Myoglobin-Mediated Oxygen Delivery to Mitochondria of Isolated Cardiac Myocytes (Electron Transport/Heart Cells/Cytochrome Oxidase) BEATRICE A

Home , Cytochrome b

Proc. Nati. Acad. Sci. USA Vol. 84, pp. 7503-7507, November 1987 Biochemistry Myoglobin-mediated oxygen delivery to mitochondria of isolated cardiac myocytes (electron transport/heart cells/cytochrome oxidase) BEATRICE A. WITTENBERG* AND JONATHAN B. WITTENBERG Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461 Communicated by Berta Scharrer, July 20, 1987 (receivedfor review May 5, 1987)

ABSTRACT Myoglobin-mediated oxygen delivery to in- Cytochrome oxidase, half-oxidized when ambient oxygen tracellular mitochondria is demonstrated in cardiac myocytes partial pressure (Po2) is 0.07 torr (1 torr = 133 Pa) (16), in the isolated from the hearts of mature rats. Myocytes are held at circumstance described here experiences oxygen pressures high ambient oxygen pressure, 40-340 torr (5-45 kPa); 20- to 200-fold the pressure required to maintain the normal, sarcoplasmic myoglobin is fully oxygenated. In this condition largely oxidized, state seen in resting myocytes (16). Carbon oxygen availability does not limit respiratory rate; myoglobin- monoxide in this circumstance blocks oxygenation of facilitated diffusion contributes no additional oxygen flux and, sarcoplasmic myoglobin selectively without perturbing the since oxygen consumption is measured in steady states, the optical spectrum of intracellular cytochrome oxidase. We storage function of myoglobin vanishes. Carbon monoxide, conclude that cardiac mitochondria accept two additive introduced stepwise, displaces oxygen from intracellular simultaneous flows of oxygen: the well-known flow of dis- oxymyoglobin without altering the optical spectrum of the solved oxygen to cytochrome oxidase and a flow of largely oxidized intracellular mitochondria. A large part, myoglobin-bound oxygen to a mitochondrial terminus. The about one-third, of the steady-state oxygen uptake is abolished myoglobin-mediated oxygen flow supports ATP generation by carbon monoxide blockade of myoglobin oxygenation. The in the physiological range of oxygen pressure. myoglobin-dependent component of the oxygen uptake decreases linearly with decreasing fraction of intracellular MATERIALS AND METHODS oxymyoglobin, with a slope near unity. Studies using inhibitors of mitochondrial electron transport indicate- that myoglobin- Isolated Cardiac Myocytes. These were prepared from the delivered oxygen uptake depends on electron flow through the hearts of mature rats by enzymatic digestion, purified on mitochondrial electron transport chain. We conclude that Percoll (Pharmacia) density gradients, and suspended to a cardiac mitochondria accept two additive simultaneous flows of final density of 0.5-1.0 x 106 cells per ml (19, 20). oxygen: a flow of dissolved oxygen to cytochrome oxidase and Mitochondria. Mitochondria were prepared by the method a flow of myoglobin-bound oxygen to a mitochondrial termi- of Palmer et al. (21) from adult rat hearts that had been nus. Myoglobin-mediated oxygen delivery supports ATP gen- perfused with balanced saline solution to remove erythro- eration by heart cells at physiological ambient oxygen pressure. cytes. Gas Partial Pressures and Oxygen Uptake. All measure- Myoglobin is an oxygen-binding monomeric hemeprotein ments were made in steady states (7, 16) of constant oxygen found in the cytosol of cardiac myocytes and of those pressure and oxygen uptake. The measuring chamber had vertebrate muscle fibers that do sustained work (1). The both liquid and gas phases (16, 22). The gas phase composi- myoglobin content of muscles increases with exercise (1-3) tion was set by a mass flow controller (Tylan, Torrance, CA). and is proportional to the cytochrome oxidase content (2, 4, Since carbon monoxide