Redox potentials and phosphorylative efficiency of mitochondria

ARCHIVES

OF

Redox

BIOCHEMISTRY

AND

Potentials

and

BIOPHYSICS

136, 268-272 (1970)

Phosphorylative

Efficiency

of Mitochondria’

W. S. LYNN Department of Biochemistry, Duke University Medical Center, Durham, North Carolina ~7’706 Received September 4, 1969; accepted October 20, 1969 In mitochondria which exhibit high P/O ratios, >5, P/O ratios progressively increase during State 3 respiration. This is associated with progressive increases in the level of reduction of electron carriers, especially cytochrome C and flavin. With mitochondria whose P/O ratio is 3 or less, all electron carriers are oxidiied upon addition of ADP. Similarly with substrates of progressively decreasing redox potential, a parallel decrease in the steady-state level of reduction of the electron carriers also occurs. These data suggest that mitochondria, like chloroplasts, can phosphorylate efficiently only under conditions in which high levels of reduction of the electron carriers are maintained during State 3 respiration. These data are discussed in relation to the theory of the mechanism of phosphorylation previously proposed for chloroplasts.

Previous studies on chromatophores and chloroplasts (l-4) have indicated that efficient phosphorylation occurs only under conditions in which high degrees of reduction, as assayed by steady-state levels of reduction of electron carriers, is energetically maintained within chloroplasts. Studies on mitochondria, which exhibit P/O ratios of 3 or less, have shown that the addition of ADP to mitochondria in State 4 results in precipitous oxidation, i.e., the steady-state level of reduction of NAD, flavin, cytochrome b, and cytochrome c is decreased by 50 % or more (5). The purpose of this report is to demonstrate that mitochondria which exhibit P/O ratios greater than 4 (6), maintain a relatively high redox potential during State 3 respiration. In fact, the efficiency of phosphorylation appears to be linearly related to the overall internal redox potential. With prolonged State 3 respiration, progressively higher levels of reduction of cytochrome c and flavin, as well as NAD and cytochrome b, are attained and this is associated with progressively higher P/O ratios. These studies suggest that a major 1 Research supported GM 14022-02.

in part by NIH

Grant

difference between mitochondria with low P/O ratios (3 or less) and those with high P/O ratios (4 or greater) is that the more efficient mitochondria are able to maintain a high internal redox potential while synthesizing ATP. METHODS Rat liver mitochondria were prepared, using a tissue press as before (6). Under these conditions, approximately 2 out of every 3 preparations exhibit high P/O ratios. Mitochondria (8 to 15 mg of protein in 0.1 ml) were added to 2.6 ml of 0.25 M sucrose at 22” containing 0.0015 M sodium phosphate, 0.003 M MgClz, 1.0 mg bovine serum albumin, 0.0002 M EDTA and I.28 patoms of oxygen. Concentrations of all substrates were 0.0015 M. Concentrations of ADP, ATP, and uncoupling agents, where added, are indicated in the Iegends. Initial pH was 7.45. Addition of ATP and AMP at the above concentrations did not result in any increase in 0~ consumption. 02 was determined polarographically in closed vessels as before (7), and the polarograph was standardized using chloroplasts under anaerobic conditions and known amounts of ferricyanide, as before (7). Rate of formation of ATP was measured titrimetrically as before (7). ATP was also measured enzymatically on aliquots of the reaction mixtures at periodic intervals (7)) as well as spec268

STATE

OF REDUCTION

DURING

trally, using glucose, hexokinase and glucose-6phosphate dehydrogenase as before (6). All methods yielded identical results. All spectral assays were performed, using a Briton Chance Aminco dual wavelength spectrophotofluorimeter. Changes in reduction of cytochrome c were measured at four different wavelengths, 540 rnp and 550 rnp and 521 rnp, 416 rnp and 500 ml.c,and 550 rnp and 575 rnp. All the above difference spectra yielded comparable results. Cytochrome b was measured at 540 rnF and 563 rnp and 575 rnp, and 430 rnp and 505 ~QJ. Cytochrome cl was measured at 540 rnp and 554 m, and at 554 rnp and 578 q. Cytochrome a was measured at 585 w and 605 q, and 605 rnp and 620 rnp. Pyridine nucleotides were estimated at 343 m and 364 rnp. Since NADP is known to be largely reduced in liver mitochondria and to remain reduced during State 3-State 4 transitions (B), it is assumed that the observed spectral changes in this report represent changes in reduction of NAD. Flavin was estimated at 490 rnp and 505 rnp, and at 476 rnN and 505 rnp. The observed decrease in absorption upon 100% reduction of the fiavin was approximately a-fold greater at 476 rnp than at 490 rnl.r. Attempts to measure flavin fluorimetrically, under the conditions of these experiments, were unsuccessful. The fluorescent emission was too weak to obtain useful quantitative data. Zero percent levels of reduction of all the carriers was obtained by allowing the mitochondria to incubate with excess ADP and 0, in the absence of substrate. To insure that all the carriers were 100% oxidized, excess ferricyanide was also added. One hundred percent levels of reduction were obtained by allowing the mitochondria to become anaerobic in the presence of excess substrate and ADP. RESULTS

