of 3.83. Since the upper limit given bythe plots in Fig. 5 is 4.12 and thelower limit is about 3.86 (Fig. 61, it may be concludedthat the mechanistic H+/O ratio for the cytochrome oxidasereaction is very closeto 4.0. The intercept on the JOaxis is larger in the presence of antimycin than in itsabsence, indicating that the state4 rate

8260


+

I

0

E

=

1000-

I -2

I

,

, 200

I

40

I20 ' 160. ' Jo , nmol 0 / min. mg

80

'

J,,

260

FIG. 6. Lower limit of the H+/O ratio from titration with cyanide; effectof antimycin. The medium (total volume, 1.35 ml; 10 "C) was prepared as described for Fig. 5. Ferricytochrome c was added at 200 nmol and mitoplasts at 4-5 mg. After the ferrocytochrome c was completely reduced and the system was completely anaerobic and in equilibrium, 40 nmol/mg N-ethylmaleimide, 150 ng of valinomycin, and 0.1 nmol/mg antimycin A were added, followed by freshly neutralized cyanide in concentrations ranging from 0.2 to 2.2 p ~ Two . minutes after stabilization of the traces, the reactions were initiated by introducing 100 pl of medium air-saturated at 10 "C, containing 63.0 ng atoms of oxygen. 02 uptake and H+ejection were recorded, and the datawere processed and plotted as for Fig. 4.Plot A gives data obtained from two different mitoplast preparations. The mitoplasts for plot A (0)were added at 5.0 mg and for pZot B at 4.0 (e).Plot B was obtained from an identical series of experiments in which antimycin A was omitted.

is increased by antimycin, presumably the result of its known uncoupling activity. Upper and Lower Limits of the H+/ORatio Determined from Other Variables-In principle, the methods described above can yield the upper and lower limits of the H+/O ratios with any variable that can inhibit respiratoryenergy utilization or production, respectively. To show the validity of this principle, the effect of two other sets of conditions was examined. In the first, the test system was as in Fig. 5, with valinomycin constant a t 200 ng/mg of protein, but with K+ concentration varied from 0 to 150 mM, with LiCl added to keep the total concentration of uni-univalent salt constantat 150 mM. Since the rate of K+ entry on valinomycin in exchange for ejected H+ will be determined by the K+ concentration, data on JO and J H as K+ concentration is varied should set the upper limit of the H+/O ratio. The initial rate data obtained from seven different concentrations of K+ are plotted in Fig. 7. Again, the plot of JH versus Jo was found to be near-linear over a wide range of K+ concentrations. Its slope is 4.19, which is in close agreement with the value 4.12 for the upper limit of the H+/O ratio obtainedwith valinomycin varied and K+ fixed (Fig. 5). To obtain an independent confirmation of the lower limit of the H+/O ratio, some means of inhibiting electron flow other than cyanide were required. For this purpose, the temperature of the testsystem was varied from 5 to 30 "C. In this case, Ca2+was used instead of K+ and valinomycin as the charge-compensating permeant cation, but all other conditions were as for Fig. 6. The same mitoplast preparation was used for all the experiments, which could be carried out within a 2-h period by successively increasing the temperature of the water bath starting at 5 "C. Since the temperature coefficient of the 50% response time of the two electrodes was found to be about the same, nosignificant differential error was introduced. The JH versus Jo plot obtained from 13 experiments

