a study of oxidative phosphorylation with o’*-labeled inorganic … · · 2003-02-21a study of...

A STUDY OF OXIDATIVE PHOSPHORYLATION WITH O’*-LABELED INORGANIC PHOSPHATE*

BY MILDRED COHN

(From the Department of Biological Chemistry, Haward Medical School, Boston, Massachusetts, and the Department of Biological Chemistry, Washington

University School of Medicine, St. Louis, Missouri)

(Received for publication, October 9, 1952)

It has been generally accepted for some time (1) that phosphorylation processes occur concomitantly with oxidation in the electron transport system via pyridine nucleotide, flavoprotein, and cytochrome c to oxygen; indeed in recent years direct experiments by Lehninger and coworkers have demonstrated that phosphorylation accompanies the oxidation of reduced diphosphopyridine nucleotide in mitochondrial preparations (2). How- ever, there have been no experiments which give an insight into the nature of the interaction of the electron transport system with phosphate or any part of the phosphorylating system. The present paper deals with a new approach to this problem with Ols-labeled phosphate.

The rationale of the method depends upon the fact that the phosphate group does not necessarily proceed intact through a sequence of phosphorylation, transphosphorylation, and dephosphorylation reactions, but may lose one or more of its original oxygen atoms. Thus, by labeling the oxygen of the phosphate group, it may be possible to follow the path of the phosphate group through a series of reactions in which the phosphorus leaves no trace. If inorganic phosphate labeled with 01* is taken up in organic linkage by the formation of a carbon-oxygen bond as in phosphorylase reactions (3) and in the glyceraldehyde phosphate dehydrogenase reaction: the oxygen bridging the carbon and phosphorus becomes labeled with O’*. Should the organic phosphate now be cleaved by the rupture of the phosphorus-oxygen bond as in phosphatase reactions (3), the organic moi- ety remaining would contain 018. Moreover, if inorganic phosphate were formed in such a reaction, one of the four labeled oxygens would have been replaced by normal oxygen from the water. This opens up two possibil- ities, (1) the identification of phosphorylated intermediates by the presence of Oi* in the dephosphorylated products and (2) the detection of reactions otherwise unobservable by following the loss of Oi* from inorganic phosphate.

* The part of the work conducted at the Harvard Medical School was supported by a contract between Harvard University and the Atomic Energy Commission.

1 Cohn, M., unpublished data.

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736 OXIDATIVE PHOSPHORYLATION

In the current study, the second possibility, namely the loss of 018 from inorganic phosphate, has been explored in oxidative phosphorylation. It has been established that there is a reaction which causes a rapid loss of 018 in the phosphorylation, coupled with the oxidation of or-ketoglutarate, succinate, and fl-hydroxybutyrate in rat liver mitochondria. The number of cycles of reaction in which inorganic phosphate has participated can be quantitatively estimated, since only one-fourth of the labeled oxygen is lost in each cycle, on the assumption that a monoester of phosphate is split. The conditions required for this reaction to occur have been investigated and an attempt has been made to localize the reaction in the oxidation and in the phosphorylation systems.

Methods

Preparation of 018-Labeled Phosphate-2 liters of oxygen gas enriched with 018 were generously supplied by Dr. A. 0. Nier. The oxygen gas was converted to water by alternately passing hydrogen and the isotopic oxygen over copper at 300” and collecting the water in a trap cooled with dry ice. The water was converted to orthophosphate by reaction with PZOs as described previously (3). The 018 content of the phosphate was 2.60 atom per cent excess and the same batch was used in all the experiments described.

Materials Used-The ar-ketoglutarate was obtained from the Nutritional Biochemicals Corporation, and the dl-P-hydroxybutyrate from the Mal- linckrodt Chemical Works. The diphosphopyridine nucleotide (DPN) used in the experiments with dl-p-hydroxybutyrate was obtained from the Sigma Chemical Company and was approximately 90 per cent pure. The oxalacetic acid used was kindly supplied by Dr. R. K. Crane. The adeno- sinetriphosphate (ATP) and tris(hydroxymethyl)aminomethane (Tris) used were products of the Sigma Chemical Company.

EXPERIMENTAL

Isolation of Mitochondria-The mitochondria were obtained from rat liver by differential centrifugation in isotonic sucrose essentially by the method of Schneider (4), as modified by Kielley and Kielley (5). The individual preparations varied somewhat in the rate of oxidation and phosphorylation as well as in their ATPase activity. Since the supply of Ol*- labeled phosphate was limited, no experiments were discarded, regardless of the activity of the particular preparation. This variation in activity did not affect the results since all the measurements were relative. The amount of mitochondria used in 1 ml. of reaction mixture corresponded to t,he amount obtained from 0.1 gm., wet weight, of liver tissue.