Addition of ADP to freshly prepared containing pyridine-linked mitochondria, substrates, results in the changes shown in Fig. 1. With the initial addition of ADP, an initial rapid uptake of 02 occurs, but there is a delay in the onset of phosphorylation, as indicated by the pH trace. NADH and cytochrome b are rapidly oxidized and cytochrome c and flavin are reduced. Upon exhaustion of the added ADP, H+ consumption ceases, 02 consumption almost ceases, and a higher level of reduction of cytochrome c, cytochrome b, and flavin are attained. With repeated additions of ADP, less NADH and cytochrome b are oxidized, more flavin and cytochrome c become reduced, the initial fast rate of 02 consumption is no longer observed, and essentially no delay in

PHOSPHORYLATION

269

75-

Cyt. 0.

Min.

pmoles &moles

& 0.65 p moles

FIG. 1. Changes in redox state of liver mitochondria during State 4-State 3 transitions. Aliquots of the same mitochondrial suspension (2.6 mg protein/ml) were incubated at 22” as in Methods. In addition, 0.0007 M sodium ATP, 0.0012 M sodium glutamate, and 0.0006 M sodium malate were present. ADP was added when indicated. 01 consumption was measured polarographically on 2.7-ml aliquots. Rate of ATP formation was measured titrimetrically on 12 ml aliquots, and the spectral data obtained on 2.7-ml aliquots, as in Methods. Net formation of ATP after each addition of ADP was also measured enzymatically, as in Methods. The level of reduction of each of the electron carriers during and after their State 4-State 3 transitions were recorded as in Methods. The calculated ADP/O ratios are given in the figure. Similar data as depicted in the figure were obtained using either glutamate, glutamate plus malate or cu-ketoglutarate as substrate. With a-ketoglutarate the observed ADP/O ratio was 4.8 upon the first addition of ADP and increased to 6.1 with the third addition of ADP.

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LYNN

onset of phosphorylation is observed. Very little change in the level of reduction of cytochrome a600 or cytochrome agO6is observed under the conditions of Fig. 1. An increase in the ADP/O ratio is also observed with repeated additions of ADP. The highest ADP/O which has been observed, using glutamate and malate as substrate, is 5.9. The experiment shown in Fig. 1 was done in the presence of 2 moles of ATP added at 0 time. However, data essentially identical to that shown in Fig. 1 was observed in the absence of added ATP. Maximal levels of reduction of all the electron carriers (100 %) were observed under anaerobic conditions in the presence of excess substrate and ADP and minimal levels of reduction (0%) were obtained by omission of substrate in the presence of ADP. No further oxidation of the carriers could be observed under the above conditions by addition of ferricyanide. Since no change in difference absorbance at wavelengths of 575 rnp and 590 rnp, or of 590 rnp and 620 rnp were observed under the conditions of Fig. 1, it is unlikely that any of the data of Fig. 1 is in error due to change in light scattering. The data presented in Fig. 1 can be observed only in freshly prepared mitocondria. If the mitochondria are stored in sucrose at 0” for 12 hr or longer, ADP/O ratios decline to 3 or less, and a decrease in reduction of all the above electron carriers occurs upon addition of ADP. Prolonged storage, therefore, results in preparations of mitochondria which exhibit the spectral changes TABLE EFFECT

