Biochimica et Biophysica Acta, 463 (1977) 29-89 © Elsevier/North-Holland Biomedical Press BBA 86038

H+-ADENOSINE

TRIPHOSPHATASE

AND

MEMBRANE

ENERGY

COUPLING I. A. K O Z L O V a n d V. P. S K U L A C H E V

Department of Bioenergeties, Laboratory of Bioorganic Chemistry, Moscow State University (U.S.S.R.) (Received July 20th, 1976) (Revised m a n u s c r i p t received D e c e m b e r 17th, 1976)

CONTENTS 1.

Introduction. T h e a i m of the review a n d s o m e definitions . . . . . . . . . . . . . .

30

II.

T h e position of H + - A T P a s e in the system o f the m e m b r a n e - l i n k e d A T P synthesis . . .

31

A. H + - A T P a s e a n d c h e m i o s m o t i c coupling . . . . . . . . . . . . . . . . . . . .

31

B. A T P a s e as a necessary c o m p o n e n t of the m e m b r a n o u s p h o s p h o r y l a t i o n system . . .

31

C. Evidence of A/t.+ generation by the coupling m e m b r a n e A T P a s e s . . . . . . . . .

32

D. Constituents of the H + - A T P a s e complex

33

III. Soluble mitochondrial A T P a s e (factor FI)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Factor FI as a n object of oxidative p h o s p h o r y l a t i o n studies B. T h e q u a t e r n a r y structure of factor FI

. . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . .

35 35 36

C. T h e catalytic properties of factor FI . . . . . . . . . . . . . . . . . . . . . 1. M g ' A T P as the substrate o f soluble A T P a s e . . . . . . . . . . . . . . . . 2. T h e nucleotide specificity o f factor F~ . . . . . . . . . . . . . . . . . . . 3. Functional groups of the catalytic site . . . . . . . . . . . . . . . . . . . 4. O n the covalent intermediates of the H + - A T P a s e reaction . . . . . . . . . . . 5. K o r m a n ' s hypothesis o n the m e c h a n i s m of the A T P a s e (ATP-synthetase) reaction 6. T w o regions for the substrate binding in the F~ catalytic site . . . . . . . . . . 7. A D P - b i n d i n g in the non-catalytic site of factor FI . . . . . . . . . . . . . .

38 38 39 40 41 42 44 48

D. T h e role of factor F~ in the f o r m a t i o n of 1 ~ , + by the H + - A T P a s e complex . . . . . . 1. T h e hypothesis o n A T P translocation between the catalytic a n d non-catalytic F~ sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Factor F, o n the octane/water interface . . . . . . . . . . . . . . . . . . 3. T h e effect of inhibitors on A T P a s e in solution, on the octane/water interface a n d in the m i t o c h o n d r i a l m e m b r a n e . . . . . . . . . . . . . . . . . . . . . .

48

E, T h e role of FI tightly-bound nucleotides . . . . . . . . . . . . . . . . . . . 1. T i g h t l y - b o u n d A T P as a n intermediate of the H + - A T P a s e reaction . . . . . . . . 2. T h e change in the F1 position in the m e m b r a n e during its functioning . . . . . . 3. T h e m e c h a n i s m of action o f the protein inhibitor on the A T P a s e . . . . . . . . 4. T h e reactions of the isotope exchange . . . . . . . . . . . . . . . . . . .

58 58 65 67 68

48 50 51

continued overleaf Abbreviations: A-(CH2)2-P-P-P, 1-adenyl-2-triphosphorylethane; A-(CH2)3-P-P-P, 1-adenyl3 - t r i p h o s p h o r y l p r o p a n e ; A-(CHz)4-P-P-P, l - a d e n y l - 4 - t r i p h o s p h o r y l b u t a n e ; A D P - M C , mixed a n h y d r i d e of A D P a n d mesitylene carboxylic acid; A M P - M C , mixed a n h y d r i d e o f A M P a n d mesitylene carboxylic acid; A M P - P N P , 5'-adenylilimidodiphosphate; A T P - M C , mixed a n h y d r i d e of A T P a n d

continued overleaf

30 1V. The proton-conducting components of H+-ATPase . . . . . . . . . . . . . A. The mechanism of proton transfer between the F~ catalytic site and the chondrial space . . . . . . . . . . . . . . . . . . . . . . . . . . B. The mode of action of DCCD and oligomycin . . . . . . . . . . . . . I. The object of the inhibitors' action . . . . . . . . . . . . . . . . . 2. OSCP-deficient particles . . . . . . . . . . . . . . . . . . . . . V.

H+-ATPase in a range of other biological energy transducers . . . . . . . . A. Ion transport ATPases . . . . . . . . . . . . . . . . . . . . . . B. ATPase of chromaffin granules from the adrenal gland . . . . . . . . . . . . . C. Mitochondrial l/t.+ generators . . . . . . . . . . . . . . . . . . D. Bacteriorhodopsin H + pump . . . . . . . . . . . . . . . . . . .

. . . . extramito. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

70 70 72 72 73 74 74 76 76 77

VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80

Acknowledgements

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83

I. INTRODUCTION. THE AIM OF THE REVIEW AND SOME DEFINITIONS This review is devoted to the problem of the mechanism of the reversible H+-ATPase reaction. This enzyme plays a crucial role in m e m b r a n e energy coupling, as it is responsible for the A T P formation in biological m e m b r a n e s c o n t a i n i n g a respiratory oi photosynthetic apparatus. In what follows we are a t t e m p t i n g to present a general scheme for the H+-ATPase mechanism in terms of a consistent conception, which includes the m i n i m u m n u m b e r of c o m p o n e n t s needed to explain the main experimental results of research in this field. It is expedient to begin the review by defining certain concepts. We shall call H+-ATPase the lipoprotein complex which transfers H + ions through the h y d r o p h o b i c region of the m e m b r a n e in a n A T P hydrolysis-dependent fashion. In intact a n d reconstituted vesicular systems, the H+-ATPase-mediated p r o t o n transfer can occur against the electrochemical gradient of H + ions (A#H~). C o u p l i n g factor F~ is a water-soluble protein which forms the part of the H+-ATPase complex, bearing the catalytic site responsible for the A T P hydrolysis. The ATPase proteolipids are highly b y d r o p h o b i e proteins of the H+-ATPase complex which r e m a i n in the m e m b r a n e after cleavage of factor FI. Factor O S C P is a water-soluble protein which, when c o m b i n e d with ATPase proteolipids, imparts to H + - A T P a s e a sensitivity to such inhibitors as oligomycin and DCCD. mesitylene carboxylic acid; CDP-MC, mixed anhydride of CDP and mesitylene carboxylic acid; CMCD, N-cyclohexyI-N'-/4-(4-methylmorpholine)ethylcarbodiimide; CTP-MC, mixed anhydride of CTP and mesitylene carboxylic acid: DCCD, N, N'-dicyclohexylcarbodiimide; factor F~, soluble mitochondrial ATPase; GDP-MC, mixed anhydride of GDP and rnesitylene carboxylic acid; GTP-MC, mixed anhydride of GTP and mesitylene carboxylic acid: OSCP, oligomycin sensitivity conferring protein; PCMB, p-chloromercuribenzoate; l/tn+, transmembrane difference of electrochemical potentials of hydrogen ions; ] % Volta potential difference in the octane/water system; .l~p, transmembrane electric potential difference.