is not consumed, solution Pco is 5). Myoglobin, by facilitating oxygen diffusion, maintains an known from the composition of the gas phase. Solution ample free oxygen concentration at the muscle mitochon- oxygen pressure was monitored by a sensitive polarographic drion (2, 4, 6). Blockade of myoglobin function decreases oxygen electrode and is the balance of oxygen entering and oxygen uptake (7, 8), work output (8, 9), and ATP generation oxygen consumed by the myocytes. At constant temperature (10) of cardiac and skeletal muscle. and stirring rate, oxygen uptake is known from the difference Facilitation of oxygen diffusion through the sarcoplasm of between solution Po2 in the absence of myocytes (equal to the myocyte has often been taken as the sole function of gas-phase Po2) and the actual solution Po2 in the presence of myoglobin in muscles in the steady state. Here we show an myocytes, using a mass transfer coefficient that is determined additional function: myoglobin-mediated oxygen delivery to daily (16, 22, 23). The temperature was 30'C in all experi- mitochondria. This is most clearly demonstrated in isolated ments. cardiac myocytes flooded with superabundant oxygen (11, Optical Spectra. The chamber was held in a thermostatted 12). In this circumstance, the maximum diffusive flow of block placed in the sample light beam of a Cary model 17 dissolved oxygen across the sarcoplasm exceeds the mito- recording spectrophotometer (Varian) equipped with a Cary chondrial oxygen demand at least 100-fold (13, 14); sarco- scattered transmission accessory. Data were acquired digi- plasmic myoglobin is essentially fully oxygenated, and facil- tally from 650 to 350 nm, and difference spectra were itated diffusion contributes no additional oxygen flux (2, 4, 6). constructed by subtraction, using an Aviv data acquisition Since oxygen uptake is in steady states, the storage function system (Aviv Laboratories, Lakewood, NJ). The spectral of myoglobin vanishes (7). Intracellular gradients of oxygen contribution of myoglobin dominates the myocyte optical pressure, always shallow (15-18), now are small relative to spectrum (16). Under the conditions of our experiments, ambient oxygen pressure. Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; MbO2, oxymyoglobin; MbCO, carbon monoxide myoglobin; Po2, The publication costs of this article were defrayed in part by page charge partial pressure of oxygen; Pco, partial pressure of carbon monox- payment. This article must therefore be hereby marked "advertisement" ide. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

7503 Downloaded by guest on October 1, 2021 7504 Biochemistry: Wittenberg and Wittenberg Proc. Natl. Acad Sci. USA 84 (1987) intracellular myoglobin is essentially completely ligated; oxy- a and carbon monoxide myoglobin are the dominant species present. The contribution of myoglobin was calculated from w the spectral changes occurring at 409 and 424 nm, when By intracellular oxymyoglobin was converted fully to carbon 0u monoxide myoglobin, and was subtracted from myocyte spectra to obtain a residuum dominated by the spectral z contribution of the mitochondria. The fraction of carbon w monoxide myoglobin at any Pco was calculated from spectral x changes observed at 409 and 424 nm. Excellent isosbesticity 0 was maintained at 415 and 438 nm. Energetics of Cardiac Myocytes. Suspensions of myocytes were incubated in the measuring chamber for 60 min. Oxygen ATP, intracellular phosphocreatine, and b uptake, intracellular 0 1.0I accumulated lactate (final minus initial) were determined as 0 previously (16, 20). me - _ _