OF SUBSTRATES

upon addition of ADP as those previously reported (5). Likewise addition of uncoupling agents at concentrations sufficient to reduce the ADP/O ratio to between 2 and 3, e.g., dinitrophenol, 1.5 X 10B5 M, salicylanilide, 7 X 10V8 M, or valinomycin 0.5 r*gram/ml plus KCl, 2.5 X 10-t M, to freshly prepared mitochondria, also results in a marked decrease in the steady-state level of reduction of all the carriers with additions of ADP. Incubation of fresh mitochondria under adverse conditions, i.e., omission of sucrose or MgCl, from the reaction solutions, also results in low ADP/O ratios and an associated decrease in level of reduction of all the carriers upon addition of ADP. Under all the above adverse conditions the steady-state level of reduction of NADH falls most precipitously upon addition of ADP. These mitochondria respond to electron transport inhibitors in the customary manner, i.e., antimycin A, 1 pgram/ml, causes NAD, flavin, and cytochrome b to become 100% reduced and the other carriers to become 0 % oxidized. Similarly, rotenone, 2 X 1O-5 M, causes all the carriers, except NAD, to become 0 % oxidized (data not shown). The effects of substrates of varying redox potential on steady-state State 4-State 3 transitions are tabulated in Table I. With all substrates, addition of ADP results in increases in the steady-state level of reduction of flavin and cytochrome c, however, the extent of reduction is progressively less with substrates of lower redox potential. Similarly, with substrates of lower redox I

STATE ACCOMPANYING STATE ~-STATE 3 TRANSITIONS

ON REDOX

Per cent reduction Substrate

DPN

p/o

+ DMPD

5.8 4.9 1.8 0

Cyt. B

Cyt. c

state

state

state

state

state

state 3

state 4

state 3

109 94 88 52

91 67 36 0

21 15 4 0

39 32 11 0

46 51 41 26

36 30 20 0

29 29 10 7

46 41 14 0

4

Glutamate Succinate Ascorbate None

Flavin 3

4

3

4

NOTE: Experimental conditions as in Fig. 1. Maximal changes in redox state served after the third addition of ADP (see Fig. 1) are tabulated. Concent.ration 0.0915 M; sodium ascorbate, 0.6035 M; and dimethylphenylenediamine, DMPD, with the above substrates were measured as in Fig. 1. The degree of reduction in the absence of substrate was recorded 15 set after addition of mitochondria

of electron carriers obof sodium succinate was 0.00006 M. P/O ratios of the electron carriers to the reaction mixture.

STATE

OF REDUCTION

DURING

potential, there is a progressive decline in the steady-state level of reduction of NAD and cytochrome b, and especially so for NAD. ADP/O ratios decline with these three substrates in a manner which parallels the decline in the level of reduction of NAD and cytochrome b observed in State 3. The State 3 levels of reduction of cytochrome c and flavin, however, do not change with various substrates in a manner which parallels the change in ADP/O ratios. DISCUSSION

Liver mitochondria, prepared in this laboratory over the past 4 years, have been repeatedly shown to exhibit P/O ratios of higher values than the generally accepted ones. Most, but not all laboratories (9-11) have been unable to confirm the results of this laboratory.2 In fact, the results of Cockrell et al. (12), and Slater (13) on the measurements of phosphate potentials in mitochondria have been interpreted to indicate that P/O ratios higher than 3 are not thermodynamically possible. Their measurements, however, were done on mitochondria in which the P/O ratio was approximately 3. Since the phosphate potential is simply an expression of the mass law for the reaction, H+ + ADPm3 + Pim25 ATPL4 + HzO, it is clear that in the steady-state or at equilibrium, the concentrations of the above reactants will be determined by the apparent equilibrium constant of the above reaction. Therefore, under conditions in which the input of energy into the above reaction is 51,000 Cal/mole and the observed P/O ratio is 3, obviously 17,000 cal/mole are required to synthesize 1 mole of ATP. Calculations of the AG, using the above apparent equilibrium constant of the above energy-coupled reaction must also equal 17,000 cal/mole. This is the value experimentally obtained by Cockrell et cd. (12). The phosphate potential then is simply another way to express the P/O ratio and the two terms must be equal. The above data (12) on phosphate potentials do not imply that P/O rati.os higher than 3 are impossible 2 Sarkissian and Srivastava have recently observed high P/O ratios in wheat mitochondria, Proc. Nat. Acad. Sci. 63, 302, 1969.

