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Nonheme iron protein as a possible site of rotenone inhibition of mitochondrial NADH dehydrogenase
ARCHIVES
OF
BIOCHEMISTRY
Nonheme
Iron
AND
BIOPHYSICS
Protein
as a Possible
of Mitochondrial ROSINE Department
NADH
Site of Rotenone
September
Inhibition
Dehydrogenase’
BOIS2 AND RONALD
of Biophysics and Physical University of Pennsylvania, Received
362-369 (1969)
129,
Biochemistry, Philadelphia,
9, 1968; accepted
W. ESTABROOK3 Johnson Research Foundalion, Pennsylvania 1910,$ September
19, 1968
The inhibition by rotenone of NADH oxidase activity of submitochondrial particles (ETP) has been studied, and a titer of 0.07 mumoles rotenone/mg protein has been found for 50% inhibition. The extent of reduction of flavoproteins has been examined spectrophotometrically using the wavelength pair 470 minus 500 nm, during the slow oxidation of NADH in the presence of varying concentrations of rotenone. With concentrations of rotenone of 0.2 mpmoles/mg protein or more, a pigment was observed which showed a decrease in absorbance at 470 nm relative to 500 nm. This pigment remains reduced after the oxidation of NADH by the rotenone inhibited system. The optical difference spectrum of this pigment is characterized by two broad troughs with minima at about 415 and 455 nm. The extent of reduction of the nonheme iron protein associated with NADH dehydrogenase has been evaluated by electron paramagnetic resonance spectroscopy. These experiments showed the presence of an EPR signal at 9 = 1.94 after the slow oxidation of NADH in the presence of rotenone. From these results it is suggested that the nonheme iron protein associated with NADH dehgdrogenase is involved in the mechanism of inhibition of NADH oxidation by rotenone. -
Rotenone is a heterocyclic alkaloid containing two dihydropyran rings and a dihydrofuran ring. Although rotenone is recognized as a potent inhibitor of NADH oxidation as catalyzed by mammalian mitochondria or submitochondrial particles, little if nothing is known about the mechanism of its inhibition. Since it does not inhibit succinate oxidation under similar experimental conditions (l-3), it is assumed that the site of rotenone inhibition is associated with those respiratory chain carriers functional in 1 These studies were supported in part by a USPHS Research Grant GM 12202. 2 Fellow of the Centre National de la Recherche Scientifique and Recipient of a Fulbright Travel Grant,. Permanent, address: Laboratoire de Chimie Biologique, Faculte des Sciences, 96 Boulevard Raspail, Paris, France. 3 Present address: Department of Biochemistry, Southwestern Medical School at Dallas, University of Texas, Dallas, Texas 75235.
the transfer of reducing equivalents from NADH to coenzyme Q. During the last 10 years there has been some controversy as to whether the site of inhibition by rotenone is on the substrate side or on the oxygen side of NADH dehydrogenase (see 2-4 for references), but it seems more and more accepted now that its site of inhibition is after the flavoprotein (5) and nonheme iron protein (KHI protein) (6, 7) components of NADH dehydrogenase and before the coenzyme Q (Co&). Recently Chance et al. (4) have proposed that the site of rotenone inhibition is situated between two postulated flavoprotein components of NADH dehydrogenase, namely 17~~1 and FED, . Horgan et al. (8) have concluded from studies measuring the binding of 14C labelled rotenone to submitochondrial particles, that there exists a diversity of sites interacting with rotenone. On the basis of their (S) experiments, they 362
SITE
OF ROTENONE
suggest that lipid may be involved in the inhibition by rotenone. The present report describes experiments suggesting an involvement of the NHI protein associated with the NADH dehydrogenase in the inhibitory effect of rotenone. This conclusion is supported by optical spectral measurements as well as electron paramagnetic resonance (EPR) studies of submitochondrial particles. MATERIALS
AND
METHODS
Electron transport particles (ETP) were prepared from beef heart mitochondria according to the method of Crane et al. (9). Reactions were carried out with ETP suspended in potassium phosphate buffer 0.1 M, pH 7.4. Protein concentrations were determined by the biuret method (10) with bovine serum albumin used as the standard. All experiments were performed at room temperature (25’). The NADH oxidase activity of ETP was measured polarographically using a Clark oxygen electrode (11). The extent of reduction of the endogenous cytochromes and flavoproteins of ETP was determined with an Aminco-Chance dual wavelength (split beam) spectrophotometer, using the following wavelengths pairs: GO5630 nm for cyt,ochrome a, 562-575 nm for cytochrome b, 550-540 nm for cytochrome cl + c, and 47@500 nm for flavoproteins. The wavelength pair for flavoproteins (12) was selected since spectral studies comparing solutions of FMN with solutions of cytochrome c showed a minimal interference from cytochrome absorption. Optical difference spectra were recorded either with the wavelength scanning recording spectrophotometer described earlier (13), or with the Aminco-Chance spectrophotometer working in the split-beam mode. Rotenone was purchased from K and K Laborat,ories Inc., Plainview, New York. Solutions were prepared as concent,rnted ethanolic solutions, and then diluted and added in small volumes (O.Ol&O.OZ ml) to the samples to obtain the final desired concentrations. Appropriate co~ltrols with equivalent volumes of ethanol were carried ollt. A solrltion of NADH (Sigma Chemical Company) was freshly prepared at a concentration of 50 1,111 in dihlte sodium bicarbonate buffer, pI1 8.5. Electron paramagnetic resonance (EPR) spectra were obtained as the first, derivative spectra with a Varian E-3 S band spectrometer. The conditions of the experiments are described in the figure legends.
363
INHIBITION RESULTS
Titration
of Inhibition of NADH Activity by Rotenone
Oxidase
Although a variety of titrations have been published (1, 5, 8) showing the effect of increasing concentrations of rotenone on the NADH oxidase activity of mitochondrial and submitochondrial particles, it was considered necessary to repeat these titrations under the experimental condiCons employed in the present study. As shown in Fig. 1, a concentration of about 0.07 mpmoles rotenone/mg protein causes a 50 % inhibition of the oxidase activity as measured by the oxygen electrode. This titer, as well as the shape of the inhibition curve, agrees fairly well with the results of Burgos and Redfearn (5) as well as with those of Horgan et al. (8). In agreement with the earlier studies of Redfearn et al. (lJ), a finite time course of inhibition rl-as observed when low concentrations of rotenone were employed as inhibitor. Spectrophotometric 2Ueaswements of Pigment Reduction and Ozidatim Attempts flavoprotein
to determine t’he extent of reduction of ETP during the
IOOL-
0
0.08
Rotenone
0.16
0.24
Concentration
0.32 mpmole/mg
Pmt.