31 The coupling membrane is a membrane structure of low proton conductivity, containing the systems of formation and use of drill+. It ensures the coupling of the functioning of dfin+ generators with production of useful work. Membrane potential (A~) is the electrical part drills. Proton control is the AfiH+-induced inhibition of H+-ATPase, respiratory or photosynthetic redox-chain activities. When applied to the respiratory chain the proton control is identical to respiratory control.

lI. THE POSITION OF H*-ATPase IN THE SYSTEM OF THE MEMBRANE-LINKED ATP SYNTHESIS IIA. H+-ATPase emd chemiosmotic coupling All the reactions supplying the cell with ATP fall into two main groups. One of these groups unites the phosphorylations requiring no insoluble membrane structures (ATP formation during glycolysis, fermentation and the substrate-level phosphorylation in Krebs' cycle). Other mechanisms of ATP synthesis are membranelinked phosphorylations localized in the inner membrane of the mitochondria, in the membranes of thylakoids of chloroplasts and in bacterial membranes. In mechanisms of the first type, the formation of ATP is the result of the transfer of a high energy phosphoryl group from the product of the substrate oxidation to ADP. In the second case ATP is formed from ADP and phosphate through reversal of the hydrolytic, i.e. the ATPase, reaction. This is why the study of membranous ATPases from mitochondria, chloroplasts and bacteria is directly related to the problem of the mechanism of respiratory and photosynthetic phosphorylation. Mitchell [1,2] put forward the chemiosmotic principle of membrane-linked phosphorylations, according to which the energy released by the respiratory (or photosynthetic) redox chain is utilized to generate a gradient of electrochemical potential of H +, directed across the coupling membrane. Then AfiH+ is used by a special ATP synthetase and results in the formation of ATP:

Respiration or ~Light

"-+ Afin+ +-

--~ ATP

(1)

According to Mitchell, the interconversion of A fin+ and ATP energy is reversible, i.e. the process of the ATP synthesis consumes ~lfiH+ whereas the process of the ATP hydrolysis generates AfiH+. This latter process is essentially no more than the ATPase reaction coupled with the transport of hydrogen ions against the electrochemical gradient. Consequently, the enzyme, which catalyzes such a process, may be defined as H+-ATPase (by analogy to Ca2+-ATPase or Na +, K+-ATPase) [3]. liB. A TPase as a necessary component o f the membranous phosphorylation system There is much evidence suggesting that one of the enzymes, participating in

32 membrane-linked phosphorylation, demonstrates ATPase activity in certain conditions. In this connection, the following well known facts may be noted : (I) In all the types of coupling membranes (mitochondria from various sources, chloroplasts, respiring and photosynthesizing bacteria) there is an enzyme (Factor F~) which hydrolyses ATP to ADP and P~ when it is detached from the membrane. (2) Coupling membranes, devoid of factor FI, lose the ability to synthesize ATP during respiratory or photosynthetic electron transfer. Tbe ATP-supported reverse electron transfer and osmotic work are also lost. All the lost properties may be reconstituted when the membrane is saturated with F~. (3) The antibodies to factor F~ inhibit oxidative and photosynthetic phosphorylation. (4) De-energization of the coupling membrane is accompanied by the appearance of ATPase activity which is arrested by DCCD, and also in the case of mitochondria and some bacteria, by oligomycin. The same quantities of DCCD and oligomycin inactivate the ATP synthesis supported by the energy of respiration or light (for reviews, see refs. [4-10]).

llC. Evidence of ,3fin+ generation by the coupling membrane ATPases Although the need for ATPase in energy coupling was realized several years ago, Mitchell's postulate on the generation of ,/fill+ by the ATPase complex of mitochondria, chloroplasts and bacteria was only proved comparatively recently. The fact that the ATP energy can be converted into drill+ became evident after the discovery of the very existence of I~H+ on the energized coupling membranes [11-13]. In this connection we should like to quote the following observations: (1) The hydrolysis of ATP in any closed vesicles formed by a coupling membrane, supports transmembrane fluxes of natural or synthetic penetrating ions, cations and anions which are transported in opposite directions. This effec! can be demonstrated both on the native and on the reconstructed membranes containing a DCCD-sensitive ATPase. This very fact points to the electrogenic character of ATPase action [12,14-16]. (2) The mitochondrial ATPase complex incorporated into a planar artificial phospholipid membrane from one of its sides generates a transmembrane electric potential difference across this membrane. This membrane potential can be directly measured with a voltmeter connected to two electrodes, which are immersed in electrolyte solutions of identical composition, separated by lhe artificial membrane [17]. (3) The transport of the cations into the mitochondria, supported by the ATP hydrolysis, is accompanied by acidification of the incubation mixture, whereas the transport of anions into the inside-out submitochondrial particles results in alkalization of the incubation medium. In both cases about 2 H + are transferred per each ATP hydrolyzed [18-22]. The above results confirm Mitchell's hypothesis (1966) about mitochondrial

33 ATPase as an electrogenic proton pump. Additional support of this conclusion can be obtained by analysing the action of various ions and ionophores on the ATPase activity of the coupling membrane. In closed membrane systems ATP hydrolysis can be activated by a protonophorous uncoupler. ATPase activated in this manner does not require any metal ions other than Mg z+ (in some coupling membranes of non-mitochondrial origin, Ca z+ rather than Mg z+ is needed). The role of an ion of a divalent metal in such a system is limited by the formation of the complex with ATP (for review, see ref. 23). No indications have been obtained of transmembrane Mg 2+ flux supported by ATPase in coupling membranes. On the contrary, it has been firmly established that intact mitochondria, energized by ATP hydrolysis or respiration, do not accumulate Mg 2+ ions [24]. The uncoupler-activated ATPase does not display a need for any anion. Some stimulation of ATPase by anions described in a number of laboratories, may be explained by the direct effect of these anions on the active site of factor FI (see p. 47, below). In any case, there are no grounds for attributing the coupling membrane ATPase to the class of ATPases which transport cations of metals and anions. Thus, the proton gradient formation seems to be the direct result of the action of this ATPase rather than a consequence of any cation/H + antiport or anion L H + symport. There are many indications that the ability to generate A p , ~ is inherent in the ATPase complex as such and does not require the participation of the respiratory generators of membrane potential. This is demonstrated in particular by the results of experiments on proteoliposomes reconstructed from the oligomycin-sensitive ATPase complex and phospholipids [16,25-27], as well as by the data on promitochondria in anaerobic yeasts, which are deficient in enzymes of the respiratory chain [28]. As regards the effects of respiratory poisons and substrates, as well as artificial oxidizing and reducing agents, on the ATP energy conversion, they may be accounted for by the action of such factors as (1) the change in the affinity of the protein inhibitor to factor Ft, depending on the magnitude of ,4ft,+ (see p. 68); (2) a change in the proton conductivity of the coupling membrane during conformational changes in the redox enzymes; (3) disturbance of the organization of the H+-ATPase complex in response to a change in the native confol mation of the neighbouring proteins [28a].

liD. Co;lstituents of the H+-ATPase complex The oligomycin-sensitive H+-ATPase complex is the smallest structural unit capable of carrying out the catalysis of all the processes accompanying ATP energy transformation in the system of oxidative phosphorylation. Related to these processes are the following: (a) the A~TH+-supported synthesis of ATP from ADP aild Pi in the closed membrane vesicles; (b) ATP hydrolysis coupled with the transmembrane H + transfer; (c) the catalysis of the 3zp~_ATP exchange, The characteristic feature of the reactions (a), (b) and (c) is sensitivity to the specific inhibitors of oxidative phosphorylation, oligomycin [26,29-33] and DCCD [34-36]. According to the data of various authors [37 42], purified oligomycin-sensitive ATPase is composed of phospholipids (from 4 ~ to 15 ~/o by weight) and 5-6 individual

