Ubiquinone-binding proteins

Biochimica et Biophysica Acta, 639 (1981) 9 9 - 1 2 8

99

Elsevier/North-Holland Biomedical Press

BBA 86078

UBIQUINONE-BINDING PROTEINS CHANG-AN YU * and LINDA YU *

Department o] Chemistry, State University of New York at Albany, Albany, NY 12222 (U.S.A.J (Received March 3rd, 1981)

Contents I. I1.

IlL

Introduction

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

100

Q-binding proteins in t h e mitochondrial system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Historical implication o f the existence o f Q-binding proteins in the mitochondrial system . . . . . . . . . . . . . . . B. QPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Properties o f QPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Solubility and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Purity o f QPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Molecular weight and a m i n o acid composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. Sensitivity o f QPs to c h y m o t r y p s i n treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. The role o f phospholipids in QPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f. Binding o f Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. Interaction between QPs and soluble succinate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . h. The nature o f the interaction between QPs and succinate dehydrogenase . . . . . . . . . . . . . . . . . . . . . C. QPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Properties of QPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. EPR spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. F o r m a t i o n o f the ubisemiquinone radical and t h e reduction of c y t o c h r o m e s b and c 1 . . . . . . . . . . . . . c. Effect o f antimycin A on QPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. Effect o f thenoyltrifluoroacetone on QPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. T h e r m o d y n a m i c properties o f QPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f. The nature o f t h e ubisemiquinone radical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. The nature of Q binding on QPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. QPn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q-binding proteins in p h o t o s y n t h e t i c systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Q-binding proteins in photosynthetic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Q-binding proteins in the reaction center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Q-binding proteins in the electron-transport chain o f the c y t o c h r o m e b-c2 complex 3. Free Q in the bacterial p h o t o s y n t h e t i c c h r o m a t o p h o r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: ETP, a nonphosphorylating inner m e m b r a n e preparation from beef heart mitochondria; PQ, plastoquinone; Q, ubiquinone; QoCl oNAPA, 2,3-dimethoxy-5-methyl6- (10-[ 3-(4 -azido -2-nitroanilino )propionoxy] decyl)-1,4-benzoquinone; QoC1 oTMPOC, 2 , 3 - d i m e t h o x y - 5 - m e t h y l - 6 - 0 0 [ 3-(2,2,5,5-tetramethyl-3-pyrrolin-l-oxy-3-carboxy)]decyl)1,4-benzoquinone; SDS, sodium dodecyl sulfate; PS, photosystem.

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

101 101 105 105 107 107 107 107 108 108 109 109

111 112 112 115 115 115 116 116 116

117 118 118 119 119 119 120 120

* Present address: Department of Biochemistry, Oklahoma State University, Stillwater, OK 74078, U.S.A.

0 3 0 4 - 4 1 7 3 / 8 1 / 0 0 0 0 - 0 0 0 0 / $ 0 2 . 5 0 © 1981 Elsevier/North-Holland Biomedical Press

100 B. Q-bindingproteins in the photosynthetic apparatus of green plants . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12 I

IV. Reaction mechanism of Q in the electron-transport reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122

V.

123

Otherpossible functions of Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VI. Concludingremarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

123

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124

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

124

I. I n t r o d u c t i o n

The participation of ubiqinone (Q) in the mitochondrial respiratory chain [1-4] and in photosynthetic electron-transport systems [5 9] has been well established, mainly through studies of chemical extraction of Q followed by restoration of enzymatic activity upon reincorporation of the extracted Q or its analogues [10-13]. The position of Q in the electron-transfer sequence in the mitochondrial system is, more or less, established at the oxygen side of the rotenone-sensitive site and at the substrate side of the antimycin A-sensitive site [14]. In photosynthetic systems, Q is believed to act as the primary electron acceptor in the light energy-harvesting system or reaction center [6 8] as well as in cyclic or noncyclic electron transport in the cytochrome b-c2 region. Although the essential role of Q in electron transport is generally accepted, the reaction mechanism is far from being well understood. Q is the only small lipophilic molecule involved in the electron-transport reaction, and is abundant (in molar excess compared to other electron-transport components) in the inner mitochondrial membrane, and photosynthetic bacterial chromatophores or chloroplasts. These facts have led to the idea that Q is a mobile component [15-17], which 'swims' in the phospholipid milieu and shuttles electrons among the electron-transfer lipoprotein complexes [20-23], such as NADH-Q reductase [20], succinate-Q reductase [21] and ubiquinol-cytochrome c reductase [22, 23]. This idea has been attractive to many investigators in the field and has promoted some popular hypotheses, such as 'pool function of Q' [18] and 'proton-motive Q cycle' [19,24]. Based on analysis of the redox state of Q obtained by extraction with organic solvent and its correlation with respiratory activity in the submitochondrial par-

ticle, Kr6ger and Klingenberg [18] have proposed a 'Q pool' model to explain the pseudo first-order kinetics of Q reduction [18] and the fact that the observed reduction rate of Q is slower than that of other redox components. The slow reduction rate of Q in the presence of terminM inhibitor was once viewed as evidence supporting the idea that Q is a 'bypass' or 'blind alley' and not an obligatory component [25] of the respiratory chain. The pool function of Q was later extended to explain the stoicbiometry of proton translocation in the Site II region [26] by emphasizing the dismutation reaction of the free ubisemiquinone radical, and assuming that ubisemiquinone was the product of dehydrogenase. The necessity for ubisemiquinone to interact with other components as the acceptor of reducing equivalents as postulated by the Q cycle was questioned. In contrast to the other redox components of tile respiratory chain, such as iron-sulfur proteins or cytochromes, Q can carry out electron transfer either by one two-electron step or by two one-electron steps. The unstable nature of the one-electron reduction product of Q, the ubisemiquinone radical [27], has somewhat obscured its importance as an active molecular species. The ubisemiquinone radical in mitochondria was detected as early as 1958 [28], yet the possibility of its involvement in electron-transfer reactions was not suggested until 1967 [29--31]. At tllat time a hypothetical redox component, X (k'm about I00 mV), which governs the redox behavior ofcytochrome b, was postulated to be present in Complex Ili to explain the unusual reduction of cytochrome b [30], and tile conformational change of the complex upon reduction. X was believed to be the oxidant ot the ubisemiquinone radical. Another hypothetical component, Y [32,33], the redox state of which controls the kinetic rate of reduction of cytochrome b, was postulated to be in

101 the cytochrome b-ca segment at the oxygen side of the antimycin A-sensitive site. The involvement of the ubisemiquinone radical in the electron-transfer reaction in the cytochrome b-cl region was also postulated by WikstrSm and Berden [34] to explain the oxidant-induced reduction of cytochrome b in submitochondrial particles in the presence of antimycin A. The oxidation of ubisemiquinone was kinetically comparable to the oxidantinduced reduction of cytochrome b in the presence of antimycin A [35], and the detection of ubisemiquinone in the mitochondrial membrane [31] was used to formulate a two-stage oxidation of QH2 in the cytochrome b-cl region, with QH2/QH" and QH'/Q as reductants for cytochrome b and cytochrome c~, respectively. Mitchell [19,24] extended the previous observations [30,31,34], with consideration of the unique nature of Q, the redox state change of which is associated with proton release and uptake [37-43], and proposed a well received proton-motive Q cycle. This hypothesis not only explained the unusual observed behavior of cytochrome b reduction but also solved the long-missing proton carrier of the chemiosmotic coupling theory [36,37]. The essence of the Q cycle is the participation of ubisemiquinone radicals at both the substrate and oxygen sides of cytochrome b in the electron-transfer sequence. The stability constant of the ubisemiquinone radical in phospholipid at neutral pH was estimated to be about 10 -12 [19], which is far too low to account for the electrontransfer reaction in the Site II region. Therefore, stabilization of QH" through complexation with one or more redox components is needed in order to bring the E m of QH'/Q and QH2/QH" to the kinetically competent levels of the other redox components in the Site II region and to inhibit 'uncontrolled' dismutation. This implies the binding of Q to protein during the electron-transfer reaction. The idea of Q bound to protein in the electrontransfer chain was also derived from orthodox biochemical studies of isolation and reconstitution of the electron-transfer chain [44]. Evidence for the existence of Q-binding proteins in the mitochondrial respiratory chain as well as in photosynthetic systems is currently accumulating. One of the Q-binding proteins has been isolated and characterized. In this review, we intend to discuss only the recent advances

in isolation and characterization of the isolated Q-binding protein, and properties of Q-binding proteins in the complex form. For the function of Q in mitochondria [1,45,46], and photosynthetic [5-8, 4 7 ~ , 9 ] and clinic applications [50], excellent reviews and monographs are available. II. Q-binding proteins in the mitochondrial system

IIA. Historical implication of the existence of Q-binding proteins in the mitochondrial system The implication of a specific Q-protein interaction in the mitochondrial electron-transfer chain appeared as early as the beginning of 1960, when the electrontransfer chain was resolved into four electron-transfer complexes. The uneven distribution of Q among the lipoprotein complexes could have been used as evidence for the specific interaction of Q with redox components. Perhaps the hydrophobic nature of Q, the molar excess of Q (compared to the redox components) present in the inner mitochondrial membrane, and the ease with which most of Q is extracted from the mitochondrial particle [10-12] have misled investigators to emphasize that Q acts as a mobile molecule in the membrane and to underestimate the specific binding between Q and protein. Furthermore, the lack of Q in the isolated Complex II [21] might have provided additional support for Q as a nonbinding entity. In fact, the lack of Q in Complex II is simply due to the organic solvent treatment during isolation. However, sufficient Q was observed in the succinate-cytochrome c reductase complex. As indicated in Table I, the amount of Q present in resolved succinate-cytochrome c reductase varies significantly among the preparations. The Q content decreased as the purity of the preparation increased, to a minimum ratio of 1 : 1 with cytochrome cl in a fully active succinate-cytochrome c reductase preparation [4]. When the concentration of Q decreased below the level of cytochrome Cl a stimulation of activity was observed upon the addition of exogenous Q [44]. This was the first indication of the existence of a Q-binding protein in succinate-cytochrome c reductase. If Q were merely dissolved in the phospholipid phase of succinate-cytochrome c reductase, one would not expect a fixed stoichiometry. In the less pure preparations of succinate-cytochrome c

102 TABLE I SUCCINATE-CYTOCHROMEc REDUCTASEPREPARATIONS Preparations of

Purity (cytochrome c I : nmol/mg)

Activity 0zmol cytochromc c reduced/rain per rag)

Q content (nmol/mg)

Ref.