1. Titration of inhibition of NADH oxidation by roteuone. Samples of ETP (0.15 0.25 mg protein) were added to 3.0 ml of 0.1 M phosphate buffer, pH 7.4. One minute after the addition of rotenone the reaction was initiated by the addition of 0.02 ml of a 50 rnxf sol\lt,ion of NADH. The rate of oxygen utilization was determined polarographically with an oxygen electrode. FIG.
364
BOIS
AND
FIG. 2. Spectrophotometric studies on the effect of varying concentrations of rotenone on the absorption changes at 470 nm minus 500 nm. Submitochondrial particles (ETP) were suspended in 3.0 ml of 0.1 M phosphate buffer, pH 7.4, to a concentration of 4.0 mg protein/ml. One minute after the addition of rotenone, a 0.015-ml aliquot of a 50 mM NADH solution was added. A downward deflection of t)he trace represents a decrease in absorbance at 470 nm relative to 500 nm. The concentrations of rotenone employed in each experiment are indicated on the appropriate tracing, and are expressed as mrmoles/mg protein. The dat.a have been retraced and superimposed from a series of independent experiments.
oxidation of NADH, in the presence of varying concentrations of rotenone, were carried out by measuring the change in absorbance at 470 nm relative to 500 nm. The results of a typical series of experiments are shown in Fig. 2. As illustrated, the addition of NADH results in a cyclic decrease in absorbance at 470 nm, presumably associated with the reduction of flavoprotein. It can be observed that after oxidation of the added NADH, in the absence of rotenone, a small residual absorbance change remains, since the tracing does not return to the original baseline level. This small absorbance remaining after NADH oxidation is attributable to the spectral contribution of a cytochrome b like pigment (compare Fig. 5A) previously described by Minakami et al. (15). More interesting is the observation that with increasing concentrations of rotenone present, i.e., a concentration producing an inhibition of the NADH oxidase activity of 95 % or more, there is a considerable increase in the magnitude of this residual absorbance change remaining after the very slow oxidation of NADH. The results of a series of many such experiments, made at different protein concentrations (l-5 mg protein/ml),
ESTABROOK
FIG. 3. The relationship between rotenone concentration and the magnitude of the spectral changes observed at 470 nm during NADH oxidation. A suspension of ETP was diluted to 3.0 ml in 0.1 M phosphate buffer, pH 7.4, at a protein concentration between 1 and 6 mg protein/ml. One minute after addition of varying concentrations of rotenone, 0.015 ml of a 50 mM solution of NADH was added, as described in Fig. 2. The maximal decrease in absorbance at 470 nm during the steady state oxidation of NADH is illustrated in Curve A. The contribution of absorption remaining after a cycle of NADH oxidation is indicated in Curve B. The duration of the redox cycle observed spectrophotometrically is presented in Curve C. Since the series of experiments were carried out at different protein concentrations, the results have been normalized to indicate the absorption differences observed at a concentration of 1.5 mg protein/ml.
are summarized in Fig. 3. The addition of rotenone prior to NADH causes no significant spectral change at this wavelength pair. Two points are apparent from this data. First, with low concentrations of rotenone (less than 0.1 mcLmoles/mg protein) a decrease in the magnitude of the absorbance decrease occurs, as observed during the steady state of NADH oxidation. This decrease corresponds to a decreasing contribution at this wavelength region of the cytochromes, as can be observed if the steady state levels of reduction of the cytochromes are followed at the wavelength pairs of their CZJ bands. During the steady state of NADH oxidation in the absence of rotenone, the decrease in absorbance at 470 nm is about 15 % higher than is seen when an excess of rotenone is present. Second, with increasing concentrations of rotenone the amount of pigment bleached irreversibly; i.e., the portion of the absorbance change remaining after oxidation of the added NADH, in-
SITE
OF ROTESONE
creases to a maximum. At concentrations of rotenone greater than 0.2 mpmoles/mg protein, about one-half of the initial absorbance change seen during the steady state oxidation of NADH remains. To determine that this residual absorbance remaining after XADH oxidation in the presence of rotenone was not a spectrophotometric artifact, experiments were carried out routinely by adding a second aliquot of NADH. As illustrated in Fig. 4, the addition of NADH causes a cyclic decrease in absorbance at 470 nm as described above. The second addition of NADH produces a similar but somewhat longer cycle of pigment reduct’ion to the same extent of absorbance change, but in this instance t’he oxidat’ion of NADH results in a return of the absorbance trace to the level prevailing before the second addition of NADH; i.e., the contribution of reRotenone NADH d--t
oJ$\+
\
NADH’,
~7.~~5 \
F 0002
.-----
I$vmo~eQ w 6Osec
0.;75 Absorbonce
COsec I*4
f FIG. 4. A comparison between the kinetics of oxygen utilization and the spectral changes observed at 470 minus 500 nm during the oxidation of NADH in the presence of rotenone. A suspension of ETP in 3.0 ml of 0.1 M phosphate buffer, pII 7.4, at a protein concentration of 0.95 mg/ml was employed for both types of experiments. At the points indicated, a 0.025.ml aliquot of a 50 mM solution of XADH was added. The upper curve represents the polarographic measurement of oxygen utilization, while the lower set of curves represents t,he spectrophotometric measurement of absorbance changes. Varying concentrations of rotenone were added one min prior to addition of NADH. Rotenone concentrations are indicated as mpmoles/mg protein.
INHIBITION
365
sidual pigment irreversibly bleached does not increase after further periods of NADH oxidation. As shown in Fig. 4, where both oxygen electrode tracings of oxygen utilization and spectrophotometric tracings of pigment bleaching are illustrated, there is a parallel time course for the rate of oxygen ut’ilizntion and the reoxidation of the pigments bleached on addition of SADH. Nature
of th,e Components Contributing Spectral Changes
to the
In order to better evaluate the spectral contributions of those pigments undergoing reduction during XADH oxidation, in the presence or absence of rotenone, a series of experiments were carried out using the wavelength scanning recording spectrophotometer. As shown in Fig. 5,4, after oxidation of an aliquot of XADH in the absence of rotenone, a cytochrome b-like component remains reduced, contributing to the difference spect,ra by absorbance maxima at 430 and 562 nm. When a similar sample of ETP is first pretreated with a high concentration of rotenone (3.1 mpmoles,/mg protein) and then NADH added, the spectral studies shorn that the extent’ of cytochrome reduction during the steady state of NADH oxidation is very low indeed, if not negligible (Fig. 5B). This is most apparent if the contribution of the cyt’ochrome b-like component is accounted for during these studies. The spectral changes att,ributable to the cytochrome h-like component can be subtracted from the spectral changes observed during IYADH oxidation in the presence of rotenone by pretreating t,he ETP sample present in the reference cuvette of the spect,rophotometer with a small aliquot of NADH (cf. Fig. 5A). In this way the spectra of those pigments, other than the cytochrome b-like component, can be determined as shown in Fig. 5B (dashed curve). This difference spectrum shows t’hat, during the steady st>ate oxidation of NADH in t’he presence of rotenone, a pigment or pigments with absorption maxima at about 455 and 415 nm are bleached. After oxidation of the XADH added to the rotenone containing sample of ETP, the pigments contributing to the residual absorbance changes described above,
366
BOIS
AND
exclusive of those spectral changes attributable to the cytochrome b-like component, can be evaluated as shown in Fig. 5 C, D, and E. With relatively low concentrations of robenone (0.1-0.3 mpmoles/mg
A
~\
-
A
protein) one observes a component with two rather broad troughs in the difference spectrum. The minima observed at 415 and 470 nm are not typical of flavin absorbance. In fact, these spectral changes are highly suggestive of the spectral changes observed during the reduction of nonheme iron proteins such as adrenodoxin and ferredoxin.