Measurement of Oxygen Consumption ano? Phosphorylation-The reaction


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M. COHN 737

was allowed to proceed at 25’ unless otherwise stated. When analyses for phosphate and 018 in the phosphate were desired for one time period only, the oxygen uptake was measured in 3 ml. of reaction mixture with air as the gas phase. At the time the experiments with cu-ketoglutarate as substrate were performed, a Scholander microrespirometer apparatus (6) was available and the oxygen uptake could be followed. In other experiments, a conventional Warburg apparatus was used for manometric measurements. The measurement of oxygen uptake was merely used as an approximate index of the oxidative activity of the mitochondrial preparation. In those experiments in which a series of samples was withdrawn at different times, oxygen uptake was not measured.

Phosphorylation was followed by the measurement of inorganic phosphate and 10 minute-hydrolyzable phosphate by the Fiske-Subbarow method (7). Since adenylic acid was used as the acceptor of the phosphate, the amount of ATP formed is used as a measure of the phosphorylation. This method for determining phosphorylation is valid with this type of mitochondrial preparation, at least for short periods when ATPase activity is relatively unimportant.

Isolation of Inorganic Phosphate-At the end of the reaction, 1 ml. of ice-cold 20 per cent trichloroacetic acid was added to 3 ml. of the reaction mixture. The precipitate was centrifuged and an aliquot of the supernatant fluid was analyzed for inorganic phosphate and 10 minute-hydrolyzable phosphate. To insure maximal recovery of phosphate, the reaction vessels were then rinsed with 1 ml. of ice-cold 5 per cent trichloroacetic acid and the rinsings were added to the original trichloroacetic acid precipitate and centrifuged. The supernatant fluid was added to the original supernatant fluid from which an aliquot had been removed for phosphate analysis. The ATP and adenylic acid were removed from the solution at this point by treatment with Norit. The supernatant fluid from the Norit treatment and the two washings of the Norit yielded a volume of approximately 8 ml. The inorganic phosphate was precipitated with Ba++ in alkaline solution and treated as described previously (3). The amount of phosphate present in 3 ml. of reaction mixture, approximately 30 to 60 PM,

is insufficient for an 018 analysis, and unlabeled phosphate must be added to the sample. After the phosphate had been precipitated twice with Ba++ and the Ba++ had been removed in an acid solution with KzS04, the solution containing the labeled inorganic phosphate was brought to an accurate volume of 2 ml. An aliquot was removed for analysis of inorganic phosphate and an accurately weighed amount of ordinary KHzPO~ (about 300 PM) was dissolved in the solution. The dilution ratio is equal to the total number of moles of phosphate divided by the number of moles of labeled

2 Personal communication from R. K. Crane and F. Lipmann.


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phosphate, and in these experiments was of the order of 10. The diluted sample was treated as described previously (3) to obtain the monobasic potassium phosphate salt for analysis of 018.

Analysis of Phosphate for Or*--The amount of phosphate available for isotopic analysis of oxygen in these experiments, even after the addition of ordinary phosphate, was approximately 10 times smaller than in former experiments (3). The method, therefore, had to be modified somewhat, although the principle of the method is the same; namely, the decomposi- tion of anhydrous KHzP04 to yield HzO, which is subsequently equilibrated with COZ. To avoid transfer of such small amounts of water, the decom- position of the KHzPOd and the equilibration with COZ were all carried out in the same vessel. The solid salt was placed in a tube about 1.5 ml. in volume, which had a break seal and a side arm. The vessel was sealed by its side arm to a vacuum line and 1.3 ml. of CO2 were transferred into the tube by cooling the tube with liquid Nz. With the CO2 still frozen, the tube was sealed. The salt was then heated gently until all the phosphate had been decomposed. The water and COZ gas were allowed to equili- brate at room temperature for 3 days.

In this modification of the procedure, it is not possible to measure the amount of water formed directly, a quantity which is needed to calculate the dilution of the 018 by the COZ. A satisfactory method for the deter- mination of water formed is by an analysis of the residual metaphosphate since the reaction of orthophosphate to metaphosphate and water is quantitative. The small tubes containing the residual metaphosphate were placed in larger test-tubes and covered with 1.0 N H&SO4 and heated at 100’ with frequent stirring for about 20 minutes to insure complete hydrolysis. The contents were then transferred with many washings to a volumetric flask and an aliquot taken for analysis of inorganic orthophosphate by the Fiske-Subbarow method. The over-all error for the Or* concentration, as determined by the method outlined, has been found empiri- cally to be about 10 per cent, although the error in the mass spectrometric analysis is about 1 per cent. This is not surprising in view of the many steps involved, including two phosphate analyses.