Trumpower et al. Erecinska et al. Yuetal. Tisdale et at.

1.5 1.3 2.3 1.3

6 (30°C) 0.3 (250(") 8 (25°C) 25 (38°C)

7.2 0.36 2.3

3,51 52 53 54

reductase a higher Q content was generally detected, indicating that in such preparations not all the Q present functions in the electron-transfer reaction. Some of the Q apparently was not specifically bound to protein. The Q content in Complex I [20] is in molar excess as compared to flavin ( 3 - 4 tool Q/mol FMN). Whether or not all the Q present is needed in the electron-transfer reaction is not known. Reconstitution studies of Complex I and Complex III [55] showed that the maximum rate of electron transfer from NADH to cytochrome c required less than 3 mol Q/mol NADH-cytochrome c reductase (Complex I-Complex IlI). The heterogeneity of Q is also observed in submitochondrial particles during organic solvent extraction and replenishment experiments. A small portion of Q could not be extracted without damaging the protein [18]. Restoration of original succinate oxidase activity required incorporation of only 15% of the original content of Q. A significant advance in the study of Q in mitochondrial particles was the detection of the ubisemiquinone radical. Raikhman and Blyumenfel'd [29] found an EPR signal with a gvalue of 2.00 and a band width of 10-15 G which was substrate and protein dependent. Using inhibitors, they were able to localize the generation site for this EPR signal between malonate or amytal and the antimycin A-sensitive site of the NADH or succinate oxidase systems, respectively. The main electron carriers in the region between these inhibitors are flavins and Q. By extraction and reincorporation of Q, they concluded [29] that at least part of the semiquinone signal was due to Q. The detectable ubisemiquinone concentration is rather low as compared to the total detectable amount of Q in the system. It amounts to no more

than 2% of the total Q in mitochondria. The detection of ubisemiquinone in submitochondrial particles was further confirmed by B/ickstr6m et al. [31 ]. Formation of ubisemiquinone is pH dependent (pH 6.5-8.5) and can be distinguished from tile flavin quinone radical because the latter has a larger band width (12 G) than the former (10 G). Using the succinate/furnarate couple to control the redox potential and the power saturation behavior technique, Konstantinov and Runge [56] were able to separate the ubisemiquinone signal observed in submitochondrial particles into two types and postulated that they are localized at different sides of tile mitochondrial membrane. The detection of the ubisemiquinone radical in the electron-transport chain of submitochondrial particles or mitochondria implies a specific Q-protein interaction because the equilibrium constant for the formation of QH" from QH 2 and Q is far too low for any detectable amount of QH" to be present in the free form or even in the phospholipid bflayer. The specific interaction between Q and protein in order to stabilize QH" was stressed in the Q cycle of" Mitchell [19,24]. The introduction of the proton-motive Q cycle indeed stimulated a new tide of investigation on Q and its possible role in protein translocation [38 431. The implication of the existence of a Q-binding protein in the isolated electron-transfer complexes derived from a completely different experimental approach. The isolated succinate-cytochrome c reductase contains about 200 nmol phospholipids and 2.3 nmol Q/mg protein (see Table I). Both Q and phospholipids can be removed under mild conditions [4,44] - repeated ammonium sulfate precipitation m

103 the presence of 0.5% cholate. However, the removal of Q by this method does not proceed with the same ease as that of phospholipids. The maximal removal of phospholipids required two cycles o f precipitation whereas that of Q required five cycles of precipitation [4]. The different behavior o f phospholipids and Q toward ammonium sulfate-cholate precipitation is illustrated in Fig. 1. Since Q is a hydrophobic molecule, it would be expected to be removed from succinate-cytochrome c reductase with phospholipids if there were no specific interaction between Q and protein. The separate removal of phospholipids and Q, as shown in Fig. 1, supported the idea o f a specific Q-protein interaction in succinate-cytochrome c reductase. Also indicated in Fig. 1 is the fact that succinate-Q reductase activity requires no phospholipids, as most of the activity was retained when the phospholipids were removed. The idea o f a Q-protein interaction or the existence of a specific Q-binding protein was further strengthened by the fact that a special sequence of addition o f Q and phospholipids was required during the restoration o f enzymatic activity from the Q- and phospholipid-depleted succinate-cytochrome c reductase [4]. The maximal or

original activity was restored only when Q was added prior to the addition o f phospholipids (see Fig. 2). Addition of phospholipids prior to or even concurrent with Q restored only partial activity, unless a great excess o f Q was used. This observation suggested that there are specific Q-binding sites in succinate-cytochrome c reductase and that such binding sites are very easily covered up by phospholipids when Q is absent. Following alkaline treatment of extensively dialyzed succinate-cytochrome c reductase at pH 10.5 in the presence o f succinate and in the absence o f 02,

8.0

7O 6.O

i~

~.o 4.0

"~ 3,C i

i

i

I

i

m

1OC

2.C 1.0

8O

0,0

7O

,

0

~ 60

30

,

60

90

120

Incubation t i m e , rain

5O 4C 3O 2O 10 0

1

2

3

4

5

No. of cycle Fig. l. Removal of Q and phospholipid from succinate-cyto-

chrome c reductase by cholate-ammonium sulfate fractionation. Aliquots were withdrawn after each cycle of cholateammonium sulfate precipitation and assayed for activities of succinate-cytochromec reductase (A A), succinate-Q reductase (o o), and Q2H2-cytochromec reductase (D =); Q content (× X); and phospholipid (determined as phosphorus content) (oo). From Ref. 4.

Fig. 2. Effect of sequence of phospholipids and Q additions to the phospholipid- and Q-depleted succinate-cytochrome c reductase on the restored activity. The Q6 used was made by dispersing 20 ~tl of 10.6 mM Q6 in 0.2 ml of 50 mM phosphate buffer containing 0.5% cholate, pH 7.4. The asolectin used was at 10 mg/ml in 50 mM phosphate buffer containing 0.5% cholate, pH 7.4. (e e) 0.1 ml of the depleted preparationwas mixed with 0.05 ml of Q6 and then incubated at 0°C for 5 min; 0.05 ml of asolectin was subsequently added. (A A) 0.1 ml of the depleted preparation was mixed with a mixture containing 0.05 ml of asolectin and 0.05 ml of Q6 and then incubated at 0°C for 5 min. C A - - ) < ) 0.1 ml of the depleted preparation was mixed with 0.05 ml of asolectin and then incubated at 0°C for 5 min; 0.05 ml of Q6 was finally added. All reaction mixtures were incubated further for 3 min before assay. At the indicated times, each system was diluted with 0.3 ml of 50 mM phosphate buffer and the enzymatic activity was assayed. From Ref. 4.

104 a soluble fraction which contains reconstitutively active succinate dehydrogenase can be separated from the cytochrome b-c~ complex by centrifugation [57]. The crude succinate dehydrogenase fraction is further purified to a homogeneous state. Pure soluble succinate dehydrogenase shows only two polypeptides with molecular weights of 7 0 0 0 0 and 2 7 0 0 0 [ 5 7 59]. It contains neither phospholipids nor Q yet can recombine with the cytochrome b-c1 complex either in particle form or after solubilization with deoxycholate to form an antimycin A-sensitive [60] succinate-cytochrome c reductase [61]. Since succinate dehydro~enase contains no Q, it is not responsible for Q binding in this particular segment of the electrontransport chain. The Q-binding sites must be in the cytochrome b-c~ complex. Chemical analysis of the soluble cytochrome b-c~ complex (see Table II) shows that all Q which was present in succinate-cytochromec reductase was recovered in the cytochrome b-c 1 complex, indicating that the Q-binding sites were retained in the soluble cytochrome b-cl complex [57,61]. As indicated in Table III, soluble cytochrome b-c~ complex is not the same as the well known Complex III [22,23] even though their chemical compositions are very similar to each other. Soluble cytochrome b-c~ complex is able to reconstitute with soluble succinate dehydrogenase to form succinate-Q reductase and succinatecytochrome c reductase whereas Complex III cannot. The latter can reconstitute only with Complex II [20] to form succinate-cytochrome c reductase. This indicates that a protein or factor which is needed to

convert succinate dehydrogenase into succinate-Q reductase is present in the soluble cytocbrome b-c1 complex but absent in Complex Ill. Since the function of such a protein is to convert soluble succinate dehydrogenase into succinate-Q reductase [66] and it was found to bind Q, it was named QPs (for Q protein involved in succinate-Q reductase) to distinguish it from other possible Q-binding proteins. QPs was found to be sensitive to treatment with chymotrypsin. When soluble cytochrome b-c1 complex was treated with chymotrypsin at a concentration of 5 ttg/mg protein, QPs activity was destroyed within i h of incubation at room temperature and the cytochrome b-c~ complex was functionally converted into Complex III. Since Complex llI, as indicated in Table II, contains significant amounts of Q which were not removed concurrently with the removal of phospholipids, it was assumed that a different type of Q-binding protein may exist and this protein was named QPc (for Q-binding protein in the cytochrome b-c1 region) [68,69]. The deduction of the existence of QPc received further support when the soluble cytochrome b-el complex was further fractionated to crude QPs and a highly purified ubiquinol-cytochrome c reductase (the cytochrome b-cl-lll complex) [57]. The Q was found to be evenly distributed between the two fractions, yet only the QPs fraction is able to convert succinate dehydrogenase to succinate-Q reductase. The Q associated in the cytochrome b-c1-lll complex [57] is totally inactive il~ reconstitution with soluble succinate dehydrogenase. In addition to QPs and QPc, evidence for another

TABLE II CHEMICAL COMPOSITION OF CYTOCHROME b-c I COMPLEX PREPARATIONS Components

Cytochromeb Cytochromecl Nonheme iron Flavin Q Pbospholipids a From Ref. 65.