EPR Spectra
i--
z------” E
400
ESTABROOK
450
& Wavelength
550
660
imp1
FIG. 5. Optical difference spectra of ETP reduced with NADH in the presence or absence of rotenone. Aliquots of ETP were suspended in 0.1 M phosphate buffer, pH 7.4, to a protein concentration of 4 mg/ml. The diluted ETP was divided equally into two cuvettes and a baseline of equal light absorbance recorded. Curve A illustrates the difference spectrum of the cytochrome b like pigment which remains reduced after oxidation of 0.02 ml-aliquot of 50 mM NADH. A similar experiment was carried out with another aliquot of diluted ETP, except that the contents of the experimental cuvette were pretreated by the addition of a rotenone solution (3.1 maoles/mg protein). The subsequent addition of 0.02 ml of 50 mM NADH causes the spectral change shown by Curve B (solid line). Addition of 0.02 ml of 50 mM NADH to the contents of the reference cuvette results in the appearance of the cytochrome b like component, and the resultant difference spectrum is shown by Curve B (dashed line). A similar series of experiments was carried out using lower concentrations of rotenone, thereby permitting the oxidation of NADH added. The difference spectrum of the pigment remaining reduced after oxidation of NADH in the presence of rotenone corrected for the cytochrome b like component seen in the absence of rotenone, is shown in Curve C, D, and E. The results with 0.1 (E), 0.2 (D), and 0.3 (C) mfimoles rotenone/mg protein are indicated on the figure.
Submitochondrial particles are known to have at least three different types of nonheme iron proteins detectable at low temperature by EPR spectroscopy. These nonheme iron proteins are believed to be associated with NADH dehydrogenase, succinate dehydrogenase, and the cytochromes b and cl complex of the respiratory chain. The reduced nonheme iron proteins are distinguished by their characteristic EPR spectra showing principal absorption (in the first derivative spectra) in the region of gL equals 1.90-1.94, and g/j equals 2.0. Experiments were therefore carried out to investigate by EPR spectroscopy the state of the NH1 proteins during the oxidation of NADH with rotenone present. As shown in Fig. GA, the EPR spectrum of ETP in the absence of either inhibitor or substrate shows a broad signal attributable to the copper associated with oxidized cytochrome oxidase. When an excess of NADH is added to a sample of ETP, sufficient to utilize the oxygen present in the reaction mixture, a complex EPR spectrum is obtained (Fig. 6B) which is dominated by an absorption at about g = 1.94. As shown by Palmer et al. (7) and Beinert et al. (16, 17) as well as by Tyler et al. (6)) this EPR spectrum illustrates the contributions of the three types of nonheme iron proteins present in ETP. In confirmation of the earlier studies by Tyler et al. (6), the EPR spectrum observed in the presence of an excess of rotenone and NADH during the aerobic steady state (Fig. 6C) shows a significant decrease in the magnitude of the g = 1.94 signal relative to that observed anaerobically. Addition of rotenone in the absence of NADH caused no significant change in the EPR spectrum. Previous studies have shown (6) that about 50% of the g = 1.94 signal is contributed by the nonheme iron protein associated with NADH
SITE
3;50 Magnet,c
OF KOTENONE
3$50 Field
iGouss)
FIG. 6. Electron paramagnetic resonance (EPR) spectra of ETP reduced with NADH in the presence or absence of rotenone. Each sample consisted of a 0.5-ml suspension of ETP diluted in 0.1 M phosphate buffer, pH 7.4, to a protein concentration of 10 mg/ml. Curve A is the spectrum obtained with untreated ETP. Curve B represents the spectrum obtained when a sample of ETP was treated with 0.025-ml aliquot of a 50 mM solution of PiADH and the sample permitted to attain anaerobiosis. For Curve C, rotenone (2.7 mpmoles/mg protein) was added to ETP two min prior to the addition of a 0.025.ml aliquot of a 50 rn.\l solution of NADH. The sample was then frozen 1 min after addition of NADH. The EPR spectrum of those pigments observed after NADH oxidation in the presence of rotenone are shown in Curve D. In this experiment the reaction was followed in the dual wavelength spectrophotomet,er at 470 nm minus 500 nm, and samples withdrawn at different times after addition of NADH and rapidly loaded into the EPR tubes and frozen. In the experiment illustrated in Curve D, 0.5 mpmoles rotenone/mg protein were added to 3 ml of ETP suspension containing 10 mg of protein/ml. A 0.015-ml aliquot of a 50 rnM solution of NADH was then added. Six minutes after the addition of NADH, the sample was removed and frozen. The EPR spectra were recorded at liquid nitrogen temperature using a microwave power of 25 mW, modulation amplitude of 16 G, a scanning rate of 125 G/min with a time constant of 1 set and a receiver gain of 2.5 X 10-b.