,

,

400 600 nrnol 0 1 rninarng

800

FIG. 7. Effect of KC1 concentrationon the ratesof 0 2 uptake and H+ ejection: the upper limit of theH+/O ratio. The reaction medium for the seven experiments contained 0,5,12,22,52,122, and 152 mM KC1 plus 2.0mM LiHepes buffer, pH 7.05. In all systems, the total concentration of uni-univalent salt was made 155 mM with LiC1. The temperature was 10 "C. The systems were supplemented with 2 pM rotenone, 200 nmol of cytochrome c, and mitoplasts (3.0 mg). After the oxygen was exhausted and thecytochrome c was fully reduced, N-ethylmaleimide (40 nmol/mg) and valinomycin (150 ng/ mg) wereinjected. After 2 min, 100 pl of air-saturated medium (10 "C) were injected, and the changes in 02 and H+ were recorded at 120 cm/min. When each system came to full equilibrium, after theejected H+ had been fully reabsorbed via the leak, antimycin A (0.1 nmol/ mg) wasadded, and additions of 02 were made to oxidize the remaining ferrocytochrome c and to calibrate the O2electrode.

I

IO0

260

,

300 400 500

J o , nmol.O/min.mg

FIG. 8. Lower limit of the H+/O ratio obtained from rate data at different temperatures. The test system was exactly as for Fig. 4 with 4.0 mg of mitoplasts and with 3.65 mM Ca2+replacing valinomycin. Antimycin A was present in all systems at 0.05 nmol/ mg of protein. The temperature was varied from 9 to 36.5 "C. The 02 solubility data atdifferent temperatures were obtained from Ref. 40. The line was fitted to r = 0.994.

at five different temperatures is shown in Fig. 8. The plot gave a slope of 3.96, compared to thevalues 3.83 and 3.86 for the lower limit of the H+/O ratio obtained on varying cyanide concentration (Fig. 6). This experiment was of special interest because the rate of H+ backflow is greatly depressed as the temperature is lowered. Thus, thevalidity of the upper limitlower limitrationale was sustained by these two sets of experiments employing independent means of altering the rates of electron flow and of utilization of the respiratory energy. Other Observations-The permeant divalent cations Ca2+ and Sr2+can replace K+ (+ valinomycin) for exchange with the extruded H+; in fact, the datain Fig. 6 were obtained with Ca2+.The only requirement is that theconcentration of Ca2' or Sr" must be sufficiently high to yield maximal rates of O2 uptake over the entire reaction period so that the rate of