Concentration (nmol/mg protein) Soluble cytochrome b-c 1 complex

Complex III a

Cytochromc b-el -Ill complex

Crude QPs

6.5 4.1 6.0 0 3.7 200

6.8 3.4 6.2 0.15 1.0 257

10 -10.5 5.7- 6.0 6.6- 7.2 0 2 3 250

2.5 0.6 5.0 0 4.0 350

_+_0.4 ±0.2 ± 0.5 - 0.5 - 4.0

105 TABLE IlI ELECTRON-TRANSFER COMPLEXES AT THE SITE II REGION OF THE MITOCHONDRIAL RESPIRATORY CHAIN Succinate-cytochrome c reductase = complex II+ complex III [64] or succinate dehydrogenase+ soluble cytochrome b-c 1 complex [61] or succinate dehydrogenase + QPs + cytochrome b-cl-III complex [77]. Succinate-Q reductase or complex II = soluble succinate dehydrogenase + QPs [77 ]. ISP, iron-sulfur protein. Preparations

Electron transfer

Essential redox components

Reconstituted with

Ref.

Complex II

Succinate --* ubiquinone

FAD, ISP, (cytochrome b), QPs

Complex III

21

Complex Ill

Ubiquinol --, cytochrome c

Cytochrome b, cytochrome c l , ISP, QPc

Complex II

64

Cytochrome b-c1 particle

Ubiquinol ~ cytochrome c

Cytochrome b, cytochrome c l , ISP, QPs, QPc

Succinate dehydrogenase corn plex II

62

Soluble cytochrome b-c 1 complex

Ubiquinol ~ cytochrome c

Cytochrome b, cytochrome c l , ISP, QPs, QPc

Succinate dehydrogenase complex II

61

Cytochrome b-c 1 -lil complex

Ubiquinol ~ cytochrome c

Cytochrome b, cytochrome c l , ISP, QPc

Complex II (succinate dehydrogenase, QPs)

57

Cytochrome b-c I complex

-

Cytochrome b, cytoclarome c I

-

63

Q-binding protein, QPn (for Q-binding protein involved in NADH-Q reductase) [70], in the mitochondrial electron-transport chain has been accumulating and will be discussed. The existence of QPn was established by partial fractionation as well as proteolytic digestion and EPR detection of the ubisemiquinone radical in Complex I.

IIB. QPs QPs is a protein which binds Q and converts soluble succinate dehydrogenase into succinate-Q reductase [71 ].

IIB-1. Isolation Since the first brief report on the isolation of QPs [67], a protein which is capable of converting soluble succinate dehydrogenase into succinate-Q reductase, several methods have been developed for the isolation of QPs from different sources and under different terminologies [73-75]. Essentially, all of them discuss the same protein. The most purified QPs was obtained from soluble cytochrome b-c~ complex by a procedure (Method I) developed in our laboratory [71]. The method involves treatment of soluble cyto-

chrome b-Cl complex with 1% Triton X-100 in the presence of 2 M urea, calcium phosphate column chromatography and ammonium sulfate fractionation. This procedure gave an almost pure protein with a molecular weight of 15 000. It contains less than 1 nmol cytochrome b/mg protein. The specific activity for this preparation was 17 ~tmol succinate oxidized/min per mg protein, which is lower than the expected value. An improved method (Method II) which gave better specific activity and recovery was introduced by our laboratory [71 ]. The method involves ammonium acetate fractionation of soluble cytochrome b-cl complex in the presence of deoxycholate, ammonium sulfate fractionation in Tris buffer in the presence of 2 M urea and 0.67 M sucrose, ammonium sulfate fractionation in phosphate buffer containing 2 M urea, 0.25 M sucrose and 0.3% ~-mercaptoethanol, and differential centrifugation. The purified QPs obtained by this method has a specific activity 4-5-times higher than that of the protein obtained by Method I, but has lower protein purity. The main contamination was found to be a denatured cytochrome b protein of molecular weight 17000 [72]. The QPs obtained by Method II contains 8 nmol

106 cytochrome b/nag protein [71 ]. By a completely different approach, a protein fraction, which has properties very similar to those of QPs obtained by Method I, was obtained by Vinogradov et al. [73] from submitochondrial particles by Triton X-100 extraction after removal of succinate dehydrogenase by alkaline treatment. The Q-binding protein thus obtained was estimated to be about 80% pure from SDS-polyacrylamide gel electrophoresis. This Q-binding protein preparation was termed PF (for protein fraction) by the original author [73]. The molecular weight of the major polypeptide of PF was estimated to be less than 13 000. This protein was believed to be identical to that obtained by Method I described above. Simplicity and good recovery are the advantages of this method, but only a dilute protein can be obtained. A two-polypeptide (CII-3,4) protein preparation, which is active in reconstitution with soluble succinate dehydrogenase to form succinate-Q reductase and is similar to the QPs obtained by Method I1 described previously, was obtained from Complex lI by Ackrell and co-workers [74]. Essentially, Complex II was first treated with 0.8 M sodium perchlorate in the presence of succinate and dithiothreitol to remove succinate dehydrogenase [58] and then extracted with 0.5% (w/v) Triton X-100 or deoxy-

cholate. The preparation thus obtained shows two major polypeptides in SDS-polyacrylamide electrophoresis (using the system of Swank and Munkres [109]) with molecular weights of 13 500 and 7000. The preparation contains significant amounts of phospholipids and trace amounts o f cytochrome b (0.04 tool/tool CII-3,4), but does not contain Q. Q is absent from the preparation because the starting material, Complex II, as prepared is depleted of Q. Another reconstitutively active preparation of QPs similar to Cll-3,4, called cytochrome b-560, was independently obtained by Hatefi and Galante [75]. Cytochrome b-560 was obtained from Complex II following the extraction of succinate dehydrogenase with 0.35 M and 0.8 M sodium perchlorate consecutively in the presence of succinate and dithiothreitol by potassium deoxycholate solubilization and ammonium sulfate fractionation. The purified cytochrome b-560 preparation contains two polypeptides in a roughly equal molar ratio. The molecular weights of these two polypeptides were estimated by SDS-polyacrylamide gel electrophoresis to be 15500 and 13 500 (using the system of Weber and Osborn [108]). The preparation of cytochrome b-560 is similar to QPs obtained by Method II and to that obtained by Ackrell et al. [74]. Table IV summarizes the QPs preparations available to date.

TABLE IV COMPARISON OF Q-BINDING PROTEIN (QPs) PREPARATIONS Preparations

QPs (Method I)

Starting material

Specific activity (#mol succinate/ mg per rain)

Polypeptides (tool. wt.) (× 10-3 )

Cytochrome b (nmol/mg)

Rcf.

Soluble cytochron~c

17 (23°C)

15


7I

79

(23°C)

15,17

8

71

3

(23°C)

b-el complex QPs (Method II)

Soluble cytochromc

b-q complex PF

Submitochondrial particles

CII-3,4

Complex Ii

Cytochrome b-560

Complex II

<13 13, 7

54.8 (37°C)

13.5,15.5

73 2

74

14 a

75

a A slightly lower molar extinction coefficient (20 • 103) was used in the calculation. The value would be about 10 if the molar extinction of 28.5 • 103 described for cytochrome b by Berden and Slater [76] were used. The reason for using the lower molar extinction coefficient was given in the original paper [75].