Results

When it was found in an exploratory experiment on the oxidative phos- phorybtion of or-ketoglutarate that less than 10 per cent of the Or* initially present in the inorganic phosphate remained after 1 hour of reaction, it was realized that the inorganic phosphate must have passed through many reaction cycles. If one envisages a sequence of reactions in which inorganic phosphate is taken up in organic linkage and then split hydrolytically with an O-P cleavage, 75 per cent of the labeled 0’8 will be retained in the in-


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M. COHN 739

organic phosphate formed after one cycle. Should this inorganic phosphate now react in another cycle, again 75 per cent of the remaining labeled 01* would be retained; that is, 56 per cent of the initial Ol*, as indicated in the scheme in Fig. 1. The relationship of the final and initial

0’“H ?‘“H 1) ROH + HO’“-b-0’“H

38

- RO-r-0’“H + H,O 0’”

, ?‘“H 0’“H I’) RO+t-0’“H + H,O” - ROH + HO%-0’“H

’ 0’8 58

0’“H ?‘“H Q’“H 2) 4 ROH + 4 HO’*-b-OEH -

i;l8

I RO-e-0’“H + 3 RO+OEH

0’” 0’” 2’) i I I I +4H,O

0 3) :

I

F= FINAL 0’” CONCENTRATION N = NUMBER OF REACTION CYCLES

INITIAL 0’” CONCENTRATION

NIO I 2 3 4 5 6 7 8 9 IO

F ’ I .75 .56 A2 .32 .24 .I8 .I3 JO .08 .06

FIG. 1. Scheme of series of reactions leading to loss of Or* from inorganic phosphate. Strictly formulated, a sample of phosphate containing 2.60 atom per cent excess 0’8 randomly distributed would contain the following fractions of the possible varieties of molecule: Fpodla = (0.974)4, Fpos~60u = 4 X (0.026) X (0.974)3, Fpo21~02~8 = 6 X (0.026)2 X (0.974)2, F~OIS,,~IB = 4 X (0.026)s X (0.974), Fpo4m = (0.026)4. How- ever, the difference between a consideration of this assembly of molecules, with approximately 4 X 2.6 per cent of the molecules in the form POar60rs and 2.6 per cent in the form Pod’*, is quantitatively negligible and makes no essential difference in the reasoning.

concentrations as a function of N, the number of reaction cycles, is given by the equation in Fig. 1, and the values are given for N (from 1 to 10).

Oxidative Decarboxylation of a-Ketoglutarate-From the observation that a rapid turnover of inorganic phosphate occurs during the over-all oxidative phosphorylation of a-ketoglutarate, it could not be concluded that the two phenomena are intrinsically interrelated. There might be an enzyme in the mitochondrial preparation which catalyzes an exchange between inorganic phosphate and water. The observed result could also


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be achieved by a non-oxidative reversible reaction of ol-ketoglutarate; namely, the addition of inorganic phosphate to the keto group, followed by enolization and dehydration. If the reverse reaction entailed a P-O cleavage, the Ols in the inorganic phosphate would be replaced by normal oxygen from water. The series of experiments summarized in Table I was designed to establish that the loss of Ols from inorganic phosphate occurred only upon transfer of electrons from ar-ketoglutarate all the way

TABLE I

Loss of 0’8 from Znorganic Phosphate under Various Conditions with a-Ketoglutarate As Substrate

Reaction mixture, phosphate buffer 0.02 M, pH 7.4; MgClz 0.005 M; adenylic acid

0.008 M; liver mitochondria 0.6 ml.; total volume 3.0 ml.; T = 25”. Initial concentration of 018 in inorganic phosphate, 2.60 atom per cent excess.

Added components Gas phase

1. None 2. “ 3. 0.01 M cY-ketoglutarate

4. 0.017 M a-ketoglutarate + 0.04 M oxal-

acetate 5. 0.01 M ol-ketoglutarate + 0.01 M malon-

ate -I- cytochrome c, 1 X low5 M

6. Same as (5) + 2,4-dinitrophenol

.I- Nitrogen Air Nitrogen

“

Air “

- I Atom per cent excess 0’8 in

inorganic phosphate

Experi- ment 1

20 min.

2.00

2.13

2.23

1.00 1.99

I Cxperimenl 2

60 min.

1.94

1.77* 1.69t

2.38

0.24

I- -l-

Experi- ment 3

70 min.