34 proteins with molecular weights ranging from 1 I 000 to 380 000. Among them factor F1 represents the largest component of this complex, which can exist in water-soluble form, demonstrating the ATPase activity. The second water-soluble component is the so-called "oligomycin sensitivity-conferring protein" (OSCP) [43]. This is an alkaline protein with a molecular weight of 18 000. Besides factor Ft and OSCP, oligomycin-sensitive ATPase consists of three or ['our very hydrophobic waterinsoluble proteins (proteolipids) which are needed, together with OSCP, for ATPase to be sensitive to oligomycin and DCCD [8,44,45]. One of these hydrophobic proteins was identified as the target of the DCCD attack on the ATPase complex. Some preparations of oligomycin-sensitive ATPase also contain the protein inhibitor of mitochondrial ATPase [46] (see p. 67). The characteristics of the protein components of the ATPase complex are described in greater detail in the review [8]. The ability of factor F1 to hydrolyze ATP and the colrelation between the effectiveness of the 32p-ATP exchange and the amount of FI in submitochondrial particles [4] indicate that this enzyme participates directly in the catalysis of the ATPase and ATP-synthetase reaction. The remaining protein components of the ATPase complex apparently play a role in the proton transfer fi'om the outer side of the mitochondrial membrane to factor F~. This conclusion is mainly based on the fact that removal of factor F~ from the submitochondrial particles leads to the appearance of high proton conductance through their membrane. Oligomycin and DCCD in the concentrations in which these compounds inhibit oxidative phosphorylation, suppress proton conductance in submitochondrial [14,47-50] and subbacterial particles [51,52] devoid of factor F~. The latter observation suggests the inhibiting effect of oligomycin and DCCD to be due to the inactivation of an H+-conducting system included between the external surface of the mitochondrial membrane and factor Fj. Racker [53] was the first to obtain an indication that the inclusion of the proteolipid component of H+-ATPase and OSCP in the membrane of liposomes induces the penetrability of this membrane to H + ions. He reconstituted proteoliposomes from proteolipids, OSCP and phospholipids in the presence of phenol red, and the dye outside the proteoliposomes was washed out. Then HCI was added to the proteoliposome suspension, and the time needed for the colour of the intraproteoliposomal dye to change was estimated, assuming that the less time is taken, the greater the proton conductance of the liposome membrane ought to be. It was found that proteoliposomes responded to the addition of acid by changing the colour of the indicator much more rapidly than liposomes without proteins. The effect of the proteins revealed itself only in the presence of valinomycin. It was partially inhibited by ruthamycin. Further study along this line has been undertaken by Shchipakin et a1.[54]. Liposomes prepared in the presence of K ~ were incubated in a medium without K +. The addition of a protonophorous uncoupler (2,4-dinitrophenol) and valinomycin was shown to result in K + ions being released from the liposomes in exchange for H +. When added separately, dinitrophenol and valinomycin were not effective, since

35 DNP

A

B

VALINO

r ' ~

.o_~ T

VALINO

~c

VALINO~ f

bNP

C

3I 05ecVALN IO

Fig. 1. Effect of H+-ATPase proteolipids ( - OSCP) on the ion conductance of the liposomal membrane (from Shchipakin et al. [54]. A, B: liposomes From soy-bean phospholipids; C: proteoliposomes reconstituted from the mixture of mitochondrial H+-ATPase proteolipids ( + OSCP) and soy-bean phospholipids.

the former did not create K + conductivity, and the latter heightened only the conductivity fo~ K +, not that for any other ion whose movement through the membrane could cause the collapse of the diffusion potential of the K + ions. The proteolipids -OSCP which were included in the liposome membrane during the reconstitution of the vesicles were found to replace dinitrophenol. The addition of valinomycin without dinitrophenol to such proteoliposomes caused the exchange of K + for H + (Fig. 1). The DCCD treatment of the proteolipids + OSCP before the proteoliposome reconstruction caused a sharp decrease in the H + conductivity created by these proteins. Factor F,, when added to the proteoliposomes in small quantities, also induced a certain decrease in H + conductivity. At high concentrations, factor F~ by itself created H ÷ conductivity which was not sensitive to DCCD. The data cited leave no room for doubt that proteolipids ( + OSCP) participate in the formation of some kind of H+-conducting pathway in the phospholipid bilayer. This pathway cannot be a simple chink in the hydrophobic part of the membrane since it demonstrates ion specificity and is only penetrable by H +.

III. SOLUBLE M1TOCHONDRIAL ATPase (FACTOR F,)

IliA. Factor F, as an object o f the oxidative phosphorylation studies As has been noted previously, water-soluble mitochondrial ATPase is an enzyme with a molecular weight of 380 000 of a complex quaternary structure [9,10, 55-57]. It has a comparatively high content of hydrophobic amino acids [55,58].

36 Despite its high molecular weight, complex structure and its tendency to selfaggregation, factor Ft lends itself to study much more easily than insoluble oligomycin-sensitive ATPase. However, the possibilities for studying the mechanism of oxidative phosphorylation on soluble ATPase are limited by the fact that this enzyme, when it is detached from the mitochondrial memblane, loses some very important features. Thus, the rate constant of the ATP-synthetase reaction catalyzed by the solubilized factor F~ is so small that it cannot be measured even in conditions providing for a favourable change in the reaction equilibrium (e.g. in the presence of hexokinase-glucose-6-phosphate-dehydrogenase system). Factor F~ does not catalyze the reaction of the azP~-ATP exchange. Among the properties of solubilized factor F1, apparently related to the mechanism of oxidative phosphorylation, one can note that the enzyme bydrolyzes ATP at a high rate (k~t 104 rain -~ at 25°C [59]) and is competent in very specific binding of ADP. For a long time, factor F~ was assumed to be unable to perform any type of ATP-supported work in the absence of proteolipids and OSCP incorporated into a closed membrane vesicle. However, experimental data was recently obtained, showing that this idea might be incorrect. It appeared that factor F,, situated on the interface of two immiscible liquids, octane and water, positively charged the octane phase in response to the addition of ATP. The presence of dinitrophenol was necessary to demonstrate this effect [59a] (for details, see p. 50).

IIIB. The quaternary structure of Factor F~ Preparations of soluble mitochondrial ATPase, isolated by the method of Horstman and Racker [60] or Knowles and Penefsky [55] contain five types of subunits. According to the data of Knowles and Penefsky, the molecular weights of these subunits are 54 000 (a), 51 000 (fl), 33 000 (Y), 16 000 (6) and 11 000 (s) [55]. A similar subunit composition was shown in ATPase from the membrane of chloroplasts (molecular weights ofsubunits are 59000, 56000, 37 000, 17 500 and 13000) [61], ATPase from Escherichia coli [62] (57 000, 51 000, 30 000, 20 000 and 11 000). it was proposed that the subunit composition of these ATPase molecules corresponds to the formula (-~3/5~3)/(}/?[55] or 6{2~2~22()8 [55a]. Formula (z3fia)~t~e, however, seems to us to be preferable. It is in good agreement, in particular, with the molecular weights of the subunits and of factor F~ in toto [55]. ATPase from Micrococcus lysodeikticus [62a] was found to contain four types of subunit (52 500, 47 000, 41 000 and 28 000). When comparing the amino acid composition of the F~ subunits, we noted that these parameters of (,, fl and (y + ¢}) subunits are similar, while the y and () subunits differ from one another in their composition as well as from a and fl subunits. This observation prompted us to the idea that y and 6 subunits are the products of the cleavage of a single subunit with a molecular weight of 50 000 due to a single break in the polypeptide chain [57]. In this case the F~ molecule can be presented as a complex of seven large subunits of three types similar in size and composition, and

37 one small (e) subunit. In this connection it is interesting to note that Munoz et al. [64] observed seven protein globules of similar size in micrographs of M. lysodeikticus ATPase. Nieto et al. also showed that there is "a weak link" in the polypeptide chain of the ~t-subunit of M. lysodeikticus ATPase, which is hydrolyzed in fairly mild conditions [64a]. Using the method of Horstman and Racker for F~ isolation [60] modified at the stage of ultrasonic treatment of the mitochondrial suspension, we obtained factor FI which did not contain 7 and ~ subunits [57]. It was found that this FI preparation did not differ in its catalytic properties from the enzyme containing the complete set of subunits. The results obtained prove that Ct and/3 subunits play the main role in the ATPase activity of the enzyme. Such a viewpoint can be supported by the results of the study of some bacterial ATPases. Mirsky and Barlow [65] obtained ATPase from Bacillus megaterium, containing two types of subunits with molecular weights of approximately 65 000. Active preparations of ATPase without minor subunits were obtained by Salton and Schor [66] from M. lysodeikticus and also by Bragg et al. [67] and Smith and Sternweis [68] fi'om E. coli. The latter authors note that the 6-subunit is necessary for the correct association of factor F~ with the membrane. Some indications of the structural role of minor subunits were obtained in this group [69]. There is convincing evidence that the smallest subunit of facto~ F~ acts as a natural inhibitor of ATPase activity (see below, p. 67). Taking this fact into account and assuming that the seventh large (~, + 6) subunit takes part in the attachment of factor F~ to the membrane, we may conclude that the catalytic site of factor F~ should either be located on the ct or/3 subunit, or is formed as a result of the interaction of these two types of subunits. McEvoy and Lynn [70] obtained some data that may be regarded as an indication that the presence of a subunits in factor F~ from chloroplasts is not absolutely necessary for ATPase activity. The authors treated factor F~ with trypsin which activated FI if the duration of the treatment was not too long (this activation was, in all probability, due to the cleavage of the protein inhibitor). The maximum rate of 1he ATPase reaction was reached after a 90-s incubation with trypsin. By that time the content of all the subunits was sharply decreased except for t3, which was the most resistant to the tryptic digestion. This result agrees with the suggestion that the catalytic site is situated on the /3 subunit. However, it cannot be ruled out that a small amount of the complex of/3 and ~ subunits, which may still be present in the trypsin-treated system, is responsible for the ATPase activity measured. The suggestion that/3 subunits participate in the formation of the catalytic site of factor F~ agrees with the data of Nelson and others [71]. The latter showed that 7-chloro-4-nitrobenzo-2-oxa-l,3-diazol, the inhibitor of soluble H+-ATPases, mainly modifies the tyrosine residues localised on the /3 subunits of ATPase from E. coli. Ferguson et al. recently obtained similar results for mitochondrial ATPase [72,73]. The problem of the localization of the catalytic site on the mitochondrial F~ subunits has been studied in this group by V. G. Budker. It was found that an alkylating substrate analog, p-N-(2-chloroethylmethyl)-aminobenzylamide 7-P