107

lIB-2. Properties of QPs a. Solubility and stability. All the preparations for QPs described involved detergents, either deoxycholate or Triton X-100, in the final step of purification. The bound detergent is apparently enough to disperse the isolated proteins in aqueous solution. When the detergent was removed by exhaustive dialysis or gel column filtration, the protein became aggregated and insoluble but could be resolubilized by Triton X-100 or deoxycholate. This protein has been shown to be stable at neutral to slightly alkaline pH values [71 ]. It suffers very little loss in activity when stored at -70°C, and can tolerate freeze-thawing several times with little loss of activity. However, at higher temperature, isolated QPs denatured easily. At 38°C, QPs has a half-life of only 30 min. QPs is stable in high concentrations of detergents, especially at low temperatures. No activity was lost when QPs was incubated with 1 mg SDS/mg protein or 1% cetyltrimethylammonium bromide at 0°C for 30 min [77]. Purified QPs is also very stable toward treatment with urea and nonionic detergents such as Triton X-100 or Tween 80 when they are used separately [73,77]. For example, no significant loss of activity was observed when QPs at a protein concentration of 6 mg/ml was incubated with urea at concentrations as high as 4 M or Triton X-100 at 2%. However, when QPs was incubated with 2 M urea and 2% Triton X-100, a significant loss in reconstitutive activity was observed [77]. b. Purity of QPs. The most purified QPs was the preparation obtained by Method I which was believed to have more than 90% purity of the major protein of molecular weight 15 000 [71]. The protein fraction obtained by Vinogradov et al. [73] was reported to have a purity of 80%. Trace amounts of cytochrome b could be detected in these two preparations when the proteins were concentrated. The other QPs preparations have much lower purities, determined from the reported densitometric tracings of SDSpolyacrylamide gel electrophoresis patterns, either by the blue color after staining and destaining or directly by ultraviolet absorption. Cytochrome b was clearly present in the QPs preparations obtained by Method II of our laboratory [71], by Ackrell et al. [74] and by Hatefi and Galante [75]. The absorption spectra of cytochrome b have been reported [71,75]. In fact, Hatefi and Galante [75] believe that cytochrome b is the protein responsible for the reconstitution of suc-

cinate-Q reductase from soluble succinate dehydrogenase. They showed that the cytochrome b in their preparation was succinate reducible, although the degree of reducibility was very low [75]. Other investigators [71,73,74], however, believe that the cytochrome b in the preparation has no direct catalytic fuction in converting soluble succinate dehydrogenase into succinate-Q reductase. The evidence against the participation of cytochrome b came from the observation that there is no direct correlation between the cytochrome b content of QPs and its reconstitutive activity: no more than 5 tool% of cytochrome b was detected in CII-3,4 which was able to reconstitute with soluble succinate dehydrogenase to form succinate-Q reductase with the same activity as that of Complex II [74]. The nonfunctional role of cytochrome b in QPs was further supported by the fact that no effect on the reconstitutive activity was observed when cytochrome b in a QPs preparation (Method II) was denatured by treatment with 1% Zwittergent in the presence of 0.1% ~-mercaptoethanol [71], followed by dialysis overnight against 50 mM phosphate buffer containing 0.25 M sucrose. The possibility of a structural or stabilizing function of the cytochrome b protein [78,79] in QPs or in Complex II cannot be ruled out because the higher purity preparations of QPs (Method I) show lower specific activity. More experimental evidence will be required to determine whether the decrease in activity in purer preparations is due to the removal of cytochrome b protein or to the conditions used in the isolation procedure. Until a fully active, single-protein QPs preparation is obtained, argument as to the role of cytochrome b will persist [80-82].

c. Molecular weight and amino acid composition. The minimum molecular weights of QPs reported by the four different laboratories were in the range 1 2 0 0 0 - 1 5 0 0 0 depending upon the SDS-polyacrylamide gel electrophoresis conditions. It seems clear that the different groups all deal with the same polypeptide, which appeared as the third major polypeptide of Complex II [71,83] when the electrophoresis was carried out using the system of Swank and Munkres [109], and as the fourth major polypeptide when the gel system of Weber and Osborn [108] was used. The precise minimum molecular weight of QPs has yet to be determined by a method other than

108 electrophoresis. As isolated, QPs is in a highly aggregated form. Its apparent molecular weight, as determined using a gel filtration column (BiooGel A 5m), was found to be about 10 6 in the absence of added detergent [71 ]. The amino acid composition of purified QPs has been reported by two laboratories [71,75], one using isolated QPs (Method I) and the other using the protein obtained from the sample eluted from an SDSpolyacrylamide gel column after electrophoresis. Significant differences between the two sets of amino acid composition were reported. The different results may be attributed to three possible sources: first, the QPs used contained about 10% impurities; second, the separation of protein bands during gel electrophoresis may not be absolutely complete, leaving the possibility of cross-contamination; and third, the rather high concentration of glycine and serine in the amino acid composition obtained from the sample eluted from a polyacrylamide gel [75] might result from a slight contamination by the gel, which has been shown to produce significant amounts of glycine and serine after hydrolysis [53]. Although more accurate and complete data on the amino acid composition of pure QPs are needed for the further study of QPs, it is clear that QPs is not identical to the other small subunits of the cytochrome b-c~ complex [531. Although one very important amino acid, cysteine, was not quantiatively determined in the amino acid analysis, the involvement of an -SH group in the formation of succinate-O reductase from QPs and succinate dehydrogenase was indicated, both in the isolated QPs [71] and in the cytochrome b-c1 complex [61]. The reconstitutive activity of QPs was abolished upon treatment with p-hydroxymercuribenzoate [71]. Similar results were obtained when soluble cytochrome b-c~ complex was treated with p-hydroxymercuribenzoate. Whether a specific -SH group or a collective action of-SH groups is responsible is not known because no kinetic study of the inactivation of isolated QPs by alkylation has been made.

d. Sensitivity of QPs to chymotrypsin treatment. The sensitivity of QPs toward chymotrypsin treatment was first observed in the soluble cytochrome b-ca complex [61]. This was used as evidence for the existence of QPs [67] in the cytochrome b-c1 complex in the earlier stages of investigation because

digestion of soluble cytochrome b-c~ complex with limited chymotrypsin impaired the activity in recon. stitution with soluble succinate dehydrogenase to form succinate-Q reductase but had little effect on ubiquinol-cytochrome c reductase activity. When isolated CII-3,4 was treated with chymotrypsin, only one protein, CII-3, was digested to a molecular weight smaller than 7000. The digestion of ClI-3 was found to proceed in more than one stage [74]. The initial phase of digestion caused the disappearance of Cll-3 and the formation of a new band with a molecular weight greater than 7000 but did not affect most of the activity. The later phase of digestion inrpaired the reconstitutive activity. The protein was protected from this later phase of digestion when CII-3,4 was complexed with succinate dehydrogenase. Under the same conditions, when Complex II was digested with chymotrypsin the degradation stopped at a stage with CI1-3 showing a molecular weight of about 9000, with no observed loss of succinate-Q reductase activity. These results support the idea that QPs or CII-3 is a binding site for succinate dehydrogenase [74]. The reason that no digestion occurs beyond a molecular weight of 9000 is that when QPs is complexed with succinate dehydrogenase, an aromatic amino acid residue which is susceptible to chymotrypsin digestion on QPs is covered up by succinate dehydrogenase. This amino acid residue was exposed upon removal of succinate dehydrogenase. e. The role of phospholipMs in QPs. The QPs prepared by Methods 1 and II [71] and by Ackrell et al. (CII-3,4) [74] has been reported to contain significant amounts of phospholipids. The presence of phospholipids in the preparation of Hatefi and Galante [75] and ofVinogradov et at. [70] was not specifically reported. The phospholipid in QPs is mainly phosphatidylcholine [71 ]. Whether or not this phospholipid has any functional role in the electron-transfer reaction is a matter of discussion: evidence both for and against is available m the literature [78,79. 84]. We favor the idea that phospholipids play no direct functional role in succinate-Q reductase because the reconstitutive activity of QPs was not sensitive to treatment with phospholipase A2 [71 ]. A similar result was observed in Complex II [85]. Furthermore, removal of phospholipids from succinatecytochrome c reductase destroyed the electron-transfer activity from Q to cytochrome c but left 60% of

109 the original succinate-Q reductase activity. Removal of phospholipids from Complex II also indicated that phospholipids have no catalytic role in succinate-Q reductase [86]. This was accomplished by ammonium sulfate precipitation in the presence of 0.5% cholate after replacing deoxycholate by sodium cholate with Sephadex gel column filtration in the presence of succinate. f. Binding of Q. The QPs preparations obtained by Methods I and II of our laboratory [71] and by Vinogradov et al. [73] contain considerable amounts of Q, but less than the calculated stoichiometric amount of protein. This was evidenced by the stimulation of reconstitutive activity on addition of exogenous Q, especially in the case of QPs obtained by Method I. The deficiency of Q in the PF preparation of Vinogradov et al. [73] was also indicated by the lower activity reported. No exogenous Q was used in their assay system. A lower concentration of PF is required to abolish the low-Km ferricyanide reductase activity than that required to reach full reconstitutive succinate-Q reductase activity. This also indicates the deficiency of Q in the preparation, since masking the lowKm ferricyanide reductase site requires only a protein fraction whereas succinate-Q reductase activity requires a significant amount of Q. The presence of relatively high concentrations of Triton X-100 in PF may interfere somewhat with the Q-protein interaction. The cytochrome b-560 fraction obtained by Hatefi and Galante [75] and CII-3,4 by Ackrell et al. [74] lack Q because the starting material used, Complex II, contains no detectable Q. The lack of Q in Complex II is easily explained by the fact that the isolation procedure involves treatment with organic solvent. QPs obtained from Complex II requires the • addition of Q or Q analogues to show reconstitutive activity. The binding kinetics of Q to QPs or Complex II have not been reported. The slow progress of the binding studies results from the hydrophobic nature of Q. The competition between protein and detergent for Q is difficult to delineate, and the presence of detergent in QPs and Complex II is absolutely necessary to disperse the protein. The use of radioactively labeled and spin-labeled Q analogues [87] in binding studies has indicated a specific interaction between Q and lipid-depleted QPs or Complex II. Under specific conditions, a 1 : 1 stoi-chometry between flavin and Q has been observed in