PHOSPHORYLATION

271

under other conditions. The data state only that the equilibrium constant is an expression of the energetics of the phosphorylating reaction and that the energy which is coupled to the phosphorylating reaction can be measured either as a P/O ratio or as a phosphate potential. The high P/O ratios observed in this laboratory using different mitochondria are possible, provided that the phosphate potential within the mitochondria can be maintained at values different from those existing in the external environment. Alterations by several fold of the concentrations of ATP, ADP, or Pi in the external environment has little, if any, effect on either the observed P/O ratios or the internal redox state (data not shown). As noted in Fig. 1 and Table I, these freshly prepared mitochondria exhibit only two crossover points (5). A crossover point between cytochrome c and cytochrome a is observed in these mitochondria after aging or in fresh mitochondria which are uncoupled either by uncoupling agents or by exposure to adverse conditions (see Results). Thus, it appears that efficient phosphorylation in mitochondria occurs when flow of electrons between cytochrome c-chrochrome cl and cytochrome a is relatively slow, i.e., the rate-limiting step. The data in this report support the concept of the mechanism of phosphorylation previously proposed for phosphorylation by chloroplasts (14). Phosphorylation, i.e., H+ + ADPe3 + Pi-2 zz ATP-4 + H20, is the consequence of the reduction and protonation of an ADP kinase-ATPase catalytic site. This enzyme is apparently tightly bound at three sites along the electron transport chain of mitochondria, i.e., NADH + AK + Flavin-Non Heme Fe2+ s Cyt. b $ AK + Cyt. c-cl = AK = Cyt. a -+ 02. (AK is reduced ADP kinase.) In tightly coupled mitochondria, electrons can leave the ADP kinase only under conditions which deprotonate the enzyme, i.e., in the presence of phosphorylation, which consumes H+, or of uncoupling agents, which facilitate Hf-cation exchange across the mitochondrial membrane, or adverse conditions, such as detergents or hypotonicity which allow external OH- to deprotonate

272

LYNN

the enzyme. Since the equilibrium of the above reaction appears to be controlled by the H+ pressure about the enzyme and the H+ pressure is controlled by the extent of reduction which can be maintained while H+ is being consumed by phosphorylation, it is clear that efficient phosphorylation can proceed only under conditions in which the internal H+ and electron pressures are maintained at high levels. Although no unequivocal measurements of H+ activity within mitochondria are available (15, IS), it appears that uncoupling agents, e.g., Ca2+, dinitrophenol, valinomycin, nigericin, salicylanilides, etc. (12, 15, 17), generally cause a loss of H+ from mitochondria by increasing H+ permeability (18, 19). Thus it is likely that protonation, a consequenceof electron flow (3) also supplies energy for phosphorylation in mitochondria as it has been proposed to do in chloroplasts (14). REFERENCES 1. JAGENDORF, A. T., Surv. Biol. Progr. 4, 181 (1962). 2. LYNN, W. S., AND BROWN, R. B., J. Biol. Chem. 242, 426 (1967). 3. LYNN, W. S., Biochem. 7, 3811 (1968).

4. CUSANOVITCH, M. A., AND KAMEN, M. D., Biochim. Biophys. Acta 163, 418 (1968). 5. CHANCE, B., AND WILLIAMS, G. R., Advances in Enzymol. 17, 65 (1956). 6. LYNN, W. S., AND BROWN, R. B., Biochim. Biophys. Acta 106, 15 (1965). 7. LYNN, W. S., AND BROWN, R. B., J. Biol. Chem. 242, 418 (1967). 8. KLINGENBERG, M., SLENCZKA, W., AND RITT, E., Biochem. 2. 332, 47 (1959). 9. DALLAM, R. D., AND HOWARD, R. B., Biochim. Biophys. Acta 37, 188 (1960). 10. SMITH, A. L., AND HANSEN, M., Biochem. Biophys. Res. Commun. 16, 431 (1964). 11. GURBAN, C., AND CRISTEA, E., Biochim. Biophys. Acta 96, 195 (1965). 12. COCKRELL, R. S., HARRIS, E. J., AND PRESSMAN, B. C., Biochem. 6, 2326 (1966). 13. SLATER, E. C., Unpublished data. 14. LYNN, W. S., AND STRAUB, K. D., Proc. Nat. Acad. Sci. U. S. A. 63, 540 (1969). 15. CHANCE, B., AND MELA, L., J. Biol. Chem. 242, 830 (1967). 16. AZZONE, B. F., PIEMONTE, G., AND MASSARI, S., European J. Biochem., 6, 207 (1968). 17. MITCHELL, P., AND MOYLE, J., Nature 208, 147 (1965). 18. MITCHELL, P., AND MOYLE, J., Biochem. J. 106, 1147 (1967). 19. PACKER, L., AND UTSTJMI, K., Arch. Biochem. Biophys. 131, 386 (1969).