0 U *- - - S~ RESULTS w A.~ I- 0.5 /U/ The partition of intracellular myoglobin between carbon a. ,0 monoxide and oxygen, M = ([MbCO] x Po2)/([MbO2] x z // Pco), was determined graphically as the slope of the line in 0/ Fig. 1. 'The partition coefficient, M = 20 at 30'C, is not largely / different from that of purified rat heart myoglobin, M = 29 at 0 00 I40 30'C, which we determined by conventional procedures (24, 0 10 20 30 40 25). The reasonable value of the slope, together with the Pco torr linearity of the relation found, indicates near equilibrium between sarcoplasmic myoglobin, oxygen, and carbon mon- FIG. 2. Steady-state oxygen uptake of suspensions of cardiac oxide. myocytes as a function of carbon monoxide partial pressure. Res- The effect of carbon monoxide on steady-state respiration piratory rate [14.4 ± 3.3 nmol of oxygen per min per ml per 106 cells of cardiac myocytes is presented in Fig. 2. Within each (mean ± SD; n = 29) in these experiments] is normalized, taking the experiment, the fraction of oxygen in the gas phase was held uninhibited rate in each experiment as unity. Data are from three experiments. m, 15% oxygen in the gas phase (solution P92 = 80-90 constant at 10-25% and the fraction of carbon monoxide was torr); e, 20% oxygen (solution Po2 = 100-120 torr); A, 25% oxygen increased stepwise. Steady-state solution oxygen pressure, (solution Po2 = 155-160 torr). (a) Pco = 0-730 torr. (b) The same steady-state oxygen uptake, and optical spectra were moni- data presented over a limited range; the fractional saturation of tored. The oxygen uptake of cardiac myocytes held at high sarcoplasmic myoglobin in one experiment is presented (o--- o; Po2 decreases initially as Pco is increased, stays nearly 20%o oxygen in the gas phase). The myoglobin-dependent component constant a in which is of the oxygen uptake is taken as the difference between the in prolonged plateau myoglobin fully uninhibited rate and the plateau value reached as sarcoplasmic saturated with carbon monoxide, and finally, above 95% myoglobin approaches saturation with carbon monoxide. carbon monoxide, in the gas phase, declines to zero as carbon monoxide combines with cytochrome oxidase (Fig. 2a). The The myoglobin-dependent component of oxygen uptake early decrease clearly follows the increasing fractional satu- decreases linearly 'with increasing fractional saturation of ration of cytoplasmic myoglobin with carbon monoxide (Fig. sarcoplasmic myoglobin with CO, with a slope near -1.1 2b). This part ofthe respiration may be called the myoglobin- (Fig. 3). This indicates that the myoglobin-dependent oxygen dependent oxygen uptake. uptake is proportional to the fraction of sarcoplasmic oxymyoglobin. The state ofintracellular mitochondria ofcardiac myocytes 3 - exposed to carbon monoxide in the presence of oxygen, the plateau region ofFig. 2, was determined from optical spectra. Myocyte spectra (Pco = 295 torr, Po2 = 36 torr) after the spectral contribution of intracellular carbon monoxide 0~~~~~~~~~~~~~~~ myoglobin is subtracted scarcely differ from spectra of the same cell suspension in oxygen alone (Po2 = 35 torr) after the 0~~~~~~~~~~~ spectral contribution of intracellular oxymyoglobin is subtracted (Fig. 4). The absence of spectral difference near 428, 445, and 606 nm between the upper and middle traces of 0o . 590, Fig. 4 indicates that cytochrome a3 of cytochrome oxidase ..0H has not ligated appreciably to carbon monoxide even at a Pco (295 torr) far greater than that required to inhibit myoglobin- dependent oxygen uptake (95% inhibition near Pco = 50 torr, Fig. 2a). The absence of spectral difference near 445 and 603 nm 0.05 0.1 0.15 indicates that the extent of steady-state oxidation/reduction of cytochrome oxidase is unchanged. The absence of the PCO/ P°2 diagnostic intense maximum of reduced cytochrome c (550 FIG. 1. Partition of intracellular myoglobin between oxygen and nm) in the upper trace of Fig. 4 indicates that the level of carbon monoxide. Data are from six experiments. Solution oxygen reduction of cytochrome c likewise is largely unchanged. pressures ranged from 40 to 340 torr in different experiments. The Taken together, these spectral findings indicate that mito- partition coefficient M = 20 at 30'C. chondria of myocytes in the plateau region of Fig. 2 (Pco = Downloaded by guest on October 1, 2021 Biochemistry: Wittenberg and Wittenberg Proc. Natl. Acad. Sci. USA 84 (1987) 7505