Complex II [86]. Due to the presence of detergents, the actual meaning of the binding data so obtained must be treated with special caution. The existence of a specific interaction between protein and Q in the succinate-Q reductase has been confirmed recently [86] through the observation of a spectral shift of Q upon interaction with lipid-depleted Complex II.

g. Interaction beween QPs and soluble succinate dehydrogenase. The physical interaction between QPs and succinate dehydrogenase [77] has been demonstrated by several lines of evidence. (i) The first is the deaggregation of QPs by soluble succinate dehydrogenase. QPs as prepared, either by Methodl or Method II [71] or by the method of Ackrell et al. [74], was in a highly aggregated form. When reacted with soluble succinate dehydrogenase, in the absence of added detergent, QPs became partially deaggregated, and the reconstituted succinate-Q reductase showed a molecular weight ranging from 12 - 104 to 5 • l0 s. This polydispersed reconstituted succinate-Q reductase can be further deaggregated to dimeric or monomeric forms if the reconstitution is made in the presence of dilute detergent such as 0.2% Triton X-100. The evidence for the deaggregation of QPs upon interaction with soluble succinate dehydrogenase was illustrated by gel filtration [77]. (ii) The second line of evidence is the diminution of the low-Km ferricyanide reductase activity of succinate dehydrogenase upon interaction with QPs. Reconstitutively active succinate dehydrogenase possesses two types of ferricyanide reductase activity: one with a Krn for ferricyanide of 3 mM and the other with a K m of about 50/aM [88-91]. The latter activity is absent in preparations such as Complex II, succinate-cytochrome c reductase or submitochondrial particles. This low-Km ferricyanide reductase activity of succinate dehydrogenase is observed only when succinate dehydrogenase is resolved from the complex, and therefore detached from QPs. This activity disappeared upon recombination with QPs. Since the low-Km ferricyanide reductase activity of succinate dehydrogenase appeared only under conditions which disrupt the structure of succinate-Q reductase, it was very labile, and denatured easily in the presence of 02 or other oxidants. Fig. 3 illustrates the effect of QPs on the low-Km ferricyanide reductase activity of succinate dehydrogenase. The ability of QPs to abolish the low-Km ferricyanide reductase activity of suc-

110 A K3Fe (CN)6

OPs x SDH

K3Fe(CN) 6~

S~H

02

Xx\

\\

1-,--30 sec---~

~A420 = 0.05

Fig. 3. Activities of succJnate dehydmgenase (SDH) and reconstituted succinate-q reductase. FerricyanMe (350 ~M) was used as electron acceptor: 5 /11 of freshly prepared succinate dehydrogenase (0.77 mg/m]) were used in the assay. In the reconstituted system, excess QPs (5-fold of succJnate dehydmgenase) was mixed with sucdnate dehydrogenase; the concentration of succJnate dehydrogenase was also adjusted to 037 mg/ml. From Ref. 66.

cinate dehydrogenase supports the physical association between the two. Interaction between succinate dehydrogenase and soluble cytochrome b-c1 complex to form succinate-Q reductase also abolished the lowK m ferricyanide reductase activity of succinate dehydrogenase. The reduction of ferricyanide after the reconstitution of succinate dehydrogenase with QPs in the presence of excess Q differs from that of ferricyanide directly reduced by succinate. (iii) The third supporting study concerns the stabilization of succinate dehydrogenase by QPs [77]. The soluble succinate dehydrogenase as prepared is very labile, especially in the presence of O2 and in the absence of succinate. Addition of QPs to soluble succinate dehydrogenase greatly stabilized the enzyme. The stabilization of succinate dehydrogenase by QPs affects not only the reconstitutive activity but also the succinate-phenazine methosulfate reductase activity of succinate dehydrogenase. The stability of succinate-Q reductase, as compared to succinate dehydrogenase, can be used to support the idea that succinate-Q reductase indeed exists as a complex in the

respiratory chain, as all other electron-transfer con> plexes are found to be stable in the isolated form. Succinate dehydrogenase should be considered as only a fragment of succinate-Q reductase. (iv) Direct evidence for the physical association between QPs and succinate dehydrogenase has been obtained by immunoprecipitation of a mixture of the two using an antibody specific for the flavin protein of succinate dehydrogenase [74]. (v) Tile physical recombination of succinate dehydrogenase with QPs was demonstrated by the reconstitution between QPs and excess succinate dehydrogenase followed by gel column filtration. The excess unbound succinate dehydrogenase was eluted later in the Bio-Gel A 0.5m column and the reconstituted succinate-Q reductase showed a 1 : 1 stoichiometry of succinate dehydrogenase and QPs [77]. Similar results were also obtained when succinate dehydrogenase was reconstituted with cytochrome b-560. When the cytochrome b-560 preparation was mixed with excess succinate dehydrogenase, succinate-Q reductase was formed and recovered in the pellet alter the mixture was dialyzed and centrifuged. The excess succinate dehydrogenase remained in the supernatant. The SDS-polyacrylamide gel electrophoresis pattern of the precipitate indicated that succinate dehydrogenase and cytochrome b-560 formed a l :1 entity [75]. (vi) In addition to the physical evidence for the association between QPs and succinate dehydrogenase, the formation of succinate-Q reductase from QPs and succinate dehydrogenase also gained support from study of the enzymatic activities of isolated QPs preparations [77]. The specific reconstitutive activities of isolated QPs preparations are sunmrarized in Table IV. None of tile five preparations, however, has a specific activity as high as that calculated for the undenatured protein. Assuming succinate dehydrogenase and QPs are present in succinate-cytochrome c reductase at a stoichiometry of 1 : I, undenatured QPs is calculated to have a specific activity of 150 200 ~nrol succinate oxidized/rain per nag of protein at room temperature in the presence of excess succinate dehydrogenase. This indicates that some denatured QPs is present in the preparations. Ackrell et al. [74] have shown that the reconstituted succinate-Q reductase has the same activity as Complex II. This may be explained by tire fact that Complex I1 as pre-

111 pared is not as intact as submitochondrial particles. Similarly, the nearly stoichiometric relationship between QPs and succinate dehydrogenase [71 ] may also be partly due to the fact that the succinate dehydrogenase used was not 100% intact, and inactive succinate dehydrogenase may have compensated for the inactive protein in QPs. Similar arguments can also be used to support the observation [75,77] that reconstituted succinate-Q reductase has a 1 : 1 stoichiometry among the subunits. The denatured proteins are inactive in interaction with succinate dehydrogenase and therefore do not precipitate after centrifugation. Since the presence of some inactive protein in the preparation does not affect the enzymatic kinetic parameters, the reconstituted succinate-Q reductase has the same Km value for succinate as does succinate-Q reductase in succinate-cytochrome c reductase [71]. Ackrell et al. [74] have also reported that the reconstituted succinate-Q reductase behaved more like Complex II than ETP [92] with respect to the K m for phenazine methosulfate and the K i for thenoyltrifluoroacetone. Reconstituted succinate-Q reductase showed an inhibition by carboxin [93] identical to that of the submitochondrial particles, and the addition of excess QPs (PF) did not change the kinetic behavior of the reconstituted succinate-Q reductase toward carboxin [73]. (vii) Thermodynamic properties of native and reconstituted succinate-Q reductase and the activation energy of interaction between QPs and succinate dehydrogenase have also been reported [77]. The activation energy of native and reconstituted succinate-Q reductase has been determined by Arrhenius plots which show a break point at 26°C. Above 26°C, the system has activation energies of 11.4 and 10.2 kcal/mol for the intact and reconstituted enzymes, respectively. Below 26°C, both systems show a higher activation energy, which was 19.7 and 26.7 kcal/mol for intact and reconstituted systems, respectively. The similar break points in the Arrhenius plots and the identical activation energy at higher temperatures indicate that the active sites are located in similar environments. The slight deviation in activation energy at lower temperatures may result from the association of different detergents in the preparations. Using the time required to reach the half-maximal formation of succinate-Q reductase after mixing QPs and succinate dehydrogenase (in excess) at vari-

ous temperatures, one can calculate the rate contant for formation of succinate-Q reductase. Plotting the logarithm of the rate constant against the reciprocal of temperature, one can determine the activation energy for the interaction between QPs and succinate dehydrogenase. Surprisingly, the Arrhenius plot of the interaction also shows a similar break point at 26°C, but with a lower activation energy (6.9 kcal/ mol) below the break point and a higher activation energy (11.2 kcal/mol) above the break point [77]. The significance of these differences remains to be explained. However, it may mean that at low temperatures QPs is in a better orientation to interact with succinate dehydrogenase.

h. The nature of the interaction between QPs and succinate dehydrogenase. The first successful reconstitution of succinate oxidase [94] was performed with soluble succinate dehydrogenase and alkalinetreated submitochondrial particles, implying an interaction of succinate dehydrogenase with a lipoprotein environment. This naturally led subsequent investigators to believe that the interaction between succinate dehydrogenase and the next component of the electron-transfer chain was hydrophobic in nature and to disregard the participation of ionic interaction. Yet interaction between QPs and succinate dehydrogenase is pH and buffer system dependent, indicating that some sort of ionic interaction must be involved in the interaction between these two components [95]. Phosphate buffer is more effective than Tris buffer in the reconstitution of succinate-Q reductase from succinate dehydrogenase and QPs. Among the Tris buffers, the efficiency in reconstitution has the following order: Tris-acetate > Tris-phosphate > TrisHC1. The poor reconstitution in Tris-HC1 buffer was taken as an indication that a cationic group may be involved in the interaction between the two components. It is reasonable to assume that at least one cationic group is present near the active site of lowKm ferricyanide reductase, so that the enzyme can efficiently bind the negatively charged electron acceptor, ferricyanide. The presence of C1- neutralized the cationic groups, decreasing the efficiency of interaction between succinate dehydrogenase and QPs. Alkylation of primary amino groups in succinate dehydrogenase by fluorescamine in the absence of succinate inactivated the low-Km ferricyanide reductase activity and decreased the reconstitutive efficiency. However,