38 dmivative of ATP inhibits the F~ ATPase activity in the solution. Measurement of the 3Zp-labelled inhibitor binding with Fj revealed 1-2 molecules of the inhibitor incorporated per F~ molecule. As SDS electrophoresis showed, this is the fl subunit(s) of F~, that combine(s) with the inhibitor. ATP protected factor Ft from the inhibiting effect of the alkylating analog of ATP. There is much data on mitochondrial factor FI testifying that decomposition into subunits causes the enzyme to lose its catalytic activity (for review, see ref. 9). To complete the consideration of the subunit composition of factor F~, the problem of the Streptococcus faecalis ATPase should be discussed. According to Schnebli et al., this ATPase differs strikingly from mitocbondrial ATPase in its subunit composition. Treatment of S. faecalis ATPase with urea led to the appearance of two types of subunit with molecular weights of approximately 33 000. It was shown that one molecule of the enzyme consists of 12 (6 t- 6) subunits of this type [74]. However, in the recent work of Abrams et al. who used SDS instead of urea as a dissociating agent, it was established that ATPase of S.faecalis consists of 6 (3 + 3) large and three minor subunits [74a]. The molecular weight of the large subunits was about 60 000, i.e. of the same order as the molecular weight of the corresponding subunits of factors FI from other sources. Treatment of S.faecalis ATPase with urea apparently leads to a single break in the polypeptide chains forming six large protein globules of the enzyme. During treatment of ATPase of S. faecalis with SDS this type of break was not observed. Thus, the results obtained not only indicate the similarity of th~ subunit composition of S.faecalis ATPase and factor F~ from other sources, bat also confirm the above suggestion that "weak bond(s)" exist in the polypeptide chains of factor F~ subaqits. In all probability the above-considered S. faecalis ATPase plays the role of F~ in the H+-ATPase complex of this bacterium. According to Harold et al. [75,76], there is a DCCD-sensitive system for pumping H + ions from S..faec,qlis cytcplasm to the external medium. In a strain of this microorganism, growing anaerobically and containing no respiratory chain, the system in question utilizes ATP, formed by glycolysis, to generate lfi,+.

IlIC. The catalytic properties of factor F1 IIIC-I. Mg" ATP as the substrate of soluble ATPase. Despite the tremendous interest in the mechanism of the action of soluble mitochondrial ATPase, there are still very few reliable data on the structure of the enzyme active site, as well as on the number and sequence of the stages in the ATPase reaction. The first question on which various authors could not agree was the role of Mg 2+ (or rather of the divalent ions of metals) in the ATPase reaction, in studying the dependence of the ATPase reaction rate on the free concentrations of Mg 2+ and of ATP, Selwyn [77] concluded that Mg • ATP is the substrate of mitochondrial ATPase. This opinion is shared by Akimenko et al. [78]. On the other hand, Hilborn and Hammes [79] believe that ATP and Mg • ATP are bound in the active site with approximately the same binding constants. According to Adolfsen and Modrianakis [80], Mg 2+ bears a greater affinity to factor F~ than to ATP at low concentrations of ATP. An analysis of the

39 enzyme-metal-substra~e system carried out in our laboratory, using London-Stock's method, confirmed the point of view that Mg" ATP is the substrate of the ATPase reaction (Kin = 0.4 raM), and free Mg 2+ and ATP act as competitive inhibitors (K~g2+ = 1 mM and K A y , = 8 raM) [82]. It was also shown that M g ' A D P is a more effective inhibitor of the ATPase reaction than ADP, and inhibition is of a competitive type ((KM,_ ADP) = 0.5 raM) [82]. IIIC-2. The nueleotide specificity of factor F1. There are several observations indicating that the ATPase reaction rate, mediated by FI in solution, decreases in the following order: ATP = ITP ~ G T P ~ UTP ~ CTP (for review, see ref. 10). According to our data [82a] this range is determined by the properties of the stage of hydrolysis of nucleoside triphosphates rather than by their binding with F~. In fact, the values of Km (apparent) for ATP and G T P were found to be similar (around 0.4 raM). At the same time, the maximal values of the ATP hydolysis rate (V) are 2-3 times greater than those of GTP. Km (app) for ATP differs 5-fold from K~ for U T P as an inhibitor of the ATPase reaction. On the other hand, the ATP hydrolysis rate is 15 times greater than that of UTP [82a]. Variations in the structure of the carbohydrate part of the nucleotide, like that of the base, affect V more than Km of the solubilized FI. So, the values of Km (apparent) for d-ATP, ATP and 3'-OCH3-ATP (1.4 raM, 0.4 mM and 0.5 raM, correspondingly) differ less than V values, which are in the ratio 20:10:3 [82a]. As was shown in this group, ATP analogs with hydrocarbon chains instead of ribose could not be hydrolyzed by Ft and strongly inhibited the ATP hydrolysis if the length of the chain was equal to 3 or 4 methylene groups (Ki about 0.2 mM). At the same time, the ATP analog with two methylene groups (A-(CHz)z-PPP) could be used as the Fj substrate. The rate of hydrolysis of this compound was only 8-fold lower than that of ATP. Such a relationship seems to be surprising since A-(CH2)3PPP and A-(CHzL-PPP are closer to ATP than A-(CHz)E-PPP if one takes into account the number of carbon atoms betweep adenine and triphosphate. This paradox may be explained by the assumption that the nucleotide must be bound by F~ in a tense conformation in order to be hydrolyzed. In ATP this tension may be due to the fact that, on the one hand, the distance between the F~ functional groups binding adenine and those binding triphosphate is fixed, and, on the other hand, the conformation flexibility of the ribose residue is rather small. It is not the case for A-(CHz)3-PPP and A-(CHz)4-PPP possessing flexible, sufficiently long hydrocarbon chains instead of a ribose ring. In A-(CH2)z-PPP, the distance between the adenine and triphosphate moieties is apparently Ioo short to allow for the flexible conformation of the bound nucleotide. As the experiments showed, the affinity of A-(CHz)3-PPP and A-(CHa)4-PPP to F t is 2-3 times greater than that of ATP. This fact is in agreement with the above concept since compounds of flexible conformation should combine with FI more easily than those of tense conformation. Moreover, the ATP derivative with the open ribose ring, 2,2'-l-(9-adenyl)-l'(triphosphoryl-oxymethyl)-dihydroxydiethylether was demonstrated to be hydro-