1.95

0.24 1.64

* Thunberg tube. t Scholander apparatus.

to oxygen. The possibility of a reversible reaction between cr-ketoglutarate itself and inorganic phosphate leading to loss of 018 is eliminated, since the addition of cr-ket.oglutarate under anaerobic conditions yields no more reaction than the control without added substrate. It should be pointed out that the controls, whether aerobic or anaerobic, always indicated a loss of 01* from the inorganic phosphate of the order of 25 per cent. This effect is not due to dilution by phosphate from the mitochondria, as will be shown in a later experiment. At the moment, the reaction occur- ring in the controls which lead to this loss of 01* is not known. However, the extent of reaction is small compared to that observed with the malonate-inhibited oxidation of cr-ketoglutarate.


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M. COHN 741

The phosphorylation which occurs at the substrate level in the oxidation of a-ketoglutarate has been demonstrated by two anaerobic reactions (8, 9), viz. the reaction with oxalacetate to yield succinate, carbon dioxide, and malate, and the dismutation reaction with ammonia to yield succinate, carbon dioxide, and glutamate. Both reactions have been studied with 01*-labeled inorganic phosphate; the results of the first reaction are given in Table I. The second reaction, with initial 018 concentration of 2.60 atom per cent excess, yielded values of 018 concentration of 2.32 and 2.22 atom per cent excess after 30 and 60 minute incubations at 25”, respectively, under the conditions described by Hunter and Hixon (9). These results indicate that the turnover of the inorganic phosphate, which mani-

TABLE II

Oxidation of dl+Hydroxybutyrate

Reaction mixture, Tris buffer 0.02 M, pH 7.4; phosphate 0.02 M; MgCl; 0.005 M;

adenylic acid 0.008 M; cytochrome c 1 X 10-S M; DPN 3 X 10e4 M; dl-&hydroxybutyrate 0.02 M; liver mitochondria 0.2 ml. per ml. of reaction mixture; T = 25”; gas phase air. Initial concentration of O’* in inorganic phosphate (PJ, 2.60 atom per cent excess.

Time APi Atom per cent excess 0’8 in inorganic phosphate

min. wf

Initial (O”) 2.59 0 -2.4 2.66 5 -6.3 2.36

20 -12.6 1.36 60 -9.2 0.56

fests itself by loss of 018 from the phosphate, is limited to the reactions associated with the oxidative steps in the electron transport system from DPNH to oxygen and is not associated with the substrate level oxidation reaction in which cr-ketoglutarate is converted to succinate and COZ.

Oxidation of ,&Hydroxybulyrate-For a study of oxidative phosphorylation associated with the electron transport system only, fl-hydroxybutyrate is a choice substrate since it is a one-step oxidation (10) and thereby obvi- ates the complication of adding an inhibitor such as malonate in the cr- ketoglutarate oxidation. The results in Table II show the disappearance of 018 as a function of time during the oxidation of dl-/Lhydroxybutyrate. The first sample was taken just after mixing all the components at 0” and the second sample was taken 5 minutes later when the reaction mixture had come to temperature equilibrium. This latter sample is considered to be at zero reaction time. It will be noted that both samples showed no loss of 018, indicating that the loss of Ols in controls at longer periods is


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real and not due to any rapid mixing with inorganic phosphate initially present or formed from organic phosphates already existing in the mitochondrial system.

Comparison of Diferent Substrates-One approach to localizing the phosphate turnover reaction at a given site or sites in the electron transport system is to study the reaction during the oxidation of substrates which do not have all steps in common through the electron transport system. Both ar-ketoglutarate and /3-hydroxybutyrate oxidation utilize the system from DPN to oxygen, but the oxidation of succinate does not involve the formation and subsequent oxidation of reduced DPN. Thus, if the phos-

TABLE III

Comparison of Different Substrates

Reaction mixture, Tris buffer 0.02 M, pH 7.4; phosphate 0.02 M; MgClz 0.005 M; adenylic ,acid 0.008 M; cytochrome c 1 X 10s6 M; liver mitochondria 0.6 ml.; total volume 3.0 ml.; T = 25“; gas phase air. Initial concentration of 018 in inorganic phosphate, 2.60 atoms per cent excess.

Substrate Time APi

None

0.01 M a-ketoglutarate -I- 0.01 M malonate

0.02 M dl-&hydroxybutyrate f DPN, 3 x lo+ M

0.01 M succinate

min.