112 when QPs was treated with fluorescamine, no apparent effect on reconstitutive activity was observed, indicating that the essential amino group involved in reconstitution is located on succinate dehydrogenase. The possibility of participation of the guanidino or imidazo groups of arginine or histidine residues in either QPs or succinate dehydrogenase remains to be experimentally verified. Although no evidence is available as to which subunit of succinate dehydrogenase is in direct contact with QPs, it is generally believed that QPs binds in the vicinity of the S-3 ironsulfur cluster [96]. With the currently available evidence, one can formulate the following picture of succinate-Q reductase in the inner membrane of mitochondria: succinate dehydrogenase and QPs form a three-subunit complex through protein-protein interaction, with the hydrophobic portion of QPs embedded in the matrix side of the membrane. Succinate dehydrogenase itself has contact only with the polar groups of phospholipid molecules and does not penetrate deeply into the hydrophobic region of the membrane. Such an arrangement would explain the solubility of succinate dehydrogenase in aqueous solution in the absence of detergent, and the ease with which it is released from the membrane under slightly alkaline conditions. The fact that the S-3 iron-sulfur cluster is located in the hydrophobic region [96] and that succinate dehydrogenase can be released from QPs by chaotropic reagents [97] indicates that a hydrophobic interaction between QPs and succinate dehydrogenase also plays an important role [98]. Both hydrophobic and ionic interactions [99] are apparently involved in the formation of succinate-Q reductase from succinate dehydrogenase and QPs, and disregard of either one leads to false conclusions about the nature of the interaction. Further studies on the protein-protein interaction between these two components by chemical modification and physical studies should provide better information not only on the specific interaction between succinate dehydrogenase and QPs but also about interactions between peripheral and integral membrane proteins in general.

HC. QPc II1C-1. Identification Complex Iii and other highly purified preparations

of ubiquinol-cytochrome c reductase (see Table 111) from mitochondria contain significant amounts of Q which cannot easily be removed, yet these preparations are not active toward reconstitution with succinate dehydrogenase to form succinate-Q reductase. The specific binding of this Q to a protein or proteins was evident from the detection of a ubisemiquinone radical [68,69] under controlled reduction of the cytochrome b-Cl-llI complex using a fumarate/succinate mixture at a fixed ratio as substrate in the presence of a catalytic amount of succinate-Q reductase or by adjusting the redox state of the enzyme with redox mediators. A ubisemiquinone radical concentration as high as 60% of the total Q present in the cytochrome b-c~-III complex has been detected at pH 9.0. The detection of the ubisemiquinone radical in the cytochrome b-c~-lll complex was attributed to the presence of a Q-binding protein by the following reasoning. In the free state o1" in phospholipid vesicles, the equilibrium for the following equation is very much toward the left. QIt2 + Q ~" 2QH' The Keq value is very small, and one would not expect to detect a ubisenriquinone radical at neutral or slightly alkaline pH. Unless Q is tightly bound to a protein, no ubisemiquinone radical should be detected. A mixture of Q and QHz in phospholipid vesicles will show the ubisemiquinone radical only above ptt 12. The ubisemiquinone radical detectable in the cytochrome b-c~-Ill complex decreased drastically when the pH was higher than 9. In fact, at pH 10 very little ubisemiquinone radical was detected, indicating that tile stability of the ubisemiquinone radical in the cytochrome b-Cl-lll complex is mainly due to protein rather than phospholipids. Digestion of the cytochrome b-el-Ill complex with trypsin abolished the ability to form the ubisemiquinone radical. However, protein in the absence of ptaospholipids cannot stabilize the ubisemiquinone radical. Following addition of Q to the Q- and phospholipid-depleted cytochrome b-cx-lll complex, no ubisemiquinone radical was detected until phospholipids were added. This indicates that phospholipids may not be directly involved in binding but may act indirectly by affecting the conformation of the protein [851 which is responsible for Q-protein (QPc) interaction. This close rela-

113 tionship between QPc and phospholipids may be the reason why QPc has not yet been purified beyond the stage of the cytochrome b-cl-III complex. The isolation of active integral lipoproteins from the mitochondrial respiratory chain is still a challenge to investigators. All the active components of the electron-transport chain isolated so far are peripheral proteins. Therefore, it may be some time before an active, pure preparation of QPc can be made. An indirect approach was taken to identify QPc using a radioactive photoaffinity-labeled Q analogue [101] to label specifically QPc in the cytochrome b-el-III complex. The proteins which were covalently linked to the Q analogue were then identified by radioactivity counting after SDS-polyacrylamide gel electrophoresis. The key steps in the synthesis of the radioactive photoaffinity labeled Q analogue, QoCloNAPA, have been reviewed recently [100]. Since QoC~oNAPA is not identical to Q~o, its affinity to QPc is not yet known. To ensure that it could be bound by the real QPc, the biological function of QoCloNAPA was tested in reconstitution. The Qlo and phospholipids were removed from the cytochrome b-e]-III complex by a procedure [85] similar to that reported for the removal of Q and phospholipids from succinate-cytochrome c reductase [4]. The depleted complex was then reacted with QoCloNAPA followed with replenishment of phos-

2°° I

I

i

I

Iltl

1

i

i

pholipids. The enzymatic activity was assayed through reconstitution with Complex II or with reconstituted succinate-Q reductase (succinate dehydrogenase + QPs) to form succinate-cytochrome c reductase. The reconstitutive activity of QoCloNAPA was found to be satisfactory; in fact, QoC10NAPA has an activity as high as that of Q2. When 14C-labeled QoC10NAPA was mixed with freshly prepared phospholipid- and Q-depleted cytochrome b-c~-III complex [85] and illuminated for 20 min with a 300 W spotlight with a Turner filter, No. 110-811 (7-60), at approx. 5°C, QoC10NAPA became covalently linked to the protein. The distribution of radioactivity among the proteins of the complex was determined by SDS-polyacrylamide gel electrophoresis. Fig. 5 shows the proteins and 14C-activity distribution among the proteins of the cytochrome b-crIII complex in the gel column. The 14C activity on each band was counted from a pool of slices collected from 23 polyacrylamide gel columns (0.5 × 8 cm). It should be mentioned that the result given in Fig. 4 was obtained only when the gel columns were destained extensively after staining. Incomplete destaining of the gel gives false results because QoC10NAPA

1oo

i

! l

8o

~ 0

q

6o

D.4

150

~ 4o

{L

¢

b

0

>_E;_ 100

D

(J

t~

C.)

50

0 0

0.2

0,4 0.6

0,8

1.0

1.2

1.4

R ELATIVE MOBILITY Fig. 4. Distribution of 14 C radioactivity a m o n g the proteins o f the c y t o c h r o m e b-c 1 -III complex. F r o m Ref. 101.

O

10 Time (min)

20

I-0.2

Fig. 5. Comparison of ubisemiquinone radical f o r m a t i o n and the reduction o f c y t o c h r o m e s b and c 1. The reduction o f b c y t o c h r o m e s (curve b) and c y t o c h r o m e cl (curve c) was followed in an Aminco DW-2 spectrophotometer in cuvette with a 2 m m light path. C y t o c h r o m e b was monitored at 561 n m and c y t o c h r o m e cl at 552 n m . The EPR m e a s u r e m e n t s (curve q) were performed at room temperature with detailed settings given in Ref. 69. Protein concentration used was 20 mg/ml.

114 itself has a mobility in electrophoresis similar to that of the small molecular weight subunits. It is clear from Fig. 4 that QoCIoNAPA is preferentially bound to the proteins with molecular weights of 37 000 and 17 000. These two proteins were previously identified as cytochrome b proteins [72]. Since the 14C activity was almost equal in the two proteins it is difficult to determine whether one or both are responsible for QPc. |t is possible, but not probable, that a protein which is sandwiched between the two cytochrome b proteins could be responsible for QPc because the photoaffinity group is on the isoprenyl side chain, and the ability of QPc to stabilize the ubisemiquinone radical indicates that the binding of Q to protein may be involved more with the benzoquinone ring than the isoprenyl side chain. Abnormal mobilities of the cytochrome b proteins in SDS-polyacrylamide gel electrophoresis and their poor dye affinity have caused some confusion on the assignment of the subunits of ubiquinol-cytochrome c reductase among the investigators [105]. The cytochrome b with a molecular weight of 37 000 can only be observed when particular care has been taken during preparation of the sample for electrophoresis [105]. Detergents used in preparation, such as deoxycholate, cholate or Triton X-100, should be removed completely, and the digestion reagents, SDS and /3-mercaptoethanol, should be freshly made. After treatment with SDS and/3-mercaptoethanol, the sample should be subjected to electrophoresis without prolonged storage. Without these precautions, the subunit of molecular weight 37 000 may move faster and merge with the band of molecular weight 30 000 or move more slowly and overlap with the second protein band of molecular weight 50 000. The cytochrome b protein of molecular weight 17 000 sometimes moves slower and merges with the Rieske ironsulfur protein. Perhaps due to this abnormal behavior in electrophoresis and to tire lack of an active isolated preparation, the molecular properties of cytochrome b are very obscure. The idea of cytochrome b with a molecular weight of 30 000 [ 102-104], possibly functioning as a dimer [106] in ubiquinol-cytochrome c reductase, has been very popular, and much genetic work also supports that protein of molecular weight 30 000 as one of the cytochrome b proteins. The most purified ubiquinol-cytochrome c reductase either from beef heart mitochondria [57] or