40 lyzed by F~ 6 times more slowly and to be bound to F~ with a 6-fold higher affinity, than the native ATP [82a]. Interesting data were obtained in experiments with CTP and its ribose ring open derivative, namely 2,2'-l-(3-cytosyl)-l'-(triphosphoryl-ox)methyl)-dihydroxydiethylether. CTP could not be hydrolyzed by F~ and in a concentration of 10 mM did not affect the rate of ATP hydrolysis. So, CTP does not form a complex with the F~ active site. On the other hand, CTP with the open ribose ring proved a powerful inhibitor of the F~-catalyzed ATPase reaction (K~ = 0.1 raM). It may be concluded that native CTP does not interact with the F~ active site due to the unfavourable conformation of this nucleotide rather than the specificity of F, to the type of heterocyclic base [82a]. There is some information on the requirements to the polyphosphate moiety of nucleotides bound in the active site of F~. It is known that A D P and adenosine tetraphosphate inhibit the ATPase activity of F~ in solution (K~ : 0.4 mM and 0.15 mM, respectively) [82a]. As was mentioned above, the alkylating p-N-(2-chloroethylmethyl)-aminobenzylamide derivative of ATP acts as an F~ inhibitor. Mixed anhydrides of nucleoside triphosphates and mesitylene carboxylate synthesized in the laboratory of Shabarova [82b] were also shown to be effective inhibitors of factor F~ in solution. At the same time, the anhydride of A D P and mesitylene car~oxylate, which bears only two negative charges, cannot be bound in the F, catalytic site (see below, p. 51). A M P is also ineffective as an F, inhibitor. Thus, the impression is created that the presence of at least three negative charges in the molecule of the nucleotide is necessary for it to bind in the F~ active site. 111C-3. The functional groups of the catalytic site. Senior [83] and Ferguson et al. [84,85,85a] have shown that modification of the tyrosine residue in the mckcule of factor F~ by 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole leads to complete inactivation of the enzyme. The results obtained suggest that tyrosine participates in the formation of the Fj active site. On the other hand, Ferguson et al. [85a] suggested that tyrosine residues play a certain role at the stage of the F~ conformation change during the ATPase reaction. The data on the pH-dependence of Km of ATPase for Mg ' ATP, and also K~ for M g ADP, obtained in this group, are indicative of the existence of a carboxyl group in the active site, which takes part in the binding of nucleotide-magnesium complexes [59]. The results of other authors also lend support to this suggestion. Thus, fi'om the data of Pedersen et al. [86], and Adolfsen and Mondrianakis [80] it follows that the affinity of the metal ' ATP complex to the enzyme is dependent on the size of the ion radius of a divalent metal rather than on the polarization of electronic shells of the metal. So, the complexes Mg" ATP, Mn • ATP, Fe" ATP and Co " ATP are hydrolysed by factor F~ at a high rate. On the other hand, in the presence of Ca 2+, the ionic radius of which is bigger than that of Mg 2+, Mn 2+, Fe 2+ or Co 2+, mitochondrial ATPase hydrolyses ATP at a very low rate [10]. These relationships may be expected if the carboxyl group is assumed to be a ligand of Me 2+ in the active site of ATPase. The value of the binding constant of factor F~ for Mg 2+ (1 raM) is also in agreement

41 with the idea that the carboxyl group is to be found in the active site [82]. Finally, it was recently found in this group that a modification of factor Fl with a watersoluble reagent to the carboxyl groups of proteins, N-cyclohexyl-N'-fl-(4-methylmorpholine)-ethylcarbodiimide (CMCD), inhibits completely the F1 ATPase activity. The inactivation rate decreased in the presence of Mg • A D P [87,88]. Treatment of factor F1 with a preparation of 3H-labelled C M C D in conditions of complete inhibition of ATPase activity leads to the modification of one amino acid residue per molecule of the enzyme. It has also been established that factor F~ treated with C M C D reacts with an aromatic amine (proflavin), forming a fluorescent derivative of the enzyme. Covalent binding of the amine to the factor F1 treated with C M C D is conclusive evidence of the fact that C M C D reacts with a carboxyl group controlling the ATPase activity of the enzyme. Studying the pH-dependence of the pseudomonomolecular constant of the reaction rate of factor FI with C M C D made it possible to estimate the pK of the CMCD-binding group in the catalytic site of ATPase, which proved to be between pH values of 6.8 and 7.0. This result is well in conformity with the data on the pH-dependence Km (app) of the F~ ATPase activity [59]. Similar app;oaches were used by us to study factor F~ from M. lysodeikticus. The pH-dependence of the rate of inactivation of this enzyme by C M C D is in agreement with the suggestion that a carboxyl group with pK ~:: 5.8 is present in the active site of ATPase [89]. IIIC-4. On the covalent intermediate of the H+-ATPase reaction. The idea that the ATP hydrolysis by mitochondrial ATPase includes a number of covalent intermediates, was put forwald long before Agn+ was identified as a component coupling oxidation and phosphorylation. In 1946 Lipmann [90] postulated a phosphorylated electron carrier as an intermediate of oxidative phosphorylation. In 1953 Slater [91] modified Lipmann's scheme, having assumed the existence of non-phosphorylated high-energy intermediate(s) on the way from the respiratory chain to ATP. It was established, in agreement with Slater's idea, that the energy, liberated by the electron transfer systems of mitochondria, chloroplasts and bacteria, is accumulated in an ATP precursor whose formation does not require the addition of inorganic phosphate. The energy stored in such a form can be used not only for the ATP synthesis, but also for the reverse electron transfer in any redox chain coupling sites or for the ion transport against concentration gradients. It was believed originally that the component in question was a high-energy chemical compound (X ~ Y), e.g. a thioester or an acylimidazole derivative of a redox chain carrier and a protein coupling factor. It is also assumed that the formation of the non-phosphorylated intermediate X ~ Y by mitochondrial ATPase is sensitive to oligcmycin, while that by the respiratory chain is not (Eqn. 1). Following the discovery of the generation of Afin+ on the coupling membranes, there was no longer any need for X ~ Y as suggested. Today there is no reason why Slater's intermediate should not be identified with AfiH+ (Eqn. 2).

42 oligomycin

Redox chain ~-~ X ~ Y

$

-~

~- ATP

(1)

~ ATP

(2)

inhibits

Ion transport oligomycin

Redox chain+~/lfiH+ inhibits

Ion transport The only stage in oxidative phosphorylation where there could be chemical high-energy intermediates is the H*-ATPase reaction, i.e. the stage 1fill+ ATP (Eqn. 3). Redox ehain+-~ .dfiH+e-~ X ~ Y ~ Y ~ P+-~ ATP (3)

$ ion transport It is a well known fact, for example, that cleavage of ATP by (Na +, K+)-ATPase [92], Ca2+-ATPase [93] or myosin [94] occurs through the formation and hydrolysis of high-energy acylphosphate, which forms as a result of the phosphoryl transfer from ATP to the free carboxyl group of one of the dicarboxylic amino acid residues in the polypeptide chain of the enzyme. However, attempts to reveal the phosphorylated derivative of H+-ATPase were inevitably unsuccessful. One of the last studies of this subject was carried out in Packer's group [95]. In several laboratories it was shown that the ATPase activity of factor F~ is not suppressed by such a compound as hydroxylamine, a reagent for acylphosphate, which inactivates (Na +, K+)-ATPase, Ca2+-ATPase and myosin ATPase (for a possible reason for the differences between H+-ATPase and the other ATPase systems mentioned above see p. 75). The expeliments carried out in this labolatory showed that factor F~ does not catalyze either the transfer of the phosphoryl residue from ATP to aromatic oximes or the incorporation of the oximes into the protein. The aromatic oximes are highly reactive nucleophilic compounds, which react with the high-energy intermediates of a number of enzyme reactions [97,98]. So, their ineffectiveness in the case of factor F~ testifies against the existence of X ~. Y and Y ~ P as a component of the F~-mediated ATPase reaction. Thus, today there are no serious grounds for complicating the scheme of H +ATPase action with high-energy intermediates. Therefore all our further considerations are made from the assumptions that (1) ATP is the only high-energy chemical product in the system of membranous phosphorylation, and (2) water interacts directly with ATP during its hydrolysis in the active site of factor F~. IHC-5. Korman's hypothesis on the mechanism of the A TPase (ATPase-synthetase) reaction. It is well known that the hydrolysis of esters and anhydrides of phosphoric acid includes the protonation of phosphoryl oxygen. Unfortunately, at present nothing definite can be said about the natme of the amino acid functional group which causes this protonation in the active site of mitochondrial ATPase. Despite this uncertainty, we would like to assume that the processes occurring in Ihe