20

60 20 60 20 60 10 30

PM

-2.1 -3.7

-17.5 -19.8 -19.0 -18.0 -19.3 -21.9

Atom per cent excess 0'8in

inorganic phosphate

2.13 0.8

1.96 1.0 0.91 3.8 0.37 6.8 0.79 4.2 0.27 8.0 1.15 2.9 0.56 5.5

phate turnover reaction measured with 018 were involved solely with the oxidation of reduced DPN, there should be no loss of 018 in the inorganic phosphate when succinate is oxidized. The results presented in Table III show that this is not the case. Since succinate is oxidized in this system much more rapidly than a-ketoglutarate or /3-hydroxybutyrate, the time of incubation was chosen to yield approximately equal amounts of oxygen consumption. The amount of phosphorylation as measured by formation of 10 minute-hydrolyzable phosphate is given in each case. It will be noted that this amount did not increase after the first period, probably due to ATPase activity overshadowing the phosphorylation. Although the hydrolysis of ATP may lead to some dilution of the 01* in the inorganic phosphate, the maximal dilution that could be introduced in this way is not quantitatively significant. The rapid phosphate turnover found with


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M. COHN 743

all three substrates shows conclusively that the reaction is not limited to the site of oxidation of reduced DPN. The somewhat higher turnover for a-ketoglutarate and /3-hydroxybutyrate than for succinate suggests that the reaction does occur concomitantly with the oxidation of DPNH as well as at other steps in the electron transport system.

Ferricyanide As Acceptor-Another type of experiment designed to localize the phosphate turnover reaction was the use of ferricyanide as an acceptor in an anaerobic system. Although it is not entirely clear at what level ferricyanide accepts electrons (II), it is undoubtedly below cytochrome c. In Table IV, the 018 disappearance from inorganic phosphate is compared with oxygen and ferricyanide as acceptors in the oxidation of

TABLE IV

Ferricyanide As Acceptor

Reaction mixture, Tris buffer 0.02 M, pH 7.4; phosphate 0.02 M; MgCL 0.005 M;

adenylic acid 0.008 M; dl-P-hydroxybutyrate 0.02 M; DPN 0.0003 M; liver mitochondria 0.6 ml.; total volume 3.0 ml.; T = 25“. Time of incubation 30 minutes. Initial concentration of 018 in inorganic phosphate, 2.60 atom per cent excess.

Substrate

None .................................... @-Hydroxybutyrate ......................

“ + cytochrome c, 1 X 10-6M .................................

0.02 M 0.02 “

N2 “

PH

0

-6.0 2.50 1.12

None Air -22.0 0.68

,

.-

Gas phase APi

Atom per cent excess 0’s in

inorganic phosphate

fl-hydroxybutyrate. There is a significant phosphate turnover with ferricyanide, though not as large as with oxygen. These results indicate that the phosphate turnover reaction is not confined to the last steps in the electron transfer system. The results of the succinate experiment, taken in conjunction with the ferricyanide experiment, would suggest that the phosphate turnover reaction occurs at every step in the electron transport system. Further attempts at localization were unsuccessful, since suitable conditions could not be found by which phosphorylation could be obtained with cytochrome c as the acceptor. Similarly no phosphorylation was observed with reduced cytochrome c as substrate with either cysteine or ascorbic acid as the reducing agent, contrary to the findings of Judah (12).

Relationship to Phosphorylation-It has been shown in the preceding series of experiments that continuous oxidation and reduction of the con- stituents of the electron transport system are necessary for the inorganic phosphate turnover reaction. It was desirable to determine whether oxi-


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dation and reduction alone were sufficient or whether phosphorylation was also required. The effect of uncoupling oxidation and phosphorylation with 2,4-dinitrophenol is shown in Table I. The concentration of 2,4- dinitrophenol in Experiment 1 was 3 X 10e4 M, and in Experiment 3 4 X 1OW M; another experiment of 1 hour’s duration with a concentration of 2,4-dinitrophenol of 4 X lop4 M resulted in a final concentration of Ol* in the inorganic phosphate of 2.12 atom per cent excess O1*. It is clear from these data that 2,4-dinitrophenol inhibits the phosphate turnover reaction observed by the disappearance of O18.

Since the mechanism whereby 2,4-dinitrophenol uncouples phosphorylation and oxidation is not yet clearly understood, other systems were used in which oxidation occurs without phosphorylation. Succinate was oxidized in the presence of 018-labeled inorganic phosphate with a preparation

TABLE V

Oxidation of Succinate with Succinic Oxidase

Reaction mixture, phosphate 0.02 M, pH 7.4; succinate 0.013 M; cytochrome G 1 X 10-b M; succinic oxidase 0.2 ml.; total volume 3.0 ml.; T = 37.2”; gas phase air. Ini- tial concentration of 0’8 in inorganic phosphate, 2.60 atom per cent excess.

I

min.

None. ..____.............................. 30 Succinate.................................. 15

“ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

c.m?n.