8261

electron flow is not impeded by the rateof entry of Ca” nor important, the initial ratesof O2consumption and H+ejection by the depletion of Ca2+ from the medium as the reaction have been determined under near “level flow”conditions, i.e. proceeds. A concentration of 1.0 mM Ca”, a very large excess, when the driving force (A;H+) responsible for the backflow of H+ is stillminimal. was generally used. used Although the highly sensitive 02 electrode (33, 34) 2-n-Nonyl-4-hydroxyquinoline N-oxide (cfi Ref. 48), myxothiazol (49), and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzo-here has allowed the estimation of the ratesof O2consumption thiazole (50),inhibitors of electron flow through site 2, when only 100 ms, or less, after the beginning of the reaction, the added in concentrations just sufficient to completly inhibit pH electrode with its rather sluggish response time (90% in succinate oxidation, did not lower the level flow H+/O trans- 600 or more ms) has posed a serious problem to the accurate location ratios for the cytochrome oxidase reaction. These determination of the H+/O ratio under nearlevel flow condiobservations, together with those of Ref. 48, would seem to tions. To overcome this problem, it has been assumed, in eliminate the claim (30)that H+ translocation observed dur- accordance with the chemiosmotic hypothesis, that the vecing the cytochrome oxidase reaction does not actually arise torial translocation of protons (whatever its mechanism) deof electron flow or from the cytochrome oxidase reaction but from interaction of pends directly on both the rate and extent ferrocytochrome c with components of site 2 on the O2 side oxygen consumption. Fig. 1 shows that the rates of O2 consumption and H+ of the antimycin block. uptake follow, for a long portion of the reaction, the kinetics DISCUSSION of the following simple exponential equation: The results of experiments described in this paper clearly ds - = k[S]o (5) show (i) that cytochrome oxidase can vectorially translocate dt H+ across the mitochondrial inner membrane during, and as a consequence, the flow of electrons from ferrocytochrome c where k represents the rate constant (in s-l) and [SI,, the to oxygen (1, 2), and (ii) that the number of H+ ejected per total concentration of either the substrate ( 0 2 ) or the product pair of electrons reducing half a molecule of 0, (H+/2e- or (H+) at the beginning and end of the reaction, respectively. H+/O) is higher than 2 and, in all probability, as high as 4. It For this type of reaction, the integrated form of Equation 5 is, however, necessary to emphasize technical and methodoln[S], = ln[S]o + k.t (6) logical difficulties inherent in this type of determination (8, 51, 52) and to comment about the measurements that have can be used to calculate the initial or level flow rate of the been takenin theseexperiments to overcome difficulties reaction by simply multiplying the rate constant by the total which otherwise could contribute either to underestimation extent of either the oxygen initially added or the protons or overestimation of the results. utilized at theend of the reaction. Underestimation of the H+/O ratio isfrequently the result However, Equation 6 cannot be applied to a reaction such of classical O2 pulse experiments in which O2 consumption is as that represented in Fig. 3 in which the total amount not determined and H+ ejection is estimated from the ApH of protons vectorially ejected is not exactly known because after the reaction is over and the added 0 2 has been totally the protons appearing in themedium represent only a net balconsumed. At this point, a large fraction of the protons have ance between two processes, one ejecting protons and the already returned to thematrix, driven by a A;+, generated in other consuming them. The Guggenheim modification of full from almost the beginning of the reaction (static head Equation 6 conditions). ln(AS/At), = ln(AS/At),, + k.t (7) The oxidation of exogenous ferrocytochrome c by the cytochrome oxidase generates 2 negative charges (2 OH-) in the can advantageously be used in these cases since a knowledge mitochondrial matrix/pair of electrons reducing oxygen to of the totalamount of substrate utilized or product formed is water. This extraincrement in bothA* and ApH constitutes not required (47). an extra driving force for the return of H+, which is not Fig. 4,A and B, shows plots of the log of AS/At versus time. present when other substrates are utilized, since the 2 scalar They intercept gives directly the initial ratesof the processes H+ released during their oxidation directly neutralize the 2 which in this case are 86.6 nmol/s for H+ ejection and 21.8 OH- formed as a product of the reduction of 0, to water. nmol of oxygen for oxygen consumed, i.e. the H+/O stoichiNeither the extrapolation of the pure backflow of H+ to an ometry at near level flowis 3.97. arbitrarily chosen time pointa t which reduction of the added Although the Guggenheim plot can provide directly the oxygen is still incomplete (46)nor the use of inhibitors to initial rate of the reaction disregarding the values of the rate prevent the facilitated return of H+ through specific carriers constants and the total extent of substrate or product, the (43)will prevent the underestimation of the results since the experiment shown in Fig. 3 poses the problem of the long driving force is present from the beginning of the reaction delay in the response of the pH electrode. It is not known and persists for a long time after the O2 has been totally what the changes in the rates of H+ ejection (AH+/At) are exhausted. There is no experimental evidence showing that during the first 500-600 ms after the reaction begins. It is the innermitochondrial membrane is impermeable to protons only assumed that the simple exponential reaction observed in the presence of a strong driving force. for over 2 s can also hold for this initial unobserved period of Other causes of underestimation of the H+/O ratio are (i) H’ ejection. A further problem in this type of calculation is the use of excessive amounts of the respiratoryinhibitor to determine the instantwhen the reaction begins. In this, as antimycin A, which also has a protonophore activity and can well as in previous experiments (15), the transition (shown stimulate H+ backflow, and (ii) the use of nonbiological re- by the pen of the recorder) from the anaerobic to theensuing ductants of cytochrome c (ferrocyanide, N,N,N’,N”tetraoxygenated state due to theinjection of 0 2 has been taken as methyl-p-phenylenediamine), which in either theirreduced or the zero time of the reaction. Obviously, this point inthe time oxidized forms may adversely influence the pathway or the course of the reaction does not necessarily represent the rate of electron flow, H+ translocation, or H+ backflow. All instant thefull reaction starts, but rather some time after it, these causes of error have been avoided in this work. Most since the response time of the recorder is apparently longer