from yeast [107] has a cytochromeb content of about 10 nmol/mg protein and a cytochrome Cl content of 5 - 6 nmol/mg protein. If these two cytochromes have a molecular weight of 30 000, then that particular band in an SDS-polyacrylamide gel should contain 50% of tile total protein. This is far from the actual observations, either from densitometric tracing of a stained SDS-polyacrylamide gel electropboresis column, or from direct analysis of the total amino acid content of each band after staining and destaining with dye [53]. Although quantitative data of the protein distribution of cytochrome b-Cl-II1 complex other than densitometric tracing are still lacking, the protein distribution among the seven major protein bands of the soluble cytochrome b-c~-complex has been carefully examined [53] and is given in Table V. Bands III and IV together comprise 25% of the total protein in soluble cytochrome b-c1 complex. It is unlikely if not impossible that this protein(s) can accommodate all the heine b and heine c. A second or even third protein must also be responsible for cytochrome b. Evidence for the protein with a molecular weight of 17 000 (band V1) as cytochrome b has been shown [72] but is less conclusive because the ratio of the heine content to protein in the isolated form of this particular cytochrome b is less than stoichiometric. Recent sequence study of the mitochondrial genome [112] indicates that the cytochrome b protein has a molecular weight of 42 700. This does not exclude the possible existence of multiple forms of cytochromesb, as posttranslational cleavage could occur before the heme b is inserted into the protein. The existence of a cytochrome b with a molecular weight larger than 42 700 is not likely. In view of the diversity in the value for the molecular weight of the subunits, it would be beneficial if investigators in tire field could use some standard reference to report the molecular weights of subunit proteins. Since protein mobility differs from gel system to gel system, we suggest the use of relative mobility to report subunit position. The use of tracking dye as reference is unsatisfactory because it is too diffuse to give a definite reference. Perhaps the use of cytochrome c or some other small protein as an internal reference would be acceptable. For instance, R~v° and RSc m represent the mobility relative to tyrochrome c in the gel systems of Weber and Osborn [108] and Swank and Munkres [109] respectively:

115 TABLE V PROTEIN DISTRIBUTION OF SOLUBLE CYTOCHROME b-c 1 COMPLEX Band No. e

R~VOa

Rsm a

I II III IV V VI VII

0.36 0.39 0.48 0.55 0.66 0.84 1.04

0.14 0.18 0.32 0.36 0.47 1.22 0.95

(VIII)

1.15

1.48 1.65

Cytochrome c

1.00

1.00

Mol. wt. (X10-3) b

53 50 37 30 28 17 <15

% protein

Redox components

By total amino acid c

By dye density d

22.9 21.9 10.0 15.1 9.2 5.5 15.8

17 17 5 18 14 9 18

xf x cytochrome b (QPc) cytochrome cl, x FeS protein cytochrome b (QPc) QPs, x x

12.5

-

-

c

a Under the conditions of Weber and Osborn [108] or Swank and Munkres [109] using cytochrome c as internal reference. b The molar weight obtained by Yu et al. [53] is generally slightly higher than that reported by other investigators [110,l 11 ]. c Total amino acid recovered from the hydrolysate of stained protein bands sliced from polyacrylamide gel column. d Densitometric tracing at 600 nm. e Band number based on increasing mobility on gel system of Weber and Osborn [ 108]. f Unknown component. with this notation the proteins o f QPc would have relative mobilities R w° o f 0.475 and 0.841 and R sm o f 0.32 and 1.22 (see Table V). This indicates clearly that the smaller protein for QPc has a faster mobility than cytochrome c in the gel system o f Swank and Munkres. Gel system-dependent mobility o f Complex III proteins was first observed by Capaldi [113].

gen temperatures, respectively. Identical EPR spectra have also been observed in succinate-cytochrome c reductase [51 ] and submitochondrial particles [114]. If the redox potential was adjusted to about 80 mV, either using a fumarate/succinate mixture as substrate or by redox dyes, the signal was stable for hours even at room temperature, lasting, in fact, until the protein became denatured.

HC-2. Properties o f QPc

b. Formation of the ubisemiquinone radical and the reduction of cytochromes b and cl. The reduction o f

Since a method for the isolation o f QPc has not yet been developed, the purest preparation available is purified ubiquinol-cytochromec reductase (the cytochrome b-Cl-III complex) [57]. The molecular properties of QPc are not yet clear at the protein chemistry level, but through chemical and physical measurements, many of the properties o f QPc (i.e., the Q-QPc e n t i t y ) h a v e been observed [69,100,105]. a. LTR spectra. EPR spectra o f the ubisemiquinone radical in QPc were observed when highly purified cytochrome b-cl-III complex was reduced by succinate in the presence o f a catalytic amount of reconstituted succinate-Q reductase or Complex II, either at room temperature or at liquid nitrogen temperature. The signal has a g value of 2.0045 [100] and line widths o f 8 and 9 G at room and liquid nitro-

c y t o c h r o m e s b and cl in the cytochrome b-c~-III complex can be detected by a spectrophotometer when a catalytic amount of succinate-Q reductase is used in the presence of succinate. As indicated in Fig. 5, a small portion o f cytochrome b was reduced before the reduction of cytochrome Cl commenced, and was reoxidized as cytochrome cl reduction proceeded. The reduction of cytochrome b commenced as soon as all the cytochrome c~ was reduced. The formation of the ubisemiquinone radical, as detected by EPR measurement, was concurrent with the reduction of cytochrome b but reached its maximum and decreased before the cytochrome b was completely reduced, indicating that the ubisemiquinone radical formed was further reduced to quinol. This result sug-

116 gests that in the cytochrome b-c I region, Q acts as a one-electron redox system, QH'/Q or Q-/Q, and that the fully reduced form, QH2, is probably not involved. These results, however, did not distinguish whether QH'/Q is acting parallel to or sequentially with cytochrome bZ+/cytochrome b 3+. c. Effect of antimycin A on QPc. Antimycin A can inhibit electron transfer at the Site II region quantitatively, with the amount of inhibitor equal to that o f cytochrome c~ [115]. When antimycin A was added to a system in which the ubisemiquinone radical was already formed, the signal diminished immediately [69] but very little oxidation of c y t o c h r o m e b occurred. If the inhibitor was added prior to the addition of substrate, no ubisemiquinone radical was detected but the rate of reduction of cytochrome b increased slightly. The effect o f antimycin A on QPc is oxygen indepedent. One plausible explanation for antimycin A inhibition is that antimycin A dislodges Q from its binding site on QPc. QPc can therefore no longer stabilize the ubisemiquinone radical, which dismutates to become reduced Q and Q. Since the photoaffinity label study o f QoCIoNAPA showed that the labeling pattern of the cytochrome b-ca-Ill complex was not significantly different in the presence or absence of antimycin A, the effect of antimycin A on Q binding may not be through direct competition for the Q-binding site. The inhibition could be exerted by a protein conformational change, a suggestion which is supported by the observed sigmoidal curve o f tile titration of antimycin A [60,I 16] and the low efficiency of reactivation o f antimycin A-inhibited enzyme by addition o f exogenous Q [ 1171.

d. EJfect of thenoyltrifluoroacetone

on QPc.

Thenoyltrifluoroacetone, an inhibitor of succinate-Q reductase, showed a surprisingly significant effect on the ubisemiquinone radical of the cytochrome b-%III complex, especially when thenoyltrifluoroacetone was added after the formation of the ubisemiquinone radical. The effect o f thenoyltrifluoroacetone on the ubisemiquinone radical is completely different from that of antimycin A, and is oxygen dependent. Addition of thenoyltrifluoroacetone to a system in which the ubisemiquinone radical was already formed immediately diminished the amount o f the ubisemiquinone radical and caused oxidation of cytochrome b (see Fig. 6). Cytochrome b was reduced and tile ubisemiquinone radical reappeared after the oxygen in

6 9 •~-

b 2

100

'

i TTFA'

l

8o i-

6C' }

<

<

ac ~5

0

o

, lO

~ 2o

, 3o Time

40

',',

"

~ ~o

~o

a _8 .jo~

(rain)

Fig. 6. Effect of thenoyltrifluoroacetone (TTFA) on the ubisemiquinone radical signal and the reduction of cytochrome b. (¢, o) EPR signal of the ubisemiquinone radical; (×-×) reduction of cytochrome b monitored at 561 nm in difference spectra using 2-ram cuvettes. At the point marked by the first arrow, thenoyltrifluoroacetone was added. The second arrow shows the time when the sample was shaken in air. Thenoyltrifluoroacetone concentration was 2.5 raM. The complex concentration was 20 mg/ml and other conditions are the same as those described in Fig. 1 of Ref. 69. the system was exhausted, but tile rate of radical formarion and c y t o c h r o m e b reduction was much slower: less than 10% of that seen in the absence of thenoyltrifluoroacetone. The slower rate of reduction may be due to the incomplete inhibition o f succinate-Q reductase, which was used as the electrondonating system for the cytochrome b-cl-lll complex. The oxygen-dependent radical formation and tile cycle of decrease in tile amount o f tile ubisemiquinone radical can be repeated several times with a subsequent decrease in tile extent o f ubisemiquinone radical formation and cytochrome b reduction. The oxygen-dependent effect on ubisemiquinone radical formation was explained as being due to the fact that thenoyltrifluoroacetone serves as a scavenger for the ubisemiquinone radical. The electron was shuttled from reduced cytochrome b to molecular oxygen through tile ubisemiquinone radical. This explanation is supported by tile observation of Nelson et al. [1 l 8] that thenoyltrifluoroacetone acts as an antagonist to Q in submitochondrial particles. e. Thermodynamic properties of QPc. The rate of formation of the ubisemiquinone radical in the cytochrome b-c~-IlI complex depends on the amount of electron-donating enzyme (succinate-Q reductase) used [120]. At a given catalytic amount of succi-