0 2.31 157 2.45 246 2.43

of succinic oxidase (13) which was kindly supplied by Dr. E. G. Ball and Miss 0. Cooper. It can be seen from Table V that no 01* was lost from the phosphate although the succinate was rapidly oxidized.

It has been shown by Lehninger (10) that fl-hydroxybutyrate is oxidized in the absence of added magnesium ions and adenylic acid but that both are required for phosphorylation. The effect of omitting these components from the system on the phosphate turnover reaction is shown in Table VI. The requirements for the phosphate turnover reaction seem to parallel the requirements for phosphorylation.

Attempts to localize the phosphate turnover reaction are only possible within the narrow limits of our present knowledge of the phosphorylating system. The absence of a requirement for Mg++ would have implied that the. reaction probably occurred prior to any phosphate transfer reaction. Similarly, if adenylic acid had not been required, it would have indicated that the phosphate turnover reaction occurred prior to the formation of ATP. However, the requirement for both these components sheds no light on the problem.


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M. COHN 745

E$ect of Added ATP-If the turnover of inorganic phosphate arises from a reaction of ATP, hydrolytic or otherwise, such a relationship should be reflected in an increased dilution of the O** in the inorganic phosphate when a large amount of unlabeled ATP is added initially. The results of such

TABLE VI

Requirement for Mg* and Adenylic Acid

Reaction mixture, phosphate 0.02 M, pH 7.4; MgClt 0.005 M; adenylic acid 0.008 M; cytochrome c 1 X lo+ M; DPN 3 X 10e4 M; dl-&hydroxybutyrate 0.02 M; liver mitochondria 0.6 ml., total volume 3.0 ml.; T = 25”; gas phase air. Time of incubation 40 minutes.

Omitted components APi Atom per cent excess

Om m inorganic phosphate

No substrate............................... 0 2.05 “ Mg++ . . . . . . . . . . . . . 0 1.93 “ adenylic acid. . . . . . . . . . . 0 1.79 “ “ “ and no Mg++. . . . . . . . . 0 2.18

Complete system........................... -15.5 0.69

TABLE VII

Comparison of Dilution of Pa and 018 from Inorganic Phosphate in Presence of Added ATP

Reaction mixture, glycylglycine buffer 0.067 M, pH 7.3; phosphate 0.02 M; MgClz 0.005 M; adenylic acid 0.003 M, cytochrome c 1 X lo+ M; malonate 0.01 M; liver mitochondria 0.2 ml. per ml. of reaction mixture; T = 23”. in inorganic phosphate, 2.60 atom per cent excess.

Added components Time APi

0.01 M ketoglutarate

0.01 M ketoglutarate i- 0.0033 M ATP

min. PM

20 -21.0 40 -28.3 20 -15.3 40 -17.0

Initial concentration of 0’8

9060 0.55 9550 0.23 7700 0.63 6840 0.34

an experiment are presented in Table VII. The inorganic phosphate used in this experiment was labeled with both 01* and P32. The dilution of the P32 in the inorganic phosphate in reaction mixtures containing added ATP is a measure of the breakdown of the unlabeled ATP, 17 per cent and 27 per cent dilution at 20 and 40 minutes respectively. The dilution of.the 018 with and without ATP is of quite a different order of magnitude and cannot be due to breakdown of ATP. The fact that the loss of Oi* from the inorganic phosphate is somewhat less in the presence of unlabeled ATP

Inorganic phosphate

Par c.p.m. 0” atom per per mg. cent excess


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precludes any reaction of ATP as the primary one which leads to th. ! urn- over of inorganic phosphate. The slightly higher value of 018 in thr ror- ganic phosphate with a high initial concentration of ATP is probabl, due to an inhibition of the phosphorylation reaction as reflected in the lowered formation of organic phosphate (cf. APi, Table VII).

These experiments also demonstrate that ATP and inorganic phosphate are not in equilibrium in this system. A comparison of Experiments 1 and 2 shows that there is no decrease of O’* in the inorganic phosphate, cor- responding to the lowered Or* concentration in the total ATP, which must result from dilution by the large amount of unlabeled ATP which has been added initially in Experiment 2. The possibility that added ATP does not penetrate into the mitechondria, and the corollary that the added ATP and the ATP formed in the reacting system are not equivalent, should not be overlooked. However, it is apparent from the dilution of P32 in the inorganic phosphate that added ATP is susceptible to ATPase activity.