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0.2 0.4 0.6 0.8 1.0 WAVELENGTH, nm FIG. 4. Optical spectra of a suspension of cardiac myocytes, [MbCo]/([ + ]) MbCo] [MbO2 compared to spectra of isolated mitochondria. Bottom trace, spectrum of an oxygenated suspension of rat heart mitochondria. Middle FIG. 3. Myoglobin-dependent oxygen uptake of suspensions of trace, spectrum ofa suspension ofcardiac myocytes (106 cells per ml; cardiac myocytes as a function of the fraction of sarcoplasmic solution Po2 = 35 torr) minus the spectral contribution of 5.2 AuM myoglobin ligated to carbon monoxide. Myoglobin-dependent oxy- MbO2. At this solution oxygen pressure the intracellular myoglobin gen uptake, defined in the legend to Fig. 2, is normalized, taking the is %% oxygenated (16). Upper trace, spectrum of the same cell maximal value in each experiment as unity. Results of five experi- suspension (solution Po2 = 36 torir; Pco = 295 torr) minus the ments are shown. *, Two experiments with solution Po2 = 40-60 spectral contribution of 5.2 ,M MbCO. This trace corresponds to a torr; *, solution Po2 = 70-90 torrP e, solution Po2 = 100-120 torr; El, point on the respiratory plateau of Fig. 2, where the myoglobin- a single experiment with solution Po2 = 340 torr (precision of dependent respiration is fully inhibited, and intracellular myoglobin measuring oxygen uptake is less in this experiment). Pco at half is fully saturated with carbon monoxide. The myocyte spectra are saturation of sarcoplasmic myoglobin in different experiments was nearly the same in the absence and presence of carbon monoxide, 3-15 torr. The solid line is a least-squares fit to the data of the four differing only slightly in the Soret region. experiments represented by solid symbols. The slope is -1.1 (r = 0.98). upper and middle traces of Fig. 4, not presented). This is the only spectral change (other than the changes ascribed to 50-300 torr) are in the state expected for myocytes in oxygen and are not affected by carbon monoxide. 430 Spectral features near 430, 537, and 563 nm, seen in the myocyte but not the mitochondrial spectra of Fig. 4, indicate that cytochrome b of ubiquinone-cytochrome c oxidoreduc- 550 603 tase [429 and 562-564 nm (refs. 26-28)], oxidized in fully 0.05 ~~~520 LiJ oxidized isolated mitochondria, is detectably reduced in the 0 aerobic isolated myocyte. Spectral contributions from z cytochrome b of succinate-ubiquinone oxidoreductase [424 and 560 nm (refs. 28-30)] or from cytochrome b5 [423 and 556 nm (refs. 31-33)] are easily distinguished and are excluded. The final decrease in respiration occurs at about 95% carbon monoxide in the gas phase (Pco = 700 torr; solution Po2 = 7 torr). Concomitant with inhibition of respiration, optical spectra first indicate ligation of cytochrome a3 to carbon monoxide. The spectral change is complete at 100% carbon monoxide in the gas phase (Fig. 5). The spectral contribution of myoglobin cancels in the difference between spectra of cells exposed to 100% carbon 350 400 450 500 550 600 650 monoxide and cells on the plateau region of Fig. 2 (Fig. 5, WAVELENGTH, nm upper trace). The spectrum is essentially similar to the difference FIG. 5. Optical difference spectrum of cardiac myocytes. Intra- spectrum of isolated mitochondria exposed to cellular myoglobin is fully saturated with carbon monoxide in each carbon monoxide minus those in air (Fig. 5, lower trace), parent myocyte sample, and the spectral contribution