43

H',O~ H

II " DNP, a l l ' O~ ns, auroverlm,

1

AMPPNP~alKylating agent

Fig. 2. Stereochemistry of the ATP hydrolysis and synthesis in the catalytic site of mitochondrial ATPase [23]. Two regions responsible for binding of the diphosphoadenosine residue (-O-ADP) are shown as shaded rectangles.

active site of factor F~ should in their main features comply wilh the general principles established for the non-enzymatic hydrolysis of esters and anhydrides of phosphoric acid. The usual mechanism of the nucleophilic displacement reaction at a pentavalent atom of phosphorus includes the formation of the pentacovalent intermediate which has the form of a triangular bipyramid [99,100]. In such a structure, three covalent bonds of phosphorus are situated on one plane (equatorial substitutes) and the two remaining ones are directed towards the apices of the bipyramid (axial substitutes). It has been postulated that the conversion of the triangular bipyrarnid into products of the reaction (or its original substtates) occurs when one of the two axial substitutes is removed. On the other hand, in the axial position of the triangular bipyramid, only those substitutes are to bc found whose bond wilh the phosphorus atom is easily ruptured as a result of chemical reactions. If four of the five substitutes at the phosphorus atom in a pentacovalent intermediate are capable of occupying axial positions, then so-called pseudorotation occurs. The essence of this phenomenon is that two substitutes in the equatorial position change places with two substitutes in the axial position. The probability of pseudorotation depends on the stability of the new triangular bipyramid formed as a result of pseudorotation. As a rule, the stability of the substitute in the axial position correlates to its electron-acceptor properties. These fundamental ideas on the stereochemistry of the nucleophilic substitution reaction at the phosphorus atom were applied by Korman et al. [101-104] to the mechanism of ATP hydrolysis by mitochondrial ATPase. According to Korman, the ATPase reaction starts with an attack on the protonated 7-phosphate residue of ATP by the water molecule, which results in the formation of the triangular bipyramid (Fig. 2). There aie three ways of further converting the pentacovalent intermediate. Firstly, water may be removed from the axial position. In Ihis case,

44 the original components (ATP and H20) are formed, and the system thereby reverts to its initial state The second possibility is the removal of the disphospho-adenosine residue (O-A DP) from the axial position of the triangular bipyramid and the formation of the products of the ATPase reaction (ADP and P~). Lastly, the pentacovalent intermediate may undergo pseudorotation, thus giving a new triangular bipyramid with two new molecules of water in the axial positions. The removal of H20 from the new triangular bipyramid (Fig. 2, intermediate 2) is accompanied by the formation of free ATP, and the result of the process as a whole will be the A T P - H 2 0 exchange reaclion. Acccrding to Korman [101-104], whose opinion we shaJe, the pseudorotation rate in the active site of mitochondrial ATPase is far g~eater than the rate at which water disappears from the first triangular bipyramid (Fig. 2). In other words, the most probable way of converting the pentacovalent intermediate 1, formed as a result of (1) the attack on 7,-phosphate of ATP by a molecule of water or (2) P~ being attacked by ADP, is the removal of O-ADP from the axial position, or pseudorotation. In this case, the mechanism of pseudorotation in the active site of factor F~ can be used to reduce the activation energy of the ATP-synthetase reaction. 111C-6. Two regions for the substrate binding in the F~ catalytic site. Obviously the pseudorotation in the active site of the enzyme should be accompanied by a change in the spatial location of the substitutes at the phosphorus atom. Since the enzyme reaction supposes the good steric conformity of the amino acid functional groups taking part in the catalysis of the process, and the functional groups of the substrates, Young and Korman suggested [104] that the change in the spatial lecation of the substitutes in the pentacovalent intermediate resulting from pseudolotation is accompanied by conformational changes in the active site of ATPase. According to Korman, these conformational changes ~an play an important role in the energy coupling mechanism. Sharing Korman's point of view, in the main, on lhe role of pseudorotation in the ATP synthesis in the active site of mitochondrial ATPase, we believe that his conclusion on the large conformational changes in the enzyme, which accompany pseudorotation, is unnecessary. We suggest that the active site of mitochondrial ATPase is arranged in such a way that it is capable of binding both the pentacovalent intermediates of the ATPase reaction without any substantial change occurring in the protein conformation [23]. Moreover, we postulate that there are two regions in the active site of factor F~, which are responsible for binding the diphosphoadenosine (-O-ADP) residue, and pseudorotation results in -O-ADP being translocated from one region to the other (Fig. 2). A focal point in the concept proposed is the assertion that the two -O-ADPbinding areas in the active site of ATPase differ both in their affinity to -O-ADP and in their specificity to the heterocyclic base of the nucleotide: the region responsible for the binding o f - O - A D P in the equatorial position (Fig. 2, region 11) is firmer and more specific. The hypothesis on two nucleotide-binding regions in the catalytic site of F1 makes it possible to explain the substrate specificity of factor F~. In particular, it was

45 found that factor F1 binds ATP, UTP and G T P wilh similar affinities [82a]. However, of all the natural nucleoside diphosphates only ADP significantly inhibits the ATPase reaction [4]. As noted above, region lI of the F1 active site (see Fig. 2) possesses a high affinity to -O-ADP and a slrong specificity to the type of heteroc~¢clic base. Just these two properties of region lI determine the effectiveness of ADP and ATP binding in the active site of factor F1. The ADP binding at region II results in the enzyme-inhibitor complex, whereas binding of ATP at the same place gives rise to the formation of an unproductive enzyme-substrate complex. Indeed, the binding of the diphosphoadenosine residue of ATP in region 1I did at the next step induce the formation of the pentacovalent intermediate, which is not capable of ADP and P~ production (Fig. 2). According to our scheme, formation of the productive enzyme-substrate complex only occurs when ATP binds at region I, of which the specificity and affinity to the nucleotides is postulated to be lower than those of region II. Thus, the experimental values of Km(app) for nucleoside triphosphates are determined solely by the properlies of region I. A1 the same time the inhibiting ability of nucleoside diphosphates is determined by the properties of region II. This should, according to our scheme, inevitably result in a situation where non-adenine nucleoside triphosphates are hydrolyzed by factor F1, whereas non-adenine nucleoside diphosphates are not inhibitory for the hydrolytic reaction. If all these suppositions are correct, then it might be expected that lhe structural analogs of ATP which cannot be hydrolyzed will inhibit the ATPase reaction, and the inhibition constant will be better than Km(app) for ATP. And this is in fact the case. It was found that adenilylimidodiphosphate (AMPPNP), i.e. the ATP analog, in which there is nitrogen instead of oxygen between the fl- and 7-phosphates, strongly inhibits the ATP hydrolysis by factor F~, and K~ of this inhibition is approx;mately two orders of magnitude lower than Km(app) for ATP [86]. According Io Filo and Selwyn, the inhibiting effect of A M P P N P on the ATPase activity of factor FI is greater than on GTPase activity [105]. As the authors showed, 80 ~o inhibition of the GTPase activity was achieved at a concentration of A M P P N P equal to 3 • 10-7 M. On the other hand, 5 • 10.6 M A M P P N P was needed to attain an 80~(, decrease in the ATPase activity. Since soluble mitochondrial ATPase has similar Km(app) values for ATP and GTP, Filo and Selwyn suggested that either the mechanisms of ATPase and the GTPase reaction are different, or the enzyme preparation is a mixlure of two fractions of ATPase, which differ in their properties. in terms of our scheme, however, the results of Filo and Selwyn can be explained without any additional postulates. ATP, able to be bound at both regions I and II of the F1 active site, compeles more effectively with A M P N P for the enzyme than GTP which binds to region I only. The other group of facts which can be explained by om concept is the promoting action of certain aromatic compounds on the ATPase reaction rate measured in the presence of ADP. It was shown that 2,4-dinitrophenol lowers the factor F~ affinity to the competitive inhibitor of the ATPase reaction, Mg- ADP, without