8262


than the response time of the O2 electrode and the time 11. Brand, M. D Harper W. G., Nicholls, D. G., and Ingledew, W. J. (1978) FEBS Leti.'95,125"129 required for the magnet to uniformly mix the O2 in the 12. Al-Shawi, M. K., and Brand,M. D. (1981) Biochen J. 200,539-546 medium. Therefore, the extrapolation of the linear portion in 13. Wikstrom, M., and Penttila, T. (1982) FEBS Lett. 144, 183-189 14. Lemasters, J. J., Gmnwald, R., and Emaus, R.K. (1984) J. Biol.Chem. Fig. 4A to thiszero time cannotoverestimate, but may under259,3058-3063 B., Alexandre, A., Davies, P., and Lehninger, A. L. (1982) Proc. estimate, the initial rate of H+ ejection since during this dead 15. Reynafarje, Natl. Acad. S e i U. S. A. 7 9 , 7218-7222 period of about 500 ms the mitochondria have already used 16. Lemasters, J. J., and Billica, W. H.(1981) J. Biol.Chem. 256, 12949190~7 some oxygen and generated enough driving force (Aji~+) to 17. Sorgato, M. C., Branca, D.., and Ferguson, S. J. (1980) Biochem. J. 1 8 8 , reabsorb a considerable fraction of the ejected protons. 945-948 Krab, K., and Wikstrom, M. (1978) Biochim. Biophys. Acta 504,200-214 18. A more accurate zero time could be calculated by using 19. Sigel, E., and Carafoli, E. (1978) Eur. J. Biochem. 89,119-123 Equation 6 and back-extrapolating the linear portion of O2 20. Casey, R. P., Chappell, J. B., and Azzi,A. (1979) Biochem. J. 182, 149156 consumption to a pointwhich exactly reproduces the amount 21. Sigel, E., and Carafoli, E. (1979) J. BioL Chem. 254,10572-10574 of O2 added. However, it was found that in O2pulse experi- 22. Sone, N., and Hinkle, P. C. (1982) J.Biol. Chem. 257,12600-12604 L. J., Bisson, R., Capaldi, R. A., Steffens, G. C. M., and Buse, ments the kinetics of O2 consumption by respiring mitochon- 23. Prochaska, G. (1981) Biochim Bioph s. Acta 637,360-373 dria is quite complex (53, 54, 55), but as long as H+ ejection 24. Proteau, G., Wriggleswortt, J. M., and Nicholls, P. (1983) Biochem. J. 210,199-205 and electronflow or O2uptake aredirectly linked, the extrap- 25. Sone, N. and Yanagita, Y.(1984) J. Biol. Chem. 259,1405-1408 olation to the point considered above is the most reliable at 26. Solioz, M., Carafoli, E., and Ludwig, B. (1982) J. BWL Chem. 2 5 7 , 15791582 the present time. 27. CoSGLL. E., Reynafarje, B., and Lehninger, A.L. (1984) J. Biol.Chem. 259.48n2-4811 --. - .- - The upper and lower limits of the H+/O ratioof cytochrome 28. Pietrodon, D., Zoratti, M., Azzone, G. F., Stucki, J. W., and Walz, D. (1982) oxidase calculated here using a completely different experiEur. J. Biochem. 127,483-494 P. (1982) in Oxidases and Related Systems (King, T.E., Mason, mental approach (Figs. 5-8) have theoretical support as long 29. Mitchell, H. S., and Morrison, M., eds) pp. 1247-1268, Pergamon Press, Oxford, as the rates of H+ ejection and O2 uptake are measured a t Eneland Guerrieri, F., LONSSO,M.. Izzo, G. Boffoli,D., Capuano, F., near level flowand/or the driving forces for both H+ ejection 30. Papa,-S., Ca itanio, N., and Altamura, N. (1680) Biociem. J. 192,203-218 and H+ reuptake are the only important factors operating 31. Mitciell, P., Mitchell, R.,Moody, A. J., West, I. C., Baum, H., and Wrigglesworth, J. M.