of MbCO is except for a feature near 563 nm in the mitochondrial cancelled in the difference. This is compared to a mitochondrial spectrum ascribed to differential reduction of cytochrome b. difference spectrum. Upper trace, spectrum of a suspension of Spectral features near 550, 445, and 605 nm seen in both cardiac myocytes (0.5 x 101 cells per ml) in carbon monoxide alone traces indicate increased reduction of cytochrome c and (Pco = 730 torr) minus the spectrum of the same suspension, taken cytochrome oxidase in the absence of oxygen. A spectral earlier (solution Po2 = 195 torr, Pco = 370 torr; this corresponds to feature near 593 nm seen in both traces is ascribed to carbon about the midpoint of the respiratory plateau of Fig. 2). Lower trace, a in monoxide-ligated cytochrome a3 of cytochrome oxidase. spectrum of suspension of isolated rat heart mitochondria the This confirms that intracellular mitochondria ofcells exposed presence of succinate and carbon monoxide minus the spectrum of the same suspension taken earlier in the presence of oxygen. The to carbon are an monoxide in air in oxidized steady state. traces are largely the same except for a greater prominence of a A small maximum near 419 nm appears in the difference feature near 563 nm in the mitochondrial trace. Cytochrome b, to between spectra of myocytes exposed to carbon monoxide which this feature is ascribed, is always detectably reduced in and oxygen and those in oxygen alone (difference between myocytes (Fig. 4) and is fully reduced in anaerobic mitochondria. Downloaded by guest on October 1, 2021 7506 Biochemistry: Wittenberg and Wittenberg Proc. Natl. Acad. Sci. USA 84 (1987) myoglobin) consistently accompanying binding of intracellular myoglobin to carbon monoxide and is observed even when myoglobin-dependent oxygen uptake is decreased in the presence of antimycin, myxothiazol, or cyanide. This feature is not observed in spectra of isolated mitochondria -- LII] exposed to 50% carbon monoxide in oxygen. Candidates for 00-~~~ ~~~Lactate the origin ofthis difference are many. It may reflect increased reduction ofcytochrome c or ofcytochrome c1 ofubiquinone- oz 1.5 cytochrome c oxidoreductase [reduced minus oxidized difference spectrum, 418, 523, and 553 nm (refs. 34 and 35)]. Or 0 it may reflect a ferryl or a low-spin ferric form of myoglobin z or possibly a minor perturbation in the steady state of cytochrome oxidase. Inhibitors of the mitochondrial respiratory chain (36) were used to elucidate the relation of myoglobin-mediated oxygen 0 uptake to mitochondrial function. Cyanide (1 mM) or myxothiazole (2 ,uM) inhibit 80-85% of the respiratory oxygen uptake ofcardiac myocytes. In the presence ofeither, myoglobin-mediated oxygen flux is abolished. Oxygen up- CO CCCP CO+CCCP take can be inhibited partially (50-60% of control) at lower concentrations of cyanide (10-50 ,uM) or of antimycin (0.6 FIG. 6. Effect of carbon monoxide and CCCP on energetics of pM). At this level of inhibition, difference spectra (50 uM cardiac myocytes. CCCP (when present) was 1.0 ,uM. Carbon cyanide minus control) exhibit features near 550 and 600 nm, monoxide pressure (when present) was 75 torr. Oxygen pressure was attributed to increased reduction of cytochrome c and to 1-5 torr in 15 experiments, 15 torr in 1 experiment, and 50 torr in 1 perturbation of cytochrome oxidase. Difference spectra experiment; the results did not differ significantly. Although (antimycin minus control) exhibit well-resolved maxima near sarcoplasmic myoglobin is partially deoxygenated at solution Po2 = and attributed to additional reduction of 1-5 torr, there is no anoxic stimulus to lactate production until 430 563 nm, solution Po2 is decreased to about 0.1 torr (16). Oxygen uptake and cytochrome b of ubiquinone-cytochrome c oxidoreductase lactate accumulation are