46 impairing K,l,(app) for M g ' A T P [106] "L This result can be easily explained, assuming that 2,4-dinitrophenol combines preferentially with~region II. A similar explanation may be true for the inhibition of oxidative phosphorylation by aurovertin. The latter, if binding with region 1I of the active site of factor F~, may block the pseudorotation, thereby increasing the activation energy of the A T P synthetase reaction. Like 2,4-dinitrophenol, aurovertin lowers the affinity of the enzyme to A D P and does not increase the K,,(app) of ATPase to A T P [108-110]. As noted above, the role of pseudorotation and o f the two regions in the catalytic site of ATPase may consist in causing a decrease in the activation energy o f the catalyzed reactions. This may be o f particular importance for the process o f A T P synthesis. The increase in the A D P phosphorylation rate can be achieved due to the rapid removal o f water from lhe axial position of the triangular bipyramid 2 (Fig. 2). Such a mechanism for decreasing the activation energy can be sufficiently effective, if the pseudorotation rate is comparable 1o the rate of removal o f - O - A D P from the pentacovalent intermediate of reaction ! shown in Fig. 2. Assuming that auroverlin prevents pseudorotation in the active site of ATPase by means o f binding with region II, one can explain an intriguing paradox, namely that aurovertin inhibits oxidative phosphorylation while the rates of the ATPase reaction and the ATP-dependent reverse electron transport remain unchanged [5,29, 108,111,112]. Indeed, if the rate o f H zO removal from the axial position o f triangular bipyramid I is low (considerably less than the pseudorotation rate and the rale of H 2 0 removal from the axial position of bipyramid II, Fig. 2), then just this stage becomes the limiting one for the overall process of the A T P synthesis in the presence of aurovertin. On the other hand, the A T P hydrolysis rate can be limited by the stage of Mg • A T P translocation from water into the active site o f factor F~, as this can probably also take place without aurovertin (see below, p. 62). Since the reaction rate is measured in non-equilibrium conditions (if one of the slages in the process is very slow, as, for instance, is the case for the A T P formation in the presence of aurovertin, then the experiment is simply not long enough for equilibrium to be attained in the system), the lowering of the rate of the limiting stage in the phosphorylation reaction without the same change in the A T P a s e reaction can lead to selective inhibition o f the former processes. It is interesting to note that a high concentration of aurovertin not only inhibits phosphorylation, but also the ATPase reaclion [29]. Probably this c o m p o u n d is able to bind with region ! o f the aciive site of mitochondrial A T P a s e but with a lower affinity than with region !1. • Cantley and Hammes [107] are sceptical about the possibility of competition tzetween ADP and 2,4-dinitrophenol for the binding site on factor F,. The authors have shown that the effectiveness of binding I 40 /~M Mg ' ADP does not change in the presence of 0.3 mM 2,4dinitrophenol. It should, however, be noted that the concentration of ADP used by the authors is considerably lower than K~ for ADP (0.35 0.5 mM [82,82a]) determined in our group. Taking into account the fact that Cantley and Hammes achieved saturation of the enzyme with the nucleotide at an ADP concentration of 40 ItM, it would seem highly probable that the authors studied an ADP binding in a region other than the F~ catalytic siie.

47 The scheme suggested for the structure of the active site of factor F1 may account for the aclivation of mitochondrial ATPase by various anions, such as bicarbonate, borate, maleate, chromate, malonate etc. According to the data of Lambeth, Ebel and Lardy [113-115] the ATPase reaction rate of factor F1 increased two- to ninefold in the presence of the anions, while the activating effect of the anions is not observed in the presence of aurovertin.* In our opinion, the anions, like 2,4dinitrophenol or aurovertin, interact with binding region lI in the active site of factor F~. As a result, the formation of an unproductive complex of A T P wtih the enzyme becomes impossible, and this induces some increase in the ATPase reaction tale [116]. In agreement with this hypothesis, it was shown that the rate of G T P hydrolysis by factor F~ does not change in the presence of anions [114,l 15]. in terms of the above scheme, this should be due to the high specificity of region 1I to the type of heterocyclic base in nucleotides. For this reason, G T P only forms a productive enzyme-substrate complex in region I, and, therefore, binding of anions in region II cannot stimulate the GTPase reaction as was shown by Lardy and Ebel [114,115]. Further informalion regarding the two nucleotide-binding regions in the catalytic site of factor F~ was obtained when studying lhe inhibiting effect of bifunctional alkylating agent, p-N-di-(2-chloroethyl)aminophenylacetic acid [117], used earlier in this group by Yaguzhinsky [118] as an inhibitor of oxidative phosphorylation. Incubation of factor F1 with this compound in the presence of Mg 2+ ions results in the formation of an irreversibly modified enyzme, which does not differ from the eriginal enzyme in its affinity to Mg • ATP, but loses its ability to be inhibited by Mg • A D P [117]. Like dinitrophenol the non-alkylating analog of the inhibitor (phenylacetic acid) reversibly stimulates the ATPase reaction of factor F~ in the presence of Mg" ADP. It seems most probable that the alkylating agent occupies region II and then alkylates a nucleophilic amino acid residue in this area of the enzyme. To conclude this section, all the evidence cited testifies to the existence of two regions in the active site of milochondrial ATPase, which are responsible for the interaction with the diphosphoadenosine residue (-O-ADP). Adsorption of ATP at the active site either leads to the formation of a productive enzyme-subs~rate complex with the participation of region I, or to the formation of an unproductive complex withthe palticipation of region II. The A D P binding occurs in region I! and leads to the formation of an enzyme-inhibitor complex. The mechanism of ATPase inhibition by ADP can be explained by one extra charge of A D P in comparison to the -O-ADP residue of ATP. There is a polar region in the active site of the enzyme which is responsible for the binding of the fl-phosphate of A D P occupying region I1, and of the 7-phosphate of nucleoside triphosphates occupying region I. Apparently, the repulsion between the charges of the A D P fl-phosphate residue and of the * It is not to be excluded that the activating effect of anions on the ATPase activity of membrane-bound factor FI is determined by the action of anions on the conformation of the enzyme. Mitchell and Moyle [ll5a] have obtained some indication of such anion-induced conformational changes in membrane-bound factor FI.

48 nucleoside triphosphates' v-phosphate residue makes the simultaneous binding of A D P and ATP in the active site of factor F1 impossible. IllC-7. ADP binding in the non-catalytic site of factor F~. As noted above, factor Fa binds one molecule of M g ' A D P in the active site, and this causes the inhibition of the ATPase reaction [82,82a]. Hilborn and H~mmes [79], and independently Catterall and Pedersen [119] described another A DP-binding site in the enzyme. The A DP sorption at this site does not affect the ATP hydrolysis by factor F 1 in the solution [120]. In this respect, such an additional ADP-binding site clearly differs from regions I and II of the catalytic site, discussed in the preceding section. Probably the non-catalytic site is located at quite a distance from the catalytic one in the factor Fj molecule. As was shown by Hammes and Hilborn [79], ADP can be sorbed at the noncatalytic site, both in free form and in the form of its magnesium salt. Hammes and Hilborn [79] and later Tondre and Hammes [120] interpreted the specific binding of A D P at the non-catalytic site as a possible way of allosteric regulation of mitochondrial ATPase activity. However, these authors' hypethesis has still not been proved experimentally. According to Penefsky [121] and Pedersep et al. [86], one of the two independent binding sites of nucleotides takes part in the catalysis of the ATPase reaction, wheleas the other catalyzes the phosphorylation reaction. The idea that two independent active sites in the same enzyme are responsible for the catalysis of the forward and reverse reaction, is incompatible with the principle of the microreversibility of all the stages in the enzymatic process. The argument that Pedersen puts forward in support of his hypothesis is the close values of the A D P dissociation constant in the noncatalytic site and the Michaelis constant of oxidative phosphorylation for ADP. This fact, however, can be interpreted in another way. The sorption of A D P and Pj in the non-catalytic site of ATPase represents, in our opinion, the first stage of the ATP-synthetase reaction in mitochondria. Then the translocation of A D P and P~ from the non-catalytic site of ATPase to the catalytic occurs with the subsequent formation of ATP. This concept will be discussed in more detail in the next section.