DISCUSSION

The results of this investigation establish the existence of a reaction of phosphate which is characteristic of oxidative phosphorylation associated with oxidation in the electron transport system. The reaction is a rapid one and manifests itself by a rapid replacement of O’* by 016 in inorganic phosphate; in fact, under the experimental conditions described, about 90 per cent of the 01* has been replaced in 1 hour. In terms of a reaction such as the hydrolysis of a monoester shown schematically in Fig. 1, the loss of 90 per cent of the 01* corresponds to approximately ten reaction cycles for every molecule of inorganic phosphate.

It should be emphasized that this rapid phosphate turnover reaction does not occur during phosphorylation accompanying the substrate level oxidation of a-ketoglutarate. Moreover, the phosphate turnover reaction does not occur in the reversible oxidative phosphorylation of 3-phosphoglyceraldehyde.’ The reaction occurs only in those processes which are sensitive to 2,4-dinitrophenol, which is consistent with the finding that 2,4-dinitrophenol suppresses the reaction.

At the present stage of the investigation, the reaction which leads to the rapid turnover of phosphate cannot be defined. Nevertheless, the experimental observations place certain restrictions on the nature of the reaction. It is obvious that a P-O cleavage must take place, since the formation and rupture of carbon-oxygen bonds cannot lead to any loss of O’* in inorganic phosphate. The reaction must be a recurring one to account for the quantity of Or* lost; that is, each phosphate molecule must have gone through many reaction cycles. However, a recurring P-O cleavage reaction is not a sufficient condition for effecting a continuous loss of Or8 in phosphate. This can best be illustrated by a sequence of known reactions; namely, the


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M. COHN 747

oxi-. $ve phosphorylation reaction of 3-phosphoglyceraldehyde followed by; ,phosphate transfer reaction to ADP with Biicher’s enzyme. In the fin +step catalyzed by glyceraldehyde phosphate dehydrogenase (Equation l), there is no change in concentration of O’* due to either the forward or

O’*H

I I HCOH OH + HO’-P+-O’sH + DPN +

I I I H&-O-W--OH 01%

I

Or*H

I ’

(1)

O=C-0-P+-OQH.

’ I OW

HCOH + DPNH + H+ OH

I H,C-0-P+-OH

I O-

the reverse reaction,’ presumably only carbon-oxygen linkages being involved. In the transfer reaction (Equation Z), there is a P-O cleavage in diphosphoglyceric acid upon transfer to ADP.3

O’*H

,,,+&+~,,

’ hp OH OH

I I HCOH + A-0-P+-0-P+-OH -+

OH

I I I

O- O- H2C-O-P+-OH

I (2) O-

O=C-O’SH OH OH OBH

I I I I HCOH OH + A-0-P+--0-P+-0-P+-OuH

I I I I I H&-O-F-OH O- O- 0*

I I

O-

* Personal communication from Dr. P. D. Boyer who, in an independent study of this reaction, found 018 in 3-phosphoglyceric acid which had been formed by transfer


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When Equation 2 is reversed, the phosphate may add to either oxygen of the carboxyl group of 3-phosphoglyceric acid (3PGA). The oxygen of the phosphate group will eventually be diluted by the amount of 016 in the carboxyl group of 3PGA. By reversal of Equation 1, the 018 concentration of inorganic phosphate after attainment of equilibrium should be lowered only to the extent that it has been diluted by 016 present in the carboxyl group of 3PGA. The anticipated result has been observed experimentally.’ Although there is a recurring P-O split in this sequence of reactions, the loss of 01* in the inorganic phosphate was limited to the amount replaceable by the component which is phosphorylated.. There was no continuous loss of 01* from the inorganic phosphate as observed in the aerobic phosphorylation associated with the electron transport system.

The quantitative aspects of the inorganic phosphate turnover reaction necessitate the involvement of water either directly or indirectly in the sequence of phosphorylation reactions; there is no other source of 016 in the system sufficiently large to account for the large amount introduced into the inorganic phosphate. The simplest type of reaction directly involving water would be the continuous formation and hydrolysis of an organic phosphate bond, as indicated schematically in Fig. 1. Such a reaction is highly unlikely from energetic considerations. The number of phosphate turnover reactions is at least ten per atom of oxygen gas consumed in the over-all reaction. Since the number of high energy phosphate bonds formed per atom of oxygen gas consumed, P:O ratio, is of the order of 3 for the oxidation of DPNH, there must be several reaction cycles of the phosphate turnover reaction for each high energy phosphate bond formed.