rates. ATP and phosphocreatine are steady- (36, 37). In these partially inhibited states of cellular respi- state concentrations. Results are expressed as fractions ofthe control ration, carbon monoxide (10-40% in the gas phase) brings values. These are as follows: oxygen uptake, 2.8 ± 0.6 nmol per min about only a small inhibition (11 + 5%; n = 7) of oxygen per mg of protein (n = 16); ATP, 29 ± 5 nmol per mg of rectang- uptake in each experiment. Optical spectra indicate no ular cell protein [protein of structurally intact cells (ref. 20)] (n = 16); further reduction of cytochrome c or perturbation of phosphocreatine, 40 ± 6 nmol per mg of rectangular cell protein (n cytochrome oxidase. An increased absorbance is noted near = 15); lactate, 2.1 ± 0.4 nmol per min per mg of rectangular cell 419 nm. protein (n = 4). To demonstrate the effect of myoglobin blockade on cell energetics, intracellular mitochondria of cardiac myocytes myoglobin was essentially fully oxygenated. Since the oxy- were partially uncoupled by the proton ionophore carbonyl gen pressure difference between the extracellular medium cyanide m-chlorophenylhydrazone (CCCP), approximately and the intracellular mitochondria of isolated cells does not doubling oxygen uptake at lower cellular ATP and phos- exceed 2-3 torr (15, 16), the mitochondria experienced large phocreatine levels (Fig. 6). In the presence of CCCP, at both oxygen pressures. Optical spectra (Figs. 4 and 5) establish physiological and higher oxygen pressures, carbon monoxide that cytochromes of intracellular mitochondria of myocytes blockade of myoglobin function does not change ATP con- exposed to carbon monoxide (50%) in the presence of much centration significantly, but it markedly decreases phospho- oxygen, conditions of the respiratory plateau of Fig. 2, are in creatine concentrations (P c 0.01) and markedly increases a largely oxidized state unaltered by carbon monoxide. A the rate oflactate accumulation (P - 0.03). This indicates that large part, about one-third, of the total oxygen uptake of blockade of myoglobin function results in a decreased rate of cardiac myocytes is abolished by carbon monoxide blockade ATP generation by mitochondrial oxidative phosphorylation. of intracellular myoglobin function (Figs. 2 and 6). The Increased oxygen uptake with CCCP indicates that oxygen myoglobin-dependent component of the cellular oxygen up- diffusion to mitochondria is not rate limiting at the oxygen take decreases linearly with increasing fractional saturation pressure used in these experiments. of sarcoplasmic myoglobin with carbon monoxide (that is, decreasing fraction ofoxymyoglobin), with a slope near unity (Fig. 3). Half inhibition was achieved at different Pco (3-15 DISCUSSION torr) in experiments at different oxygen pressures, but always We use carbon monoxide to block the oxygen-binding func- when myoglobin was half saturated with carbon monoxide. tion of myoglobin in situ in the cardiac myocyte. The Therefore, the effect of carbon monoxide is exerted on equilibrium binding of cytoplasmic myoglobin to oxygen (16) myoglobin alone and cannot be ascribed to inhibition ofother and the equilibrium partition of cytoplasmic myoglobin cellular functions. We conclude that the myoglobin-depen- between oxygen and carbon monoxide (Fig. 1) are similar to dent component of the oxygen uptake is proportional to the those of purified myoglobin, indicating that ligand binding is fraction of sarcoplasmic myoglobin combined with oxygen. not strongly modified in the intracellular environment. This Myoglobin-dependent oxygen uptake disappears when justifies the use of carbon monoxide here. We note that oxidative phosphorylation is blocked by myxothiazol (2 AM) attempts to use agents such as hydroperoxides to block or cyanide (1 mM). It becomes small when the rate ofelectron