367
INHIBITION
dehydrogenase, while the other 505G is associated with the nonheme iron protein associated with succinat,e dehydrogenase. This can be demonstrated directly by the subsequent addition of succinate to a sample containing rotenone and an excess of XADH. In addition, t’hc presence of the copper signal associated with oxidized cytochrome oxidase in the EPR spectrum sho\vn in Fig. 6C, is evidence that the sample is aerobic. If a limiting amount of rotenone is present, such as t,o allow the reoxidation of the added NADH in a few minutes, thereby simulating the conditions described above for the optical spectral measurements, EPR spectra of the type presented in Fig. 6D are obtained. It is apparent that, after oxidation of the KADH added, there remains a weak EPR signa,l at about’ g = 1.94 in addition to the signal of copper. The oxidation of limiting amounts of NADH in t,he absence of rotenone did not result in any permanent measurable change in the EPR spectrum after the oxidation cycle was complete; i.e., a spectrum comparable to t’hat shown in Fig. GA was obtained. Thus, it seems probable that the respirat,ory chain component remaining reduced after NADH oxidation in t.he presence of rotenone, and responsible for the small optical absorbance changes seen at 470 nm relat’ive to 500 nm, may be t,he nonheme iron protein associated with NADH dehydrogenase. It is not possible at the present time t*o make any quantitative estimate of the iron involved. The results presented in Fig. 6 indicate that the magnitude of the g = 1.94 signal remaining after NADH oxidation in the presence of rotenone is about one-quarter to one-third of that observed during NADH oxidation in the aerobic steady state in the presence of an excess of rotenone, and about one-eighth of the g = 1.94 signal observed after complete enzymatic reduction of the respiratory chain component’s by NADH. DISCUSSION
The results presented in t,his paper suggest an involvement of the NH1 protein associated with the respiratory chain NADHdehydrogenase in the mechanism of inhibition of electron transport by rotenone. Whether the inhibition is the result of the formation of an inactive complex between
365
BOIS
AND
reduced nonheme iron and rotenone, or the result of some disruption in the arrangement of the different redox components of the complex NADH dehydrogenase, brought out by rotenone and preventing their reoxidation .by CoQ, cannot be ascertained. The possibility of rotenone inducing a structural disorganization is supported by the recent findings that rotenone (although at considerably higher concentration than the ones used in these experiments), can inhibit yeast alcohol-dehydrogenase (18) and modify glutamic dehydrogenase (19), presumably in t’he latter case by inducing a conformational change. On the other hand, the finite time course of inhibition observed during rotenone inhibition of NADH oxidation is compatible with the formation of an irreversible complex of rotenone with the reduced nonheme iron protein. One must recall that the soluble preparation of NADH dehydrogenase showing an NADH-induced EPR signal at g = 1.94 (20) is not rotenone sensitive, at least not in the same manner as that described for the particulate bound enzyme (8). This discrepancy may be explained, however, by a marked change in the nature of the enzyme after its extraction and isolation, especially if one considers that such preparations do not have a significant NADH-Co& reductase activity. The present interpretation, that rotenone inhibits by interacting with a NH1 protein associated with NADH dehydrogenase, is consistent with the recently proposed relationship between the presence of an NADH induced EPR signal at g = 1.94 and the rotenone sensitivit,y of NADH oxidation. Studies with submit.ochondrial particles prepared from Saccharomyces cerivisiae strain of yeast show no NADH induced EPR signal and no rotenone sensitivity, whereas those derived from the yeast Candida utilis show both rotenone sensitivity of NADH oxidation and an EPR signal at g = 1.94 associated with NADH dehydrogenase (21, 22).
The data presented in this paper also indicate that NH1 proteins may contribute
ESTABROOK
significantly to the absorption changes occurring in the spectral region routinely used for the study of redox changes of flavoproteins. REFERENCES 1. ERNFIXR, L., DBLLNER, G., AND AZZONE, G. F., J. Biol. Chem. 238, 1124 (1963). 2. ERNSTER, L., AND LEE, C. P., Ann. Rev. Biochem. 33, 729 (1964). 3. PULLMAN, M. E., AND SCHJYTZ, G., Ann. Rev. Biochem. 36, 539 (1967). 4. CH~NCF,, B., ERNSTER, L., GSRLAND, P. B., LICE, C-P., LIGHT, P. A., OHNISHI, T., RAG.~N, E. I., AND WONG, D., Proc. Nat. Acad. Sci. 57, 1498 (1967). 5. BURGOS, J., AND REDFE:IRN, E. R., Biochim. Biophys. Acta 110, 475 (1965). 6. TYLER, D. D., GONZE, J., ESTABROOK, R. W., AND BUTO~, R. A., in “Symposium on NonHeme Iron Proteins: Role in Energy Conservation,” (A. San Pietro ed.), p. 447. Antioch Press, Yellow Springs, Ohio (1965). H., 7. PALMER, G., HORGAN, D., TISDALE, SINGER, T., AND BEINERT, H., J. Biol. Chem. 243,844 (1968). 8. HORGAN, D., SINGER, T., AND C~RIDA, T., J. Biol. Chem. 243,834 (1968). 9. CRANE, F. L., GLENN, J. L., AND GREEN, D. E., Biochim. Biophys. Acta 22,475 (1956). C. J., AND 10. GORNALL, A. G., BARDAWILL, DAVID, M. M., J. Biol. Chem. 177,751 (1949). in Enzy11. ESTABROOK, R. W., in “Methods and Phosmology,” Vol. X, “Oxidation phorylation”; R. W. Estabrook and M. E. Pullman, p. 41. Academic Press, New York (1967). 12. KLINGENBERG, M., AND BUTCHER, T., Biochem. 2. 331, 312 (1959). Enzymes,” 13. ESTABROOK, R. W., in “Haematin (J. E. Falk, R. Lemberg, and R. K. Morton, eds.), p. 436. Pergamon Press, New York (1961). 14. REDFEARN, E. R., WHITTAKER, P. A., AND and Related BURGOS, J., in “Oxidases Redox Systems,” (T. King, H. Mason, and M. Morrison, eds.), p. 943. Wiley, New York (1965). S., SCHINDLER, F. J., AND ESTA15. MINAKAMI, BROOK, R. W., J. Biol. Chem. 239, 2942, 2049 (1966).
SITE 16. Bersrxt~~~, II.,
W., AND PALMER, G., no. 15, 229 (1962). 17. BEINERT, H., in “Symposium on Non-Heme Iron Proteins: Role in Energy Conservation,” (il. San Pietro, ed.), p. 23. Antioch Press, Yellow Springs, Ohio (1965). 18. BALC.IT-.~GI~, W., AND MarrToor;, J. R., Nature 215, 5097 (1967). 19. Brr~ro~, 1:. iz., Biochem. 6, 1088 (1967). Brookhaven,
HEISEN,
OF ROTENONE
Symp.
Biol.,
IhYlIBITIOS
369
20. BEINERT, H., PALMER, G., CREMON.~, T., AND SINGER, T. P., J. Biol. Chem. 240,475 (1965). 21. OHNISHI, T., RACKER, E., SCHLLYER, H., AXD CHANCE, B., Symp. Flavoproteins, Nagoya, Japan (1967), in press. 22. SHARP, C. W.,~~XICLER, fs., ~)OUGLAS, H. C., PALMER, G., .IND FELTOX, S. P., Arch. Biothem. Biophys. 122, 810 (1967).
OF
BIOCHEMISTRY
Nonheme
Iron
AND
BIOPHYSICS
Protein
as a Possible
of Mitochondrial ROSINE Department
NADH
Site of Rotenone
September
Inhibition
Dehydrogenase’
BOIS2 AND RONALD
of Biophysics and Physical University of Pennsylvania, Received
362-369 (1969)
129,
Biochemistry, Philadelphia,
9, 1968; accepted
W. ESTABROOK3 Johnson Research Foundalion, Pennsylvania 1910,$ September
19, 1968
The inhibition by rotenone of NADH oxidase activity of submitochondrial particles (ETP) has been studied, and a titer of 0.07 mumoles rotenone/mg protein has been found for 50% inhibition. The extent of reduction of flavoproteins has been examined spectrophotometrically using the wavelength pair 470 minus 500 nm, during the slow oxidation of NADH in the presence of varying concentrations of rotenone. With concentrations of rotenone of 0.2 mpmoles/mg protein or more, a pigment was observed which showed a decrease in absorbance at 470 nm relative to 500 nm. This pigment remains reduced after the oxidation of NADH by the rotenone inhibited system. The optical difference spectrum of this pigment is characterized by two broad troughs with minima at about 415 and 455 nm. The extent of reduction of the nonheme iron protein associated with NADH dehydrogenase has been evaluated by electron paramagnetic resonance spectroscopy. These experiments showed the presence of an EPR signal at 9 = 1.94 after the slow oxidation of NADH in the presence of rotenone. From these results it is suggested that the nonheme iron protein associated with NADH dehgdrogenase is involved in the mechanism of inhibition of NADH oxidation by rotenone. -
Rotenone is a heterocyclic alkaloid containing two dihydropyran rings and a dihydrofuran ring. Although rotenone is recognized as a potent inhibitor of NADH oxidation as catalyzed by mammalian mitochondria or submitochondrial particles, little if nothing is known about the mechanism of its inhibition. Since it does not inhibit succinate oxidation under similar experimental conditions (l-3), it is assumed that the site of rotenone inhibition is associated with those respiratory chain carriers functional in 1 These studies were supported in part by a USPHS Research Grant GM 12202. 2 Fellow of the Centre National de la Recherche Scientifique and Recipient of a Fulbright Travel Grant,. Permanent, address: Laboratoire de Chimie Biologique, Faculte des Sciences, 96 Boulevard Raspail, Paris, France. 3 Present address: Department of Biochemistry, Southwestern Medical School at Dallas, University of Texas, Dallas, Texas 75235.
the transfer of reducing equivalents from NADH to coenzyme Q. During the last 10 years there has been some controversy as to whether the site of inhibition by rotenone is on the substrate side or on the oxygen side of NADH dehydrogenase (see 2-4 for references), but it seems more and more accepted now that its site of inhibition is after the flavoprotein (5) and nonheme iron protein (KHI protein) (6, 7) components of NADH dehydrogenase and before the coenzyme Q (Co&). Recently Chance et al. (4) have proposed that the site of rotenone inhibition is situated between two postulated flavoprotein components of NADH dehydrogenase, namely 17~~1 and FED, . Horgan et al. (8) have concluded from studies measuring the binding of 14C labelled rotenone to submitochondrial particles, that there exists a diversity of sites interacting with rotenone. On the basis of their (S) experiments, they 362
SITE
OF ROTENONE
suggest that lipid may be involved in the inhibition by rotenone. The present report describes experiments suggesting an involvement of the NHI protein associated with the NADH dehydrogenase in the inhibitory effect of rotenone. This conclusion is supported by optical spectral measurements as well as electron paramagnetic resonance (EPR) studies of submitochondrial particles. MATERIALS
AND
METHODS
Electron transport particles (ETP) were prepared from beef heart mitochondria according to the method of Crane et al. (9). Reactions were carried out with ETP suspended in potassium phosphate buffer 0.1 M, pH 7.4. Protein concentrations were determined by the biuret method (10) with bovine serum albumin used as the standard. All experiments were performed at room temperature (25’). The NADH oxidase activity of ETP was measured polarographically using a Clark oxygen electrode (11). The extent of reduction of the endogenous cytochromes and flavoproteins of ETP was determined with an Aminco-Chance dual wavelength (split beam) spectrophotometer, using the following wavelengths pairs: GO5630 nm for cyt,ochrome a, 562-575 nm for cytochrome b, 550-540 nm for cytochrome cl + c, and 47@500 nm for flavoproteins. The wavelength pair for flavoproteins (12) was selected since spectral studies comparing solutions of FMN with solutions of cytochrome c showed a minimal interference from cytochrome absorption. Optical difference spectra were recorded either with the wavelength scanning recording spectrophotometer described earlier (13), or with the Aminco-Chance spectrophotometer working in the split-beam mode. Rotenone was purchased from K and K Laborat,ories Inc., Plainview, New York. Solutions were prepared as concent,rnted ethanolic solutions, and then diluted and added in small volumes (O.Ol&O.OZ ml) to the samples to obtain the final desired concentrations. Appropriate co~ltrols with equivalent volumes of ethanol were carried ollt. A solrltion of NADH (Sigma Chemical Company) was freshly prepared at a concentration of 50 1,111 in dihlte sodium bicarbonate buffer, pI1 8.5. Electron paramagnetic resonance (EPR) spectra were obtained as the first, derivative spectra with a Varian E-3 S band spectrometer. The conditions of the experiments are described in the figure legends.
363
INHIBITION RESULTS
Titration
of Inhibition of NADH Activity by Rotenone
Oxidase
Although a variety of titrations have been published (1, 5, 8) showing the effect of increasing concentrations of rotenone on the NADH oxidase activity of mitochondrial and submitochondrial particles, it was considered necessary to repeat these titrations under the experimental condiCons employed in the present study. As shown in Fig. 1, a concentration of about 0.07 mpmoles rotenone/mg protein causes a 50 % inhibition of the oxidase activity as measured by the oxygen electrode. This titer, as well as the shape of the inhibition curve, agrees fairly well with the results of Burgos and Redfearn (5) as well as with those of Horgan et al. (8). In agreement with the earlier studies of Redfearn et al. (lJ), a finite time course of inhibition rl-as observed when low concentrations of rotenone were employed as inhibitor. Spectrophotometric 2Ueaswements of Pigment Reduction and Ozidatim Attempts flavoprotein
to determine t’he extent of reduction of ETP during the
IOOL-
0
0.08
Rotenone
0.16
0.24
Concentration
0.32 mpmole/mg
Pmt.
1. Titration of inhibition of NADH oxidation by roteuone. Samples of ETP (0.15 0.25 mg protein) were added to 3.0 ml of 0.1 M phosphate buffer, pH 7.4. One minute after the addition of rotenone the reaction was initiated by the addition of 0.02 ml of a 50 rnxf sol\lt,ion of NADH. The rate of oxygen utilization was determined polarographically with an oxygen electrode. FIG.
364
BOIS
AND
FIG. 2. Spectrophotometric studies on the effect of varying concentrations of rotenone on the absorption changes at 470 nm minus 500 nm. Submitochondrial particles (ETP) were suspended in 3.0 ml of 0.1 M phosphate buffer, pH 7.4, to a concentration of 4.0 mg protein/ml. One minute after the addition of rotenone, a 0.015-ml aliquot of a 50 mM NADH solution was added. A downward deflection of t)he trace represents a decrease in absorbance at 470 nm relative to 500 nm. The concentrations of rotenone employed in each experiment are indicated on the appropriate tracing, and are expressed as mrmoles/mg protein. The dat.a have been retraced and superimposed from a series of independent experiments.
oxidation of NADH, in the presence of varying concentrations of rotenone, were carried out by measuring the change in absorbance at 470 nm relative to 500 nm. The results of a typical series of experiments are shown in Fig. 2. As illustrated, the addition of NADH results in a cyclic decrease in absorbance at 470 nm, presumably associated with the reduction of flavoprotein. It can be observed that after oxidation of the added NADH, in the absence of rotenone, a small residual absorbance change remains, since the tracing does not return to the original baseline level. This small absorbance remaining after NADH oxidation is attributable to the spectral contribution of a cytochrome b like pigment (compare Fig. 5A) previously described by Minakami et al. (15). More interesting is the observation that with increasing concentrations of rotenone present, i.e., a concentration producing an inhibition of the NADH oxidase activity of 95 % or more, there is a considerable increase in the magnitude of this residual absorbance change remaining after the very slow oxidation of NADH. The results of a series of many such experiments, made at different protein concentrations (l-5 mg protein/ml),
ESTABROOK
FIG. 3. The relationship between rotenone concentration and the magnitude of the spectral changes observed at 470 nm during NADH oxidation. A suspension of ETP was diluted to 3.0 ml in 0.1 M phosphate buffer, pH 7.4, at a protein concentration between 1 and 6 mg protein/ml. One minute after addition of varying concentrations of rotenone, 0.015 ml of a 50 mM solution of NADH was added, as described in Fig. 2. The maximal decrease in absorbance at 470 nm during the steady state oxidation of NADH is illustrated in Curve A. The contribution of absorption remaining after a cycle of NADH oxidation is indicated in Curve B. The duration of the redox cycle observed spectrophotometrically is presented in Curve C. Since the series of experiments were carried out at different protein concentrations, the results have been normalized to indicate the absorption differences observed at a concentration of 1.5 mg protein/ml.
are summarized in Fig. 3. The addition of rotenone prior to NADH causes no significant spectral change at this wavelength pair. Two points are apparent from this data. First, with low concentrations of rotenone (less than 0.1 mcLmoles/mg protein) a decrease in the magnitude of the absorbance decrease occurs, as observed during the steady state of NADH oxidation. This decrease corresponds to a decreasing contribution at this wavelength region of the cytochromes, as can be observed if the steady state levels of reduction of the cytochromes are followed at the wavelength pairs of their CZJ bands. During the steady state of NADH oxidation in the absence of rotenone, the decrease in absorbance at 470 nm is about 15 % higher than is seen when an excess of rotenone is present. Second, with increasing concentrations of rotenone the amount of pigment bleached irreversibly; i.e., the portion of the absorbance change remaining after oxidation of the added NADH, in-
SITE
OF ROTESONE
creases to a maximum. At concentrations of rotenone greater than 0.2 mpmoles/mg protein, about one-half of the initial absorbance change seen during the steady state oxidation of NADH remains. To determine that this residual absorbance remaining after XADH oxidation in the presence of rotenone was not a spectrophotometric artifact, experiments were carried out routinely by adding a second aliquot of NADH. As illustrated in Fig. 4, the addition of NADH causes a cyclic decrease in absorbance at 470 nm as described above. The second addition of NADH produces a similar but somewhat longer cycle of pigment reduct’ion to the same extent of absorbance change, but in this instance t’he oxidat’ion of NADH results in a return of the absorbance trace to the level prevailing before the second addition of NADH; i.e., the contribution of reRotenone NADH d--t
oJ$\+
\
NADH’,
~7.~~5 \
F 0002
.-----
I$vmo~eQ w 6Osec
0.;75 Absorbonce
COsec I*4
f FIG. 4. A comparison between the kinetics of oxygen utilization and the spectral changes observed at 470 minus 500 nm during the oxidation of NADH in the presence of rotenone. A suspension of ETP in 3.0 ml of 0.1 M phosphate buffer, pII 7.4, at a protein concentration of 0.95 mg/ml was employed for both types of experiments. At the points indicated, a 0.025.ml aliquot of a 50 mM solution of XADH was added. The upper curve represents the polarographic measurement of oxygen utilization, while the lower set of curves represents t,he spectrophotometric measurement of absorbance changes. Varying concentrations of rotenone were added one min prior to addition of NADH. Rotenone concentrations are indicated as mpmoles/mg protein.
INHIBITION
365
sidual pigment irreversibly bleached does not increase after further periods of NADH oxidation. As shown in Fig. 4, where both oxygen electrode tracings of oxygen utilization and spectrophotometric tracings of pigment bleaching are illustrated, there is a parallel time course for the rate of oxygen ut’ilizntion and the reoxidation of the pigments bleached on addition of SADH. Nature
of th,e Components Contributing Spectral Changes
to the
In order to better evaluate the spectral contributions of those pigments undergoing reduction during XADH oxidation, in the presence or absence of rotenone, a series of experiments were carried out using the wavelength scanning recording spectrophotometer. As shown in Fig. 5,4, after oxidation of an aliquot of XADH in the absence of rotenone, a cytochrome b-like component remains reduced, contributing to the difference spect,ra by absorbance maxima at 430 and 562 nm. When a similar sample of ETP is first pretreated with a high concentration of rotenone (3.1 mpmoles,/mg protein) and then NADH added, the spectral studies shorn that the extent’ of cytochrome reduction during the steady state of NADH oxidation is very low indeed, if not negligible (Fig. 5B). This is most apparent if the contribution of the cyt’ochrome b-like component is accounted for during these studies. The spectral changes att,ributable to the cytochrome h-like component can be subtracted from the spectral changes observed during IYADH oxidation in the presence of rotenone by pretreating t,he ETP sample present in the reference cuvette of the spect,rophotometer with a small aliquot of NADH (cf. Fig. 5A). In this way the spectra of those pigments, other than the cytochrome b-like component, can be determined as shown in Fig. 5B (dashed curve). This difference spectrum shows t’hat, during the steady st>ate oxidation of NADH in t’he presence of rotenone, a pigment or pigments with absorption maxima at about 455 and 415 nm are bleached. After oxidation of the XADH added to the rotenone containing sample of ETP, the pigments contributing to the residual absorbance changes described above,
366
BOIS
AND
exclusive of those spectral changes attributable to the cytochrome b-like component, can be evaluated as shown in Fig. 5 C, D, and E. With relatively low concentrations of robenone (0.1-0.3 mpmoles/mg
A
~\
-
A
protein) one observes a component with two rather broad troughs in the difference spectrum. The minima observed at 415 and 470 nm are not typical of flavin absorbance. In fact, these spectral changes are highly suggestive of the spectral changes observed during the reduction of nonheme iron proteins such as adrenodoxin and ferredoxin.
EPR Spectra
i--
z------” E
400
ESTABROOK
450
& Wavelength
550
660
imp1
FIG. 5. Optical difference spectra of ETP reduced with NADH in the presence or absence of rotenone. Aliquots of ETP were suspended in 0.1 M phosphate buffer, pH 7.4, to a protein concentration of 4 mg/ml. The diluted ETP was divided equally into two cuvettes and a baseline of equal light absorbance recorded. Curve A illustrates the difference spectrum of the cytochrome b like pigment which remains reduced after oxidation of 0.02 ml-aliquot of 50 mM NADH. A similar experiment was carried out with another aliquot of diluted ETP, except that the contents of the experimental cuvette were pretreated by the addition of a rotenone solution (3.1 maoles/mg protein). The subsequent addition of 0.02 ml of 50 mM NADH causes the spectral change shown by Curve B (solid line). Addition of 0.02 ml of 50 mM NADH to the contents of the reference cuvette results in the appearance of the cytochrome b like component, and the resultant difference spectrum is shown by Curve B (dashed line). A similar series of experiments was carried out using lower concentrations of rotenone, thereby permitting the oxidation of NADH added. The difference spectrum of the pigment remaining reduced after oxidation of NADH in the presence of rotenone corrected for the cytochrome b like component seen in the absence of rotenone, is shown in Curve C, D, and E. The results with 0.1 (E), 0.2 (D), and 0.3 (C) mfimoles rotenone/mg protein are indicated on the figure.
Submitochondrial particles are known to have at least three different types of nonheme iron proteins detectable at low temperature by EPR spectroscopy. These nonheme iron proteins are believed to be associated with NADH dehydrogenase, succinate dehydrogenase, and the cytochromes b and cl complex of the respiratory chain. The reduced nonheme iron proteins are distinguished by their characteristic EPR spectra showing principal absorption (in the first derivative spectra) in the region of gL equals 1.90-1.94, and g/j equals 2.0. Experiments were therefore carried out to investigate by EPR spectroscopy the state of the NH1 proteins during the oxidation of NADH with rotenone present. As shown in Fig. GA, the EPR spectrum of ETP in the absence of either inhibitor or substrate shows a broad signal attributable to the copper associated with oxidized cytochrome oxidase. When an excess of NADH is added to a sample of ETP, sufficient to utilize the oxygen present in the reaction mixture, a complex EPR spectrum is obtained (Fig. 6B) which is dominated by an absorption at about g = 1.94. As shown by Palmer et al. (7) and Beinert et al. (16, 17) as well as by Tyler et al. (6)) this EPR spectrum illustrates the contributions of the three types of nonheme iron proteins present in ETP. In confirmation of the earlier studies by Tyler et al. (6), the EPR spectrum observed in the presence of an excess of rotenone and NADH during the aerobic steady state (Fig. 6C) shows a significant decrease in the magnitude of the g = 1.94 signal relative to that observed anaerobically. Addition of rotenone in the absence of NADH caused no significant change in the EPR spectrum. Previous studies have shown (6) that about 50% of the g = 1.94 signal is contributed by the nonheme iron protein associated with NADH
SITE
3;50 Magnet,c
OF KOTENONE
3$50 Field
iGouss)
FIG. 6. Electron paramagnetic resonance (EPR) spectra of ETP reduced with NADH in the presence or absence of rotenone. Each sample consisted of a 0.5-ml suspension of ETP diluted in 0.1 M phosphate buffer, pH 7.4, to a protein concentration of 10 mg/ml. Curve A is the spectrum obtained with untreated ETP. Curve B represents the spectrum obtained when a sample of ETP was treated with 0.025-ml aliquot of a 50 mM solution of PiADH and the sample permitted to attain anaerobiosis. For Curve C, rotenone (2.7 mpmoles/mg protein) was added to ETP two min prior to the addition of a 0.025.ml aliquot of a 50 rn.\l solution of NADH. The sample was then frozen 1 min after addition of NADH. The EPR spectrum of those pigments observed after NADH oxidation in the presence of rotenone are shown in Curve D. In this experiment the reaction was followed in the dual wavelength spectrophotomet,er at 470 nm minus 500 nm, and samples withdrawn at different times after addition of NADH and rapidly loaded into the EPR tubes and frozen. In the experiment illustrated in Curve D, 0.5 mpmoles rotenone/mg protein were added to 3 ml of ETP suspension containing 10 mg of protein/ml. A 0.015-ml aliquot of a 50 rnM solution of NADH was then added. Six minutes after the addition of NADH, the sample was removed and frozen. The EPR spectra were recorded at liquid nitrogen temperature using a microwave power of 25 mW, modulation amplitude of 16 G, a scanning rate of 125 G/min with a time constant of 1 set and a receiver gain of 2.5 X 10-b.
367
INHIBITION
dehydrogenase, while the other 505G is associated with the nonheme iron protein associated with succinat,e dehydrogenase. This can be demonstrated directly by the subsequent addition of succinate to a sample containing rotenone and an excess of XADH. In addition, t’hc presence of the copper signal associated with oxidized cytochrome oxidase in the EPR spectrum sho\vn in Fig. 6C, is evidence that the sample is aerobic. If a limiting amount of rotenone is present, such as t,o allow the reoxidation of the added NADH in a few minutes, thereby simulating the conditions described above for the optical spectral measurements, EPR spectra of the type presented in Fig. 6D are obtained. It is apparent that, after oxidation of the KADH added, there remains a weak EPR signa,l at about’ g = 1.94 in addition to the signal of copper. The oxidation of limiting amounts of NADH in t,he absence of rotenone did not result in any permanent measurable change in the EPR spectrum after the oxidation cycle was complete; i.e., a spectrum comparable to t’hat shown in Fig. GA was obtained. Thus, it seems probable that the respirat,ory chain component remaining reduced after NADH oxidation in t.he presence of rotenone, and responsible for the small optical absorbance changes seen at 470 nm relat’ive to 500 nm, may be t,he nonheme iron protein associated with NADH dehydrogenase. It is not possible at the present time t*o make any quantitative estimate of the iron involved. The results presented in Fig. 6 indicate that the magnitude of the g = 1.94 signal remaining after NADH oxidation in the presence of rotenone is about one-quarter to one-third of that observed during NADH oxidation in the aerobic steady state in the presence of an excess of rotenone, and about one-eighth of the g = 1.94 signal observed after complete enzymatic reduction of the respiratory chain component’s by NADH. DISCUSSION
The results presented in t,his paper suggest an involvement of the NH1 protein associated with the respiratory chain NADHdehydrogenase in the mechanism of inhibition of electron transport by rotenone. Whether the inhibition is the result of the formation of an inactive complex between
365
BOIS
AND
reduced nonheme iron and rotenone, or the result of some disruption in the arrangement of the different redox components of the complex NADH dehydrogenase, brought out by rotenone and preventing their reoxidation .by CoQ, cannot be ascertained. The possibility of rotenone inducing a structural disorganization is supported by the recent findings that rotenone (although at considerably higher concentration than the ones used in these experiments), can inhibit yeast alcohol-dehydrogenase (18) and modify glutamic dehydrogenase (19), presumably in t’he latter case by inducing a conformational change. On the other hand, the finite time course of inhibition observed during rotenone inhibition of NADH oxidation is compatible with the formation of an irreversible complex of rotenone with the reduced nonheme iron protein. One must recall that the soluble preparation of NADH dehydrogenase showing an NADH-induced EPR signal at g = 1.94 (20) is not rotenone sensitive, at least not in the same manner as that described for the particulate bound enzyme (8). This discrepancy may be explained, however, by a marked change in the nature of the enzyme after its extraction and isolation, especially if one considers that such preparations do not have a significant NADH-Co& reductase activity. The present interpretation, that rotenone inhibits by interacting with a NH1 protein associated with NADH dehydrogenase, is consistent with the recently proposed relationship between the presence of an NADH induced EPR signal at g = 1.94 and the rotenone sensitivit,y of NADH oxidation. Studies with submit.ochondrial particles prepared from Saccharomyces cerivisiae strain of yeast show no NADH induced EPR signal and no rotenone sensitivity, whereas those derived from the yeast Candida utilis show both rotenone sensitivity of NADH oxidation and an EPR signal at g = 1.94 associated with NADH dehydrogenase (21, 22).
The data presented in this paper also indicate that NH1 proteins may contribute
ESTABROOK
significantly to the absorption changes occurring in the spectral region routinely used for the study of redox changes of flavoproteins. REFERENCES 1. ERNFIXR, L., DBLLNER, G., AND AZZONE, G. F., J. Biol. Chem. 238, 1124 (1963). 2. ERNSTER, L., AND LEE, C. P., Ann. Rev. Biochem. 33, 729 (1964). 3. PULLMAN, M. E., AND SCHJYTZ, G., Ann. Rev. Biochem. 36, 539 (1967). 4. CH~NCF,, B., ERNSTER, L., GSRLAND, P. B., LICE, C-P., LIGHT, P. A., OHNISHI, T., RAG.~N, E. I., AND WONG, D., Proc. Nat. Acad. Sci. 57, 1498 (1967). 5. BURGOS, J., AND REDFE:IRN, E. R., Biochim. Biophys. Acta 110, 475 (1965). 6. TYLER, D. D., GONZE, J., ESTABROOK, R. W., AND BUTO~, R. A., in “Symposium on NonHeme Iron Proteins: Role in Energy Conservation,” (A. San Pietro ed.), p. 447. Antioch Press, Yellow Springs, Ohio (1965). H., 7. PALMER, G., HORGAN, D., TISDALE, SINGER, T., AND BEINERT, H., J. Biol. Chem. 243,844 (1968). 8. HORGAN, D., SINGER, T., AND C~RIDA, T., J. Biol. Chem. 243,834 (1968). 9. CRANE, F. L., GLENN, J. L., AND GREEN, D. E., Biochim. Biophys. Acta 22,475 (1956). C. J., AND 10. GORNALL, A. G., BARDAWILL, DAVID, M. M., J. Biol. Chem. 177,751 (1949). in Enzy11. ESTABROOK, R. W., in “Methods and Phosmology,” Vol. X, “Oxidation phorylation”; R. W. Estabrook and M. E. Pullman, p. 41. Academic Press, New York (1967). 12. KLINGENBERG, M., AND BUTCHER, T., Biochem. 2. 331, 312 (1959). Enzymes,” 13. ESTABROOK, R. W., in “Haematin (J. E. Falk, R. Lemberg, and R. K. Morton, eds.), p. 436. Pergamon Press, New York (1961). 14. REDFEARN, E. R., WHITTAKER, P. A., AND and Related BURGOS, J., in “Oxidases Redox Systems,” (T. King, H. Mason, and M. Morrison, eds.), p. 943. Wiley, New York (1965). S., SCHINDLER, F. J., AND ESTA15. MINAKAMI, BROOK, R. W., J. Biol. Chem. 239, 2942, 2049 (1966).
SITE 16. Bersrxt~~~, II.,
W., AND PALMER, G., no. 15, 229 (1962). 17. BEINERT, H., in “Symposium on Non-Heme Iron Proteins: Role in Energy Conservation,” (il. San Pietro, ed.), p. 23. Antioch Press, Yellow Springs, Ohio (1965). 18. BALC.IT-.~GI~, W., AND MarrToor;, J. R., Nature 215, 5097 (1967). 19. Brr~ro~, 1:. iz., Biochem. 6, 1088 (1967). Brookhaven,
HEISEN,
OF ROTENONE
Symp.
Biol.,
IhYlIBITIOS
369
20. BEINERT, H., PALMER, G., CREMON.~, T., AND SINGER, T. P., J. Biol. Chem. 240,475 (1965). 21. OHNISHI, T., RACKER, E., SCHLLYER, H., AXD CHANCE, B., Symp. Flavoproteins, Nagoya, Japan (1967), in press. 22. SHARP, C. W.,~~XICLER, fs., ~)OUGLAS, H. C., PALMER, G., .IND FELTOX, S. P., Arch. Biothem. Biophys. 122, 810 (1967).