Another possible type of reaction involving the direct participation of water is a reversible dehydration reaction. If the mechanism of the transphosphorylase reaction were of the type suggested by Baldwin (14), then the reaction shown in Equation 2 would entail the formation of a diester intermediate of ATP and 3PGA by dehydration, followed by hydration to form 1,3-diphosphoglyceric acid and ADP. 018 would be lost from the , organic phosphate in the dehydration and Ols would be introduced in the hydration. The equilibrium between the organic phosphate and inorganic phosphate as in Equation 1 would result in the replacement of 018 of the inorganic phosphate by 016. That this is not the case for the ATP-phosphoglyceric transphosphorylase has already been mentioned above, but it may be the mechanism which operates in the as yet unknown transphos- phorylating systems involved in phosphorylation associated with the electron transport system.

of phosphate from 1,3-diphosphoglyceric acid, which in turn had been formed in the oxidative phosphorylation of glyceraldehyde phosphate with Ol*-labeled inorganic phosphate.


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M. COHN 749

Water may also participate indirectly and lead to the observed result. If, for example, the 01* retained in the carboxyl group of 3PGA shown in Equation 2 were to exchange rapidly with water, the Ol* in the phosphate would eventually be replaced by O16. In a reversible system, any exchange with water of a compound containing 01* would ultimately be reflected in a loss of Ol* from inorganic phosphate.

A complete elucidation of the detailed mechanism of aerobic phosphorylation must await the chemical identification of the components of the phosphorylating system. Until the necessary information is available, one can only exclude those hypotheses which are not consistent with the rapid turnover of inorganic phosphate. It is hoped that the nature of the phosphorylation reactions will be further delimited by investigations which are now in progress, including a study of oxidative phosphorylation in water labeled with 01* and of the fate of 01* in ATP during oxidative phosphorylation and further study of model reactions.

The author wishes to thank Dr. Graham Webster and Dr. F. E. Hunter for valuable suggestions and discussions and Dr. A. Baird Hastings and Dr. Carl F. Cori for their interest and encouragement during the course of this investigation. The author also wishes to thank Mrs. E. Simpson for her able assistance, and Dr. A. K. Solomon for generously making the mass spectrometer available for the work at the Harvard Medical School.

SUMMARY

A new reaction which occurs in oxidative phosphorylation associated with the electron transport system has been observed in rat liver mitochondria with a-ketoglutarate, /3-hydroxybutyrate, and succinate as substrates. This reaction manifests itself by a replacement of Ol* with normal oxygen in inorganic phosphate labeled with Ol* and parallels the phosphorylation which is associated with the oxidation. The number of molecules of inorganic phosphate which participate in this reaction, calculated on the basis that a monoester of phosphate is involved, is several times higher than the number of high energy phosphate bonds that can be formed. The reaction does not occur at the substrate level oxidation of cr-ketoglutarate and the evidence suggests that it occurs at every step in the electron transport system.

This phosphate turnover reaction occurs only when phosphorylation is proceeding. Dinitrophenol suppresses the reaction. The omission of

Mb+ or adenylic acid also suppresses the reaction. The reaction is abol- ished when succinate oxidation is catalyzed by a succinic oxidase preparation containing no phosphorylating system. The possibility that the reaction is due to a direct reaction of ATP, hydrolytic or otherwise, is


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eliminated. Various mechanisms which are consistent with the findings are discussed.

BIBLIOGRAPHY

1. Lipmann, F., in Green, D. E., Currents in biochemical research, New York, 137 (1946).

2. Lehninger, A. L., in McElroy, W. D., and Glass, B., Phosphorus metabolism, Baltimore, 344 (1951).

3. Cohn, M., J. Biol. Chem., 180, 771 (1949). 4. Schneider, W. C., J. Biol. Chem., 176, 259 (1948). 5. Kielley, W. W., and Kielley, R. K., J. Biol. Chem., 191, 485 (1951). 6. Scholander, P. F., Claff, C. L., Andrews, J. R., and Wallach, D. F., J. Gen. Phys-

ioz., 36, 375 (1952). 7, Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 375 (1925). 8. Hunter, F. E., Jr., J. Biol. Chem.. 177, 361 (1949). 9. Hunter, F. E., Jr., and Hixon, W. S., J. BioZ. Chem., 181, 73 (1949).

10. Lehninger, A. L., J. BioZ. Chem., 178,625 (1949). 11. Copenhaver, J. H., Jr., and Lardy, H. A., J. BioZ. Chem., 196, 225 (1952). 12. Judah, J. D., Biochem. J., 49, 271 (1951). 13. Ball, E. G., and Cooper, O., J. BioZ. Chem., 180, 113 (1949). 14. Baldwin, E., Dynamic aspects of biochemistry, Cambridge, 156 (1947).


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Mildred Cohn-LABELED INORGANIC PHOSPHATE

18PHOSPHORYLATION WITH OA STUDY OF OXIDATIVE

1953, 201:735-750.J. Biol. Chem.

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