intracellular myoglobin function in cardiac myocytes are throughput in the mitochondrial respiratory chain is dimin- thwarted by rapid reduction of intracellular ferric or ferryl ished by antimycin (0.6 pM) or cyanide (10-50 pM). This myoglobin (7, 16, 38). suggests that myoglobin-mediated oxygen delivery requires In many of the experiments presented here (Figs. 2 and 3) electron flow through the cytochrome bc, complex (complex very high oxygen pressures (40-340 torr) were maintained in III) and/or cytochrome oxidase (complex IV) of the respi- the medium in which the cells were suspended. Sarcoplasmic ratory chain. Downloaded by guest on October 1, 2021 Biochemistry: Wittenberg and Wittenberg Proc. Natl. Acad. Sci. USA 84 (1987) 7507 Resting myocytes at physiological ambient Po2 (1-5 torr) 5. Lawrie, R. A. (1950) J. Agric. Sci. 40, 356-366. maintain their energy reserves in the face of blockade of 6. Murray, J. D. (1977) Lectures on Nonlinear-Differential-Equa- Partial uncoupling of mitochondrial tion Models in Biology (Clarendon, Oxford), pp. 42-82. myoglobin function. 7. Wittenberg, B. A., Wittenberg, J. B. & Caldwell, P. R. B. oxidative phosphorylation roughly doubles respiratory oxy- (1975) J. Biol. Chem. 250, 9038-9043. gen uptake and establishes a new steady state, simulating that 8. Cole, R. P. (1983) Respir. Physiol. 53, 1-14. in the working heart. The phosphocreatine reserve is less and 9. Driedzic, W. R. (1983) Comp. Biochem. Physiol. A 76, ATP concentration is near a minimum working level. Carbon 487-493. monoxide blockade of myoglobin oxygenation impairs the 10. Taylor, D. J., Matthews, P. M. & Radda, G. K. (1986) Respir. ability of these myocytes to meet the demand for ATP (Fig. Physiol. 63, 275-283. 6). The steady-state concentration of intracellular ATP is 11. Wittenberg, B. A., Wong, C. F. & Wittenberg, J. B. (1986) itselfconserved, but a sharp fall in the standing concentration Biophys. J. 49, 242a (abstr.). and a dramatic increase in the rate of 12. Wittenberg, B. A. & Wittenberg, J. B. (1987) Biophys. J. 51, of phosphocreatine 406a (abstr.). lactate accumulation indicate that the rate of oxidative 13. Kawashiro, T., Nusse, W. & Scheid, P. (1975) Pflugers Arch. phosphorylation has decreased, and the short fall in ATP 359, 231-251. generation has been compensated by a shift in the balance 14. Mahler, M., Louy, C., Homsher, E. & Peskoff, A. (1985) J. between phosphocreatine and creatine and by increased Gen. Physiol. 86, 105-134. aerobic glycolysis to meet the fixed demand for ATP. 15. Katz, I. R., Wittenberg, J. B. & Wittenberg, B. A. (1984) J. Intracellular myoglobin function, therefore, supports ATP Biol. Chem. 259, 7504-7509. generation at extracellular oxygen pressures close to the 16. Wittenberg, B. A. & Wittenberg, J. B. (1985) J. Biol. Chem. pressure expected for intact cardiac muscle. 260, 6548-6554. mitochondrial ATP generation 17. Gayeski, T. E. J. & Honig, C. R. (1986) Am. J. Physiol. 251, Oxymyoglobin supporting H789-H799. may deliver its oxygen to mitochondria. Alternatively, 18. Clark, A., Clark, P. A. A., Connett, R. J., Gayeski, T. E. J. & sarcoplasmic oxymyoglobin might accept electrons from Honig, C. R. (1987) Am. J. Physiol. 252, C583-C587. mitochondria, with concomitant reduction of heme iron- 19. Wittenberg, B. A. & Robinson, T. (1981) Cell Tissue Res. 216, ligated oxygen to water. Our evidence cannot distinguish 231-251. between these two formal possibilities. Either mechanism 20. Wittenberg, B. A., White, R. L., Ginzberg, R. D. & Spray, requires interaction between oxymyoglobin and mitochon- D. C. (1986) Circ. Res. 59, 143-151. dria. 21. Palmer, J. W., Tandler, B. & Hoppel, C. L. (1977) J. Biol. Myoglobin-dependent oxygen delivery to mitochondria, Chem. 252, 8731-8739. here in myocytes, finds a strong parallel in 22. Cole, R. P., Sukanek, P. C., Wittenberg, J. B. & Wittenberg, demonstrated B. A. (1982) J. Appl. Physiol. 