117 nate-Q reductase, the rate of formation o f ubisemiquinone and its stability depend on the potential o f the fumarate/succinate couple as indicated in Fig. 7. An increase in concentration of fumarate in the fumarate/succinate mixture caused a slight increase in the maximal radical formation but decreased the rate o f formation. When a lower ratio o f fumarate to succinate was used as substrate, the ubisemiquinone radical was formed rapidly, reached its maximal concentration and then decreased to a constant level which was controlled by the redox potential o f the system. The anaerobic redox titration [120] o f the cytochrome b-cl-llI complex in the presence o f redox mediators showed that maximal formation o f the ubisemiquinone radical occurred at a redox potential o f about 76 mV. A typical redox titration profile of a divalent redox system in which an intermediate is the EPR-detectable species is observed. The reductive titration was made with NADH and a catalytic amount o f Type II NADH dehydrogenase [121] and the oxidative titration was made with ferricyanide. Under the conditions used (pH 8.0) the maximal formation of ubisemiquinone was estimated to be about 3 0 4 0 % of the total Q found in the cytochrome b-clIII complex. This would correspond to a stability constant for the ubisemiquinone radical o f between 0.7 and 1.8. Rather close midpoint potentials (El and E2) for the two partial reactions o f Q are expected El

(Q ~ Q H "

tonated neutral radical or as a deprotonated anionic radical, depending on the pH of the solution. It is generally believed that the pKa for QH" is about pH 6 or lower [122]. Since the system described is at pH 8.0, the ubisemiquinone radical we observed is probably an anionic radical. The identity of the species o f ubisemiquinone radicals which function in native electron transfer remains to be verified experimentally. The pH profile of electron-transfer activity and ubisemiquinone radical formation showed significant overlap when the pH of the solution was higher than 7 as indicated in Fig. 8. Although formation of the ubisemiquinone radical at room temperature was not detected under the given conditions until the pH o f the solution was higher than 7, some enzymatic activity was observed below pH 7.0. This could be explained simply by the instability o f the ubisemiquinone radical at lower pH values.

}

E2

~QH2).

This is indicated in the rather narrowed bell-shaped titration curve. A more detailed discussion on the midpoint potential and stability constant o f Q is available [45]. Study of the power saturation behavior of the ubisemiquinone signal o f the cytochrome b-cl-III complex indicated that the signal started to saturate at 1 mW at liquid nitrogen temperature, whereas at room temperature the signal showed no saturation up to 20 mW. These results suggest that the ubisemiquinone radical in the complex is in a somewhat isolated environment. More detailed study o f ubisemiquinone radical behavior is needed in view o f the close relationship between QPc and cytochrome b proteins. f. The nature of the ubisemiquinone radical The ubisemiquinone radical can exist either as a pro-

Fig. 7. Time dependence of the EPR signal of the cytochrome b-cl-III complex by the addition of succinate/ fumarate mixtures. Succinate/fumarate mixtures at different molar ratios were added to the cytochrome b-cl-III complex in the presence of catalytic amounts of QPs and succinate dehydrogenase. The formation of the ubisemiquinone radical was followed by the signal change at g 2.00. Succinate-tofumarate ratios used were: A, 1 : 1 ; B, 1 : 5 ; C, 1 : 20; and D, 1 : 80. The total concentration of succinate and fumarate was 200 raM. The cytochrome b-cl-llI complex concentration was 16.3 mg/ml. The EPR signal was monitored at a peak position with a fixed magnetic field. The instrument settings were: microwave frequency, 9503 Gttz; modulation frequency, 100 kHz; modulation amplitude, 6.3 G; microwave power, 20 roW; and temperature, 23°(2; pH 8.0. The zero-time positions of these curves were arbitrarily shifted. These data were presented in the 6th International Biophys Congress, Kyoto, 1978 [119].

118 i

,

i

i

B___:/

100

i

'~

60

~ ¸

k.x•

v

-

-

i /

I / j it

hi

40

D lOG

<

0 6

7

8

l

t

9

10

E

pH

Fig. 8. Effects of pH on the formation of the ubisemiquinone radical and the enzymatic activity of the cytochrome b-c 1-III complex. The maximal EPR signal was plotted against pH (e e). The ubiquinol-cytochrome c reductase activity (o o) was measured from the same sample. The enzyme was reduced by a succinate/fumarate (1 : 80) mixture. Succinate and fumarate concentrations were 0.9 and 72 mM, respectively. The concentration of enzyme used in EPR measurement was 13 mg/ml, in which cytochrome b was 114 ,aM, cytochrome c 1 64 ~zM,and Q 30 ,uM. The pH was maintained by a 50 mM Tris-HC1system. EPR instrument settings are given in Ref. 99.

g. The nature o f Q binding on QPc. The detection of the ubisemiquinone radical in the cytochrome b-ca-IIl complex is clearly an indication that Q is bound to protein, and that the binding must involve the benzoquinone ring. Whether or not the side chain of the Q molecule also participates in specific binding is a question of interest. When a spin-labeled Q analogue, was reacted with the cytochrome b-c1-III complex, the spin label [87], which was located on the side chain, was completely immobilized in the absence of phospholipids, indicating that it was tightly bound to the protein (see Fig. 9). However, when phospholipids were replenished in the system the spin label showed some degree of mobility, indicating a more fluid environment at the Q-binding site. Since phospholipids are needed for formation of the ubisemiquinone radical, and no change in the photoaffinity labeling pattern in the presence and absence of phospholipids in the cytochrome b-c,-III complex was observed, it is certain that Q is not released in the free form in the presence

QoCloTMPOC,

Fig. 9. EPR spectra of spin-labeled Q derivative, QoCtoTMPOC, in different environments. The spectra were taken on a Varian E4 EPR spectrophotometer at room temperature, with the instrument settings as follows: field modulation frequency, 100 kHz; microwave power, 10 roW; microwave frequency, 9.5 GHz; modulation amplitude, 1 G; time constant, 0.3 s; scan rate, 12.5 G/min. Spectrum A represents QoCloTMPOC in lipid-depleted cytochrome b-cl-Ill complex, 13 mg/ml; B, same as A plus 1% SDS; C, same as A plus phospholipids, asolectin, 0.2 mg/mg protein; D, in 25~ sodium chelate. Spectra A D are in 50 mM sodium/potassium phosphate buffer, pH 7.4, containing 20~ glycerol. Spectrum E shows QoCloTMPOC dissolved in 95% ethanol. The concentrations of QoC]oTMPOC used and the receiver gain (RG) settings are: 140 ,uM, RG 2 . 103; 120 azM,RG 8 • 102; 140 ~M, RG2 • 103; 93 ,uM, RG 8 -102;87 ,2M, RG 4. 102; for spectra A to E, respectively. From Ref. 81.

of phospholipids. It is highly likely that the mobile environment, where the spin label resided, resulted from a conformational change of the protein caused by phospholipid binding rather than an environment provided solely by phospholipids [85]. The sensitivity of binding of Q to the protein's conformation is also supported by the fact that antimycin A caused a decrease in the amount of the already formed ubisemiquinone radical.

liD. QPn QPn is a Q-binding protein functioning in NADH-Q reductase [70]. Complex I, as prepared, contains 3- 4 nmol Q/rag

119 protein. Compared to other electron-transfer components, such as FMN or iron-sulfur clusters, Q is in a molar excess. Whether all the Q is bound to a specific protein or exists partially in free form is not clear. However, when a limited amount of NADH was added to Complex I (e.g., 140/aM NADH added to a sample with 40 mg protein/ml), the ubisemiquinone radical was detected by EPR measurement and the signal remained constant for about 15 min before slowly decreasing [70]. Although the signal characteristic was reported to be very similar to that obtained in the cytochrome b-cl-III complex, contamination of Complex I with the cytochrome b-cl-III complex was ruled out by the chemical analysis of redox components of the cytochrome b-ca region, and the behavior of the EPR signal toward inhibitor treatment. The signal was not sensitive to antimycin A treatment but was diminished upon the addition of rotenone. These observed facts clearly show that the Q-binding protein in Complex I (QPn) is different from QPc. The EPR signal of QPn cannot be derived from ravin because the line width of 9 - 1 0 G of the ubisemiquinone radical signal observed is different from that of the ravin radical. QPn is also distinct from QPs, as Complex I is inactive toward reconstitution with soluble succinate dehydrogenase to form succinate-Q reductase. The ability of Complex I to form the ubisemiquinone radical parallels the rotenone-sensitive NADH-Q reductase activity. Both enzymatic activity and the ability to form the ubisemiquinone radical were sensitive to proteolytic enzyme digestion and urea treatment [123].

III. Q-binding proteins in photosynthetic systems The