(1985) FEBS Lett. 1 8 8 , l - 7 these processes. However, it is not clear at the present time 32. Lehninger, A.L., Reynafarje, B., Davies, P., Alexandre, A., Villalobo, A., and Beavis, A. (1982) in Mitochondria and Microsomes (Lee, C. P. whether or not specific conductivity coefficients, closely reSchatz, G., and Dallner, G., eds) pp. 459-479, Addison-Wesley Puhlishini lated with the structural nature of the membrane, greatly Co., Reading, MA P. W., and Grenell, R. G. (1962) J. Neurophysiol. (Bethesda) 2 5 , influence the pathways of the electron flow-linked ejection 33. Davles, fiRI-fiFI.? "- "" and reuptakeof protons. Indeed, a numberof thermodynamic 34. Davies, P. W. (1962) in Physical Techniques in Biological Research(Nastuk, W. L., ed) Vol. 4, pp. !37-179, Academic Press, New York and kinetic inconsistencies have been reported between the 35. Rottenberg, H. (1979) Bmchzm. Biophys. Acta 549,225-253 observed values of AbH+,AGp, and therates of O2uptake and 36. Stucki. J. (1980) Eur. J. Biochem. 109.269-283 37. Caplan, S. R., and Essig, A. (1983) Bioenergetics and Linear Non-EquilibATP synthesis (reviewed in Refs. 5, 56, and 57; see also Ref. rium Thermodynnmics: The Steady State, Harvard University Press, 58 and 59), which have strongly suggested that mitochondrial Cambridge, MA 38. Krab, K., Soos, J., and Wikstrom, M. (1984) FEBS Lett. 178,187-192 or chloroplast energy coupling may proceed, a t least in part, 39. Affolter, H., and Sigel, E. (1979) AnaL Biochem. 97,315-319 40. Reynafarje, B., Costa, L., and Lehninger, A. L. (1985) A d . Biochem. 1 4 5 , via localized H+ currents. 406-418 Furthermore, it is very unlikely that the functional or 41. Pedersen, P. L., Greenawalt, J. W., Reynafarje, B., Hullihen, J Decker, G. L., Soper, J. W., and Bustamante,E.(1978) Methods Cell B&L 20,411effective H+/O ratio of the cytochrome oxidase reaction under 481 actual intracellular conditions is 4, owing to thefact that the 42. Speck, S. H., Dye, D., and Margoliash, E. (1984) Proc. Natl. Acad. S e i U. S. A. 81,347-351 coupling coefficient is necessarily lower than 1.0, as has been 43. Brand, M.D., Reynafarje, B., and Lehninger, A. L. (1976) J. BWL Chem. (36). Neverdeveloped in detailby others, in particular Stucki 251,5670-5679 44. Harris, E. J., and Bangham, J. A. (1972) J.Membr. Bid. 9,141-154 theless, knowledge of the mechanistic H+/O ratio has impor- 45. Gupte, S., Wu, E . 4 , Hoechli, L., Hoechli, M., Jacobson, K., Sowers, A. E., tant implications for understanding of the mechanism of both and Hackenbrock, C. R. (1984) Proc. Natl. Acad. Sci. U.S. A. 81,26062610 electron transport and energy transduction promoted by this 46. Mitchell, P., and Moyle, J. (1967) Biochem. J. 105,1147-1162 47. Cornish-Bowden, A. (1979) Fundamentals of Enzyme Kinetics, pp. 9-41, complex enzyme. LlVY

1. 2. 3. 4. 5. 6.

7. 8. 9. 10.

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