53, 1116-1124. leghemoglobin-mediated oxygen delivery to nitrogen-fixing 23. Degn, H. & Wohlrab, H. (1971) Biochim. Biophys. Acta 245, symbiotic bacteria of the soybean root nodule. Oxygen 347-355. delivered by leghemoglobin supports ATP generation in the 24. Giardina, B. & Amiconi, G. (1981) Methods Enzymol. 76, intact nodule (39) and in a broken cell system (40-43). 417-427. Dissolved oxygen, in contrast, although consumed rapidly, 25. Gill, S. J. (1981) Methods Enzymol. 76, 427-438. supports only limited ATP generation. The bacterial 26. Yu, C. A., Yu, L. & King, T. E. (1974) J. Biol. Chem. 249, oxidase(s), presumably located in the cell membrane, is 4905-4910. separated from leghemoglobin by the bacterial cell wall. We 27. Riccio, P., Schagger, H., Engel, W. D. & von Jagow, G. (1977) how leghemoglobin-delivered oxygen is Biochim. Biophys. Acta 459, 250-262. do not yet know 28. Reddy, K. V. S. & Hendler, R. W. (1983) J. Biol. Chem. 258, transferred across the bacterial wall, just as we do not 8568-8581. understand how myoglobin-bound oxygen is delivered across 29. Weiss, H. & Kolb, H. J. (1979) Eur. J. Biochem. 99, 139-149. the outer mitochondrial membrane to the inner membrane. 30. Hatefi, Y. & Galante, Y. M. (1980) J. Biol. Chem. 255, We conclude that a dominant function of myoglobin in the 5530-5537. cardiac myocyte is myoglobin-mediated oxygen delivery to 31. Strittmatter, P. & Velick, S. F. (1956) J. Biol. Chem. 221, mitochondria. The myoglobin-mediated oxygen flow sup- 253-264. ports ATP generation at physiological oxygen pressures. We 32. Strittmatter, P. (1960) J. Biol. Chem. 235, 2492-2497. suggest that myoglobin-mediated oxygen delivery may con- 33. Spatz, L. & Strittmatter, P. (1971) Proc. Natl. Acad. Sci. USA to the ability of the heart to sustain 68, 1042-1046. tribute importantly 34. Li, Y., Leonard, K. & Weiss, H. (1981) Eur. J. Biochem. 116, maximum work output. Loss ofthis function may explain the 199-205. cardiac toxicity of subacute carbon monoxide poisoning. 35. King, T. E. (1983) Adv. Enzymol. Relat. Areas Mol. Biol. 54, 267-366. We thank Drs. C. A. Appleby, D. Mauzerall, and M. Wikstrom for 36. von Jagow, G. & Link, T. A. (1986) Methods Enzymol. 126, helpful discussion and Chui Fan Wong for technical assistance. This 253-271. work was supported in part by a grant in aid from the New York 37. Wikstrom, M. & Saraste, M. (1984) in Bioenergetics, ed. Heart Association (to B.A.W.), by Grants HL 19299 and HL 33655 Ernster, L. (Elsevier, Amsterdam), pp. 49-94. from the U.S. Public Health Service (to B.A.W.), and by Research 38. Tamura, M., Araki, R., Ishikawa, T., Sagisaka, K. & Grants DMB 84-16001 and PCM 84-16016 (to J.B.W.) from the U.S. Yamazaki, I. (1980) J. Biochem. (Tokyo) 88, 1211-1213. National Science Foundation. J.B.W. is Research Career Program 39. Bergersen, F. J., Turner, G. L. & Appleby, C. A. (1973) Awardee 1-K6-733 of the U.S. National Heart, Lung and Blood Biochim. Biophys. Acta 292, 271-282. Institute. 40. Wittenberg, J. B., Bergersen, F. J., Appleby, C. A. & Turner, G. L. (1974) J. Biol. Chem. 249, 4057-4066. 1. Millikan, G. A. (1939) Physiol. Rev. 19, 503-523. 41. Wittenberg, J. B. (1980) in Nitrogen Fixation, eds. Orme- 2. Wittenberg, J. B. (1970) Physiol. Rev. 50, 559-636. Johnson, W. H. & Newton, W. E. (University Park Press, 3. Pattengale, K. & Holloszy, J. 0. (1967) Am. J. Physiol. 213, Baltimore), Vol. 2, pp. 53-67. 783-785. 42. Appleby, C. A. (1984) Annu. Rev. Plant Physiol. 35, 443-478. 4. Wittenberg, J. B. & Wittenberg, B. A. (1981) in Oxygen and 43. Bergersen, F. J. (1982) Root Nodules of Legumes: Structure Living Processes, ed. Gilbert, D. L. (Springer, New York), pp. and Functions (Research Studies Press, Wiley, Chichester, 177-199. U.K.). Downloaded by guest on October 1, 2021