IIID. The role of factor F~ in/Tn+Jbrmation by the H+-ATPase complex IIlD-1. The hypothesis on A TP translocation between the catalytic and the non-catalytic F~ sites. There are several hypotheses on the role of factor F1 in the generation of/IpH+. Chronologically Mitchell's hypothesis is the first of them [2]. When publishing his chemiosmotic conception in 1966, Mitchell attributed to factor F; the function of energy donor in the formation of a non-phosphorylated intermediale of the H+-ATPase reaction (X ~ Y, see p. 42, Eqn. 3). The X ~ Y hydrolysis by a component of the H+-ATPase complex should, according to Mitchell, produce 1/7~+, the latter process being regarded as the object of oligomycin action [51]. However, experiments on the transhydrogenase-linked zl~o formation, carried out in this group by Grinius and Severina [50,122,123] indicated that the high energy products can hardly be involved in the oligomycin- and DCCD-sensitive steps of the

49 process. The given conclusion was then supported by the results of Racker [53] and Shchipakin et al. [54], who showed that oligomycin and DCCD lower the H +conductivity of the proteolipid (+OSCP)-containing proteoliposome in the absence of any kind of energy source for the formation of a high-energy intermediate (see pp. 34,35. Generally, the very existence of covalent high-energy intermediates in oxidative phosphorylation seems today extremely doubtful (see p. 42). Recently Mitchell [124] and independently Glagolev and Skulachev [125] put forward the hypothesis that Aft,+ is formed during the ATP hydrolysis without any kind of high-energy intermediates. In terms of this hypothesis, factor Ft directly participates in the generation of A/7.+ by the ATPase complex. One of the possible schemes of the functioning of the ATPase complex consideled by Mitchell [126,127] describes the occurrence of translocation of adenine nucleotides between the mitochondrial matrix and that area of the membrane where the proteolipid system is localized, the latter organizing a proton transfer from the catalytic site of F~ to the extramitochondrial medium. Since the ionised molecules Gf ADP and of inorganic phosphate have two more negative charges than ATP, the electrogenic antiport of ATP" / ADP + P~ ,-2 between the mitochondrial matrix and the FI active site might occur, thus contributing to the generation of membrane potential. It is obvious that the mechanism postulated should differ from the translocases of adenine nucleotides and phosphate already known, which carry out the transmembranous ATP/ADP antiport and the HzPO4--H + symport in mitochondria [128,13]. There are se~,eral types of mechanism for the transport of a substance in biclogical membrane systems. One of them consists in the movement of the substancecarrier complex; another is the formation of a channel penetrable to the substance being transported; the third is like a relay race where the substance is translocated between two or several binding sites, which are situated at different distances from the start, i.e. from the surface of the membrane. In this group, a hypothesis [23] was put forward suggesting that the translocation of ATP, ADP aqd P~ by factor F~ might be carried out according to the third irelay) principle. The most important points of the hypothesis are the following: (1) The catalytic site of factor Ft attached to the membrane, is immersed in the hydrophobic layer of the membrane and is not therefore directly accessible to ATP dissolved in water. (2) In the transfer of ATP from water to the catalytic site, a special (noncatalytic) site of factor F~, which faces the water phase, is involved. (3) In one of the factor F~ conformation states, the catalytic site is localized not far from the outer surface of the mitochondrial membrane whereas the noncatalytic site is situated on the matrix surface of this membrane. (4) The antiport A T P / A D P + Pj between the non-catalytic and catalytic sites has an electrogenic character. In the following sections, data will be presented which directly justify the first three conclusions of this conception. Some pieces of indirect evidence in support of the fourth postulate will b,e given.

50 Mg'ATP

_f-1v~

Fig. 3. T h e A T P - i n d u c e d , F ~ - m e d i a t e d c h a n g e s in the V o l t a p o t e n t i a l difference ( b p ) in the o c t a n e / w a t e r s y s t e m . I n c u b a t i o n m i x t u r e : I • 10 3 M D N P , 5 • 10 2 M Tr:s ' HCI ( p H 7.4). A d d i t i o n s : 2.5 " 10 '~ M f a c t o r F~, 1 ' 10 2 M M g A T P ( c o n c e n t r a t i o n s in the w a t e r p h a s e are indicated).

IIID-2. Factor FI on the octane/water interface. The biphasic system octane/ water has proved to be a convenient model for the study of the function of isolated factor Fj. The research carried out by Dr. L. i. Boguslavsky's group at the Institute of Electrochemistry of the U.S.S.R. Academy of Sciences in cooperation with this laboratory showed [87,59a,129,130] that factor F1 is capable of transferring charges through the interphase, coupled with ATP hydrolysis. The experiment was carried out in the following manner. The octane first equilibrated with dinitrophenol in the octane/water system, was put on the incubation mixture of the corresponding composition, and factor Fi was added. Several minutes later, ATP was introduced into the water phase. Factor F~- and ATP-induced changes in the Volta potential (Iq~) on the octane/water interface were measured by the vibrating capacitor method [131]. The result of a typical experiment of this series, carried out in our group by Dr. V. M. Voytsitsky, is shown in Fig. 3. It can be seen that the factor F~ addition results in the negative charging of octane against the water phase. Mg " ATP addition gives rise to the oppositely directed charge redistribution across the interface (plus on the octane side). Both F~- and ATP-induced Volta potential changes were found to require dinitrophenol or one of the following compounds: dodecyl sulfate (1 raM), trichlorocarbonylcyanide phenylhydrazone (1 /tM), anisidin (1 raM), veratrol (1 mM). The effect of ATP was not observed if factor F~ or Mg 2+ was excluded from the system, while the change in the pH buffer concentration (3 100 mM Tris • HC1) had hardly any effect on the response. Inhibition of the ATPase activity of factor Fx by A D P was found to suppress the generation of lq:. Oligomycin did not have any effect of the phenomenon studied. Further experiments showed that similar responses were revealed when factor F~ from M. lvsodeikticus was used instead of mitochondrial F~ [89, 132]. It should be noted that the directions of the electrical vector of the field generated by ATPase in natural and model systems coincide: during the hydrolysis of ATP in mitochondria the water phase ~owards which factor FI faces (matrix) is

51 negatively charged; in the octane/water system the minus sign is also on the side of the water phase. The given data do not allow us to make a conclusive choice between the two following explanations of ATP-dependent potential changes on the octane/water interface: (1) the ,,lq~ observed is the result of the transfer of H + ions in the direction from water to octane and (2) 'lq~ is determined by the reorientation of the dipoles of ATPase molecules soibed on the interphase. However, in either of the two cases the octane/water system might have proved to be the simplest model for the study of the ATP hydrolysis-coupled electrogenic function of isolated factor F~, since in this syslem, there is only one (instead of two) lipid/water interface. For this reason, lq) measurements were used in testing the hypothesis on the role of the catalytic and non-catalytic sites. It is to be hoped that the two sites differ in lhe set of functional groups involved in the nucleotide binding. If this is the case, inhibitors could be found which would selectively modify the catalytic and non-catalytic sites. With this purpose in mirtd, we tested a number of hydrophilic protein-modifying agents, for which the octane/water interface should be an impermeable barrier. IIID-3. E{J'ect of inhibitors on A TPase in solution, on the octane/water intelface and in the mitochondrial membrane. According to the scheme proposed, the inhibitor which blocks the non-catalytic site of factor FI (or the stage of substrate translocation between the non-ca'alytic and catalytic sites) should inhibit ATPase on the octane/water interface or ATPase associated with the mitochondrial membrane. At the same time, it should not have any effect on the ATPase activity of the enzyme in aqueous solution, where ATP should have direct access to the catalytic site, making unnecessary the participation of the non-catalytic site. In accordance with the results obtained in this and other laboratories, sodium p-chloromercuribenzoate (PCMB) inhibits (at fairly high concentrations) oligomycin-sensitive ATPase and ATPase of submitochondrial particles, and has absolutely no effect on the ATPase activity of factor F1 in aqueous solution [33,87]. We tested this agent in the octane/water system. As the experiments showed, PCMB completely abolishes factor Fl-mediated ,l