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Biological electron transport
J. Theoret. Biol. (1965) 8, 214-220
Biological Electron Transport II. A Variation of the Imidazole Pump Model to Include Coupling DAN W. URRY AND HENRY EYRING Department
of Chemistry,
University
of Utah, Salt Lake City, Utah, U.S.A.
(Received 2 1 May 1964) A variation of the imidazole pump model is proposed which phosphorylates ADP and releases hydroxyl ion as the result of the passage of a pair of electrons. This is accomplished by introducing quinone between imidazoles each of which is attached to its iron porphyrin. Thus an electron flow model capable of coupling is formed which requires only electron donor, electron acceptor, ADP and P, in order to continuously generate ATP and hydroxyl ion. The mechanism is compared to experimental data and found to correlate and interpret many observations while remaining versatile enough to incorporate specific cationic and other effects. Electron flow is seen to precede and be independent of the exchange reactions. The model exhibits a stoichiometry of two electrons per coupling event while utilizing univalent changes in the oxidative states of the iron; it contains an understanding of uncoupling and of control mechanisms, and it demonstrates a reasonable way in which the energy released in the oxidative process can result in parcelling of useful energy to the organism.
Introduction The central question of coupling is how, in the process of transferring two electrons from one molecular species to another, can a molecular configuration be produced which will result in the formation of a phosphate anhydride bond with regeneration of the carriers? Progress has been made by bioThere is now substantial chemists in detailing the reactions involved. agreement on three basic steps (Chance & Hollunger, 1961; Lehninger & Wadkins, 1962), $ BHI +A N C (electron transfer step) AH,+B+C (1) A-C+Pi=A+C
N Pi (HzlsO-PO4
exchange)
(2)
C N P+ ADP $ ATP+ C (ATP-32P exchange) (3) where N stands for a high energy bond and Pi is inorganic orthophosphate. These equations are justified on the basis that the exchange reactions, which are themselves independent, are also independent of the electron transfer 214
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step, that is, under certain circumstances electron transfer can occur without eliciting the exchanges. However, as written, equation (1) is misleading in that it implies hydrogen atom transfer which certainly does not appear to be the case in the cytochrome region but may represent the situation between diphosphopyridine nucleotide and flavoprotein. In general, it is a case of two electrons transferred per high-energy phosphate bond formed. A probable sequence of the carriers of the electron transport chain, or respiratory chain as it is also called, has been given in a recent review (Lehninger & Wadkins, 1962) as succinate 3 FP2 1 DPN 3 FPr + cyt-b --t cyt-c, -+ cyt-c --) cyt-a -+ cyt-a, + OZ. V V V ATP ATP ATP The experimental evidence therein reviewed indicates that the first coupling step occurs between diphosphopyridine nucleotide, DPN, and flavoprotein, FP,, as indicated; that the second coupling step occurs between cyt-b and cyt-c, and the third between cyt-c and oxygen. Not indicated, but certainly involved at different points in the sequence, are quinones, notably coenzyme Q or ubiquinone which is a benzoquinone In fact, an entire Ciba symposium and vitamiqe K, a naphthoquinone. has been devoted to quinones in biological electron transport (Wolstenholme & O’Connor, 1960). Many useful discussions of coupled mechanisms involving quinones have appeared (Brodie, 1961; Lapidot & Samuel, 1962; Clark & Todd, 1960; Slater, Colda-Boonstra & Links, 1960), all of which show at some stage the formation of a quinol phosphate,
O+HPO; +2e-
0
R3
;--
-
-0
(4)
R4
R,
R4
Quinol phosphate is known to be a sufficiently high energy linkage to result in phosphorylation of adenosine diphosphate, i.e.
O-t R3
The quinol can now be oxidized completing
R4
electron flow.
ATP
(5)
216
D.
W.
URRY
AND
H.
EYRING
Under conditions of insufficient phosphate acceptor up to thirty ‘*O exchanges have been reported during the transfer of one pair of electrons (Chan, Lehninger & Ems, 1960). A lack of ADP would allow equation (4) to effect many Hz”0-P04 exchanges. The problems left unanswered in equation (4) are the actual source and mechanism of delivery of electrons. Some of the energy gained on reduction of the quinone must be conserved for phosphate coupling. Even with a simultaneous reduction of the quinone and attack by the quinone oxygen on the phosphorus atom, it is difficult to see why every reduction would necessarily result in quinol phosphate. In tightly coupled systems each pair of electrons results in coupling at each coupling site. Though there are problems with the above mechanism, quinone does appear to be involved in phosphorylation, and probably the best evidence for the obligatory requirement of quinone in the coupling steps is found in photosynthetic phosphorylation. Krogman demonstrated that addition of plastoquinone, a benzoquinone, is essential for photosynthetic phosphorylation in chloroplasts (Krogman, 1961). Description of a Coupling Mechanism The imidazole pump model may be varied to include coupling by interposing a quinone between two imidazoles, each of which is attached to its iron porphyrin system by a covalent bond. This molecular arrangement is given in Fig. 1 at the top left corner. The electron shifts, there indicated, result in the effective transfer of a pair of electrons from the immediate right side of the iron on the left to the right side of the imidazole which enters into covalent bonding with the quinone, reducing it to a quinol. Following the arrows, if a donor covalently interacts with the oxidized iron porphyrin, thereby reducing it, and an acceptor is properly positioned, then a second set of electron shifts is possible resulting in the oxidation of the donor and the reduction of the acceptor with return of the imidazole at the left to its iron porphyrin and the detachment of the imidazole on the right from its iron porphyrin. This, then, is the electron transfer step, and it effectively moves the quinol from the attachment at the left to attachment to the imidazole on the right. Disregarding the dashed arrows for the moment, orthophosphate, HPO,‘, may be introduced. Activation of phosphate 0 involves a phosphoryl
moiety
+P-Oand the release of OH’ which \ 0exchanges with water. Attachment of a phosphoryl moiety requires that a
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:
TRANSPORT
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EYRING
pair of electrons be supplied. Such a pair of electrons is available between the quinol and the imidazole. Therefore, an electrophilic attack on this bond by the phosphorus atom in orthophosphate can result in the release of quinol as quinone, the release of an hydroxyl ion, and the formation of a high energy phosphoryl imidazole. The phosphoryl moiety may then be passed to a negative oxygen on ADP, leaving imidazole with its pair of electrons to reattach to its iron porphyrin regenerating the initial arrangement of the carriers. The net effect of the transfer of a pair of eletrons from a donor to an acceptor is the formation of ATP from ADP and Pi, and the formation of an hydroxyl ion. The quinol-imidazole linkage would t- expected to be energy rich as there is a bridge oxygen between two delocalized systems as seen in carboxyl phosphates and polyphosphates. Also, the phosphoryl-imidazole linkage is known to be an energy-rich bond (Cramer, Schaller & Staab, 1961). What is more exciting is that Boyer et al. (1963) have actually isolated phosphoryl histidine from an actively phosphorylating mitochondrial preparation, the phosphoryl group being attached to one of the nitrogens in the imidazole of histidine. Correlation of Coupling Mechanism with Experimental Data A stringent requirement placed on a coupling mechanism is that the electron transfer step must precede and be independent of the exchange reactions. This requirement is met by the proposed mechanism. A second restriction is that of univalent changes in the oxidative states of the iron yet two electron transfers at each coupling site. This problem is solved by the imidazole pump mechanism. The H,‘sO-PO4 and ATP-32P exchange reactions are present and independent. Rate constants between the cytochromes are reported to be remarkably similar, differing by little more than a factor of ten in an actively phosphorylating system (Chance & Williams, 1956). The order of magnitude of these rates are 10’ to IO* M-l set- I. It is apparent that the coupling steps cannot differ greatly from the non-coupling steps. The rate constants in the cytochrome chain also exhibit similar temperature coefficients (Chance, 1952). The above observations are in agreement with the mechanism as both the imidazole pump model and the variation to include coupling are vibrationally limited mechanisms. In the light of the above mechanism it is not surprising that the steady state ratios of the oxidized and reduced states do not change greatly when the temperature is dropped from room temperature to liquid nitrogen temperatures (Chance & Spencer, 1959). Another point of interest is that coupling has been achieved in intact mitochondria and in mitochondrial fragments, but no aqueous systems
BIOLOGICAL
ELECTRON
TRANSPORT
219
involving isolated and purified components of the electron transport chain have displayed the coupling phenomenon (Lehninger & Wadkins, 1962). The lack of coupling could be explained on the basis of quinol imidazole instability. The quinol-imidazole. configuration would be expected to be unstable when exposed to aqueous systems at physiological pH. Hydrogen ion addition to imidazole could release the quinol as quinone. The original arrangement of the carriers at the top left of Fig. 1 is regenerated after a phosphate acceptor has removed the phosphoryl moiety. Thus electron transfer should be limited under conditions of very low ADP concentrations as is found to be the case (Chance & Williams, 1956). This affords a control mechanism. Consider the situation of abundant DPNH and a constant sum of ADP and ATP. A lack of ADP would correspond to a sufficiency or excess of ATP and oxidative phosphorylation ceases. As the ATP is utilized, the concentration of ADP increases and oxidative phosphorylation increases to meet the requirement of additional ATP. It is reasonable under conditions of rate limiting phosphate acceptor that uncouplers could increase the rate of electron transfer by initiating the electron shifts indicated by the dashed arrows in the figure. It is also understandable that some uncouplers could cause a breakdown of the quinol-imidazole configuration with resultant rates greater than observed during coupling. There are other interesting phenomena that can be interpreted in the light of the proposed model. It has been observed that during electron transport there is a separation of Hi and OH- across the mitochondrial membrane (Lehninger & Wadkins, 1962). A separation of this type can be accomplished by the proposed coupling mechanism simply by a release to one side of the membrane of the OH- which is split off phosphate and a release to the other side of the membrane of the protons resulting upon removal of two electrons from flavoprotein. The model further allows for the incorporation of specific cationic effects as the ATP, ADP, Pi and OH- are anions and associated cations would be required to allow these species to enter or leave a lipoprotein membrane. As has just been demonstrated, this model correlates and interprets many experimental observations. Its most notable features are the independence of the electron transfer step from the exchange reactions and utilization of univalent changes in the oxidative states of the iron while accomplishing two electron transfers. The mechanism answers the central question stated in the introduction of this paper and demonstrates a reasonable way in which the oxidative process can result in the parcelling of useful energy to the organism. The value of a model approach, in general, is that a proposed mechanism serves as a reservoir of known data, a point of comparison for new data and a source of experimental approaches.
220
D.
W.
URRY
AND
H.
EYRING
REFERENCES
BAYER,P. D., HULTQU~~T, D. E., PETZR, J. B., KRIEL,G., M~~CWLL, R. A., DELUCA,M., HNKSON, J. W., BUTLER, L. G. & MOYER,R. W. (1963).Fed. Proc. 22, 1080. BRODIE, A. F. (1961).Fed. Proc. 20,995. CHAN,P. C., LEHNINGER, A. L. & ERNS, T. (1960).J. biol. Chem. 235, 1790. ChWNCB, B. (1952).Nature, 169, 215. CHANCE, B. ‘& HOLLUNGER, G. (1961).J. biol. Chem. 236, 1534. OUNCE,B. & SPENCER, E. L. (1959).Disc. Faraday Sot. 27,200. CHANCE, B. & WILLLUS,G. R. (1956).Advanc. Enzymol. 17,65. CLARK,V. M. & TODD,A. R. (1960).In “Ciba FoundationSymposium on Quinonesin Electron Transport”, p. 190. (G. E. W. Wolstenhohne& C. M. O’Connor, eds.) Boston:Little, Brown & Co. CRAMER, F., SCHALLER, H. & STAAB,H. A. (1961).Chem. Ber. 94, 1612. KROGMAN, D. W. (1961). Biochem. biophys. Res. Comm. 4,275. LAPIWT,A. & SAMUEL, D. (1962).Biochem. biophys. Actu, 65, 164. LEHNINGER, A. L. & WADKINS, C. L. (1962).Ann. Rev. Biochem. 31.47. SLATER, E. C., COLDA-BOONSTRA, J. P. & LINKS,J. (1960).In “Ciba FoundationSymposiumon Quinonesin Electron Transport”, p. 161. (G. E. W. Wolstenholme& M. O’Connor,eds.)Boston:Little, Brown & Co. WOLSTENHOLME, G. E. W. & Q’CONNOR, C. M. (1960).“Ciba FoundationSymposium on Quinonesin ElectronTransport”.Boston:Little, Brown & Co.
Biological Electron Transport II. A Variation of the Imidazole Pump Model to Include Coupling DAN W. URRY AND HENRY EYRING Department
of Chemistry,
University
of Utah, Salt Lake City, Utah, U.S.A.
(Received 2 1 May 1964) A variation of the imidazole pump model is proposed which phosphorylates ADP and releases hydroxyl ion as the result of the passage of a pair of electrons. This is accomplished by introducing quinone between imidazoles each of which is attached to its iron porphyrin. Thus an electron flow model capable of coupling is formed which requires only electron donor, electron acceptor, ADP and P, in order to continuously generate ATP and hydroxyl ion. The mechanism is compared to experimental data and found to correlate and interpret many observations while remaining versatile enough to incorporate specific cationic and other effects. Electron flow is seen to precede and be independent of the exchange reactions. The model exhibits a stoichiometry of two electrons per coupling event while utilizing univalent changes in the oxidative states of the iron; it contains an understanding of uncoupling and of control mechanisms, and it demonstrates a reasonable way in which the energy released in the oxidative process can result in parcelling of useful energy to the organism.
Introduction The central question of coupling is how, in the process of transferring two electrons from one molecular species to another, can a molecular configuration be produced which will result in the formation of a phosphate anhydride bond with regeneration of the carriers? Progress has been made by bioThere is now substantial chemists in detailing the reactions involved. agreement on three basic steps (Chance & Hollunger, 1961; Lehninger & Wadkins, 1962), $ BHI +A N C (electron transfer step) AH,+B+C (1) A-C+Pi=A+C
N Pi (HzlsO-PO4
exchange)
(2)
C N P+ ADP $ ATP+ C (ATP-32P exchange) (3) where N stands for a high energy bond and Pi is inorganic orthophosphate. These equations are justified on the basis that the exchange reactions, which are themselves independent, are also independent of the electron transfer 214
BIOLOGICAL
ELECTRON
215
TRANSPORT
step, that is, under certain circumstances electron transfer can occur without eliciting the exchanges. However, as written, equation (1) is misleading in that it implies hydrogen atom transfer which certainly does not appear to be the case in the cytochrome region but may represent the situation between diphosphopyridine nucleotide and flavoprotein. In general, it is a case of two electrons transferred per high-energy phosphate bond formed. A probable sequence of the carriers of the electron transport chain, or respiratory chain as it is also called, has been given in a recent review (Lehninger & Wadkins, 1962) as succinate 3 FP2 1 DPN 3 FPr + cyt-b --t cyt-c, -+ cyt-c --) cyt-a -+ cyt-a, + OZ. V V V ATP ATP ATP The experimental evidence therein reviewed indicates that the first coupling step occurs between diphosphopyridine nucleotide, DPN, and flavoprotein, FP,, as indicated; that the second coupling step occurs between cyt-b and cyt-c, and the third between cyt-c and oxygen. Not indicated, but certainly involved at different points in the sequence, are quinones, notably coenzyme Q or ubiquinone which is a benzoquinone In fact, an entire Ciba symposium and vitamiqe K, a naphthoquinone. has been devoted to quinones in biological electron transport (Wolstenholme & O’Connor, 1960). Many useful discussions of coupled mechanisms involving quinones have appeared (Brodie, 1961; Lapidot & Samuel, 1962; Clark & Todd, 1960; Slater, Colda-Boonstra & Links, 1960), all of which show at some stage the formation of a quinol phosphate,
O+HPO; +2e-
0
R3
;--
-
-0
(4)
R4
R,
R4
Quinol phosphate is known to be a sufficiently high energy linkage to result in phosphorylation of adenosine diphosphate, i.e.
O-t R3
The quinol can now be oxidized completing
R4
electron flow.
ATP
(5)
216
D.
W.
URRY
AND
H.
EYRING
Under conditions of insufficient phosphate acceptor up to thirty ‘*O exchanges have been reported during the transfer of one pair of electrons (Chan, Lehninger & Ems, 1960). A lack of ADP would allow equation (4) to effect many Hz”0-P04 exchanges. The problems left unanswered in equation (4) are the actual source and mechanism of delivery of electrons. Some of the energy gained on reduction of the quinone must be conserved for phosphate coupling. Even with a simultaneous reduction of the quinone and attack by the quinone oxygen on the phosphorus atom, it is difficult to see why every reduction would necessarily result in quinol phosphate. In tightly coupled systems each pair of electrons results in coupling at each coupling site. Though there are problems with the above mechanism, quinone does appear to be involved in phosphorylation, and probably the best evidence for the obligatory requirement of quinone in the coupling steps is found in photosynthetic phosphorylation. Krogman demonstrated that addition of plastoquinone, a benzoquinone, is essential for photosynthetic phosphorylation in chloroplasts (Krogman, 1961). Description of a Coupling Mechanism The imidazole pump model may be varied to include coupling by interposing a quinone between two imidazoles, each of which is attached to its iron porphyrin system by a covalent bond. This molecular arrangement is given in Fig. 1 at the top left corner. The electron shifts, there indicated, result in the effective transfer of a pair of electrons from the immediate right side of the iron on the left to the right side of the imidazole which enters into covalent bonding with the quinone, reducing it to a quinol. Following the arrows, if a donor covalently interacts with the oxidized iron porphyrin, thereby reducing it, and an acceptor is properly positioned, then a second set of electron shifts is possible resulting in the oxidation of the donor and the reduction of the acceptor with return of the imidazole at the left to its iron porphyrin and the detachment of the imidazole on the right from its iron porphyrin. This, then, is the electron transfer step, and it effectively moves the quinol from the attachment at the left to attachment to the imidazole on the right. Disregarding the dashed arrows for the moment, orthophosphate, HPO,‘, may be introduced. Activation of phosphate 0 involves a phosphoryl
moiety
+P-Oand the release of OH’ which \ 0exchanges with water. Attachment of a phosphoryl moiety requires that a
BIOLOGICAL
58’ i
-9-
,,/’
ELECTRON
-e-
-IL-
:
TRANSPORT
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W.
URRY
AND
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EYRING
pair of electrons be supplied. Such a pair of electrons is available between the quinol and the imidazole. Therefore, an electrophilic attack on this bond by the phosphorus atom in orthophosphate can result in the release of quinol as quinone, the release of an hydroxyl ion, and the formation of a high energy phosphoryl imidazole. The phosphoryl moiety may then be passed to a negative oxygen on ADP, leaving imidazole with its pair of electrons to reattach to its iron porphyrin regenerating the initial arrangement of the carriers. The net effect of the transfer of a pair of eletrons from a donor to an acceptor is the formation of ATP from ADP and Pi, and the formation of an hydroxyl ion. The quinol-imidazole linkage would t- expected to be energy rich as there is a bridge oxygen between two delocalized systems as seen in carboxyl phosphates and polyphosphates. Also, the phosphoryl-imidazole linkage is known to be an energy-rich bond (Cramer, Schaller & Staab, 1961). What is more exciting is that Boyer et al. (1963) have actually isolated phosphoryl histidine from an actively phosphorylating mitochondrial preparation, the phosphoryl group being attached to one of the nitrogens in the imidazole of histidine. Correlation of Coupling Mechanism with Experimental Data A stringent requirement placed on a coupling mechanism is that the electron transfer step must precede and be independent of the exchange reactions. This requirement is met by the proposed mechanism. A second restriction is that of univalent changes in the oxidative states of the iron yet two electron transfers at each coupling site. This problem is solved by the imidazole pump mechanism. The H,‘sO-PO4 and ATP-32P exchange reactions are present and independent. Rate constants between the cytochromes are reported to be remarkably similar, differing by little more than a factor of ten in an actively phosphorylating system (Chance & Williams, 1956). The order of magnitude of these rates are 10’ to IO* M-l set- I. It is apparent that the coupling steps cannot differ greatly from the non-coupling steps. The rate constants in the cytochrome chain also exhibit similar temperature coefficients (Chance, 1952). The above observations are in agreement with the mechanism as both the imidazole pump model and the variation to include coupling are vibrationally limited mechanisms. In the light of the above mechanism it is not surprising that the steady state ratios of the oxidized and reduced states do not change greatly when the temperature is dropped from room temperature to liquid nitrogen temperatures (Chance & Spencer, 1959). Another point of interest is that coupling has been achieved in intact mitochondria and in mitochondrial fragments, but no aqueous systems
BIOLOGICAL
ELECTRON
TRANSPORT
219
involving isolated and purified components of the electron transport chain have displayed the coupling phenomenon (Lehninger & Wadkins, 1962). The lack of coupling could be explained on the basis of quinol imidazole instability. The quinol-imidazole. configuration would be expected to be unstable when exposed to aqueous systems at physiological pH. Hydrogen ion addition to imidazole could release the quinol as quinone. The original arrangement of the carriers at the top left of Fig. 1 is regenerated after a phosphate acceptor has removed the phosphoryl moiety. Thus electron transfer should be limited under conditions of very low ADP concentrations as is found to be the case (Chance & Williams, 1956). This affords a control mechanism. Consider the situation of abundant DPNH and a constant sum of ADP and ATP. A lack of ADP would correspond to a sufficiency or excess of ATP and oxidative phosphorylation ceases. As the ATP is utilized, the concentration of ADP increases and oxidative phosphorylation increases to meet the requirement of additional ATP. It is reasonable under conditions of rate limiting phosphate acceptor that uncouplers could increase the rate of electron transfer by initiating the electron shifts indicated by the dashed arrows in the figure. It is also understandable that some uncouplers could cause a breakdown of the quinol-imidazole configuration with resultant rates greater than observed during coupling. There are other interesting phenomena that can be interpreted in the light of the proposed model. It has been observed that during electron transport there is a separation of Hi and OH- across the mitochondrial membrane (Lehninger & Wadkins, 1962). A separation of this type can be accomplished by the proposed coupling mechanism simply by a release to one side of the membrane of the OH- which is split off phosphate and a release to the other side of the membrane of the protons resulting upon removal of two electrons from flavoprotein. The model further allows for the incorporation of specific cationic effects as the ATP, ADP, Pi and OH- are anions and associated cations would be required to allow these species to enter or leave a lipoprotein membrane. As has just been demonstrated, this model correlates and interprets many experimental observations. Its most notable features are the independence of the electron transfer step from the exchange reactions and utilization of univalent changes in the oxidative states of the iron while accomplishing two electron transfers. The mechanism answers the central question stated in the introduction of this paper and demonstrates a reasonable way in which the oxidative process can result in the parcelling of useful energy to the organism. The value of a model approach, in general, is that a proposed mechanism serves as a reservoir of known data, a point of comparison for new data and a source of experimental approaches.
220
D.
W.
URRY
AND
H.
EYRING
REFERENCES
BAYER,P. D., HULTQU~~T, D. E., PETZR, J. B., KRIEL,G., M~~CWLL, R. A., DELUCA,M., HNKSON, J. W., BUTLER, L. G. & MOYER,R. W. (1963).Fed. Proc. 22, 1080. BRODIE, A. F. (1961).Fed. Proc. 20,995. CHAN,P. C., LEHNINGER, A. L. & ERNS, T. (1960).J. biol. Chem. 235, 1790. ChWNCB, B. (1952).Nature, 169, 215. CHANCE, B. ‘& HOLLUNGER, G. (1961).J. biol. Chem. 236, 1534. OUNCE,B. & SPENCER, E. L. (1959).Disc. Faraday Sot. 27,200. CHANCE, B. & WILLLUS,G. R. (1956).Advanc. Enzymol. 17,65. CLARK,V. M. & TODD,A. R. (1960).In “Ciba FoundationSymposium on Quinonesin Electron Transport”, p. 190. (G. E. W. Wolstenhohne& C. M. O’Connor, eds.) Boston:Little, Brown & Co. CRAMER, F., SCHALLER, H. & STAAB,H. A. (1961).Chem. Ber. 94, 1612. KROGMAN, D. W. (1961). Biochem. biophys. Res. Comm. 4,275. LAPIWT,A. & SAMUEL, D. (1962).Biochem. biophys. Actu, 65, 164. LEHNINGER, A. L. & WADKINS, C. L. (1962).Ann. Rev. Biochem. 31.47. SLATER, E. C., COLDA-BOONSTRA, J. P. & LINKS,J. (1960).In “Ciba FoundationSymposiumon Quinonesin Electron Transport”, p. 161. (G. E. W. Wolstenholme& M. O’Connor,eds.)Boston:Little, Brown & Co. WOLSTENHOLME, G. E. W. & Q’CONNOR, C. M. (1960).“Ciba FoundationSymposium on Quinonesin ElectronTransport”.Boston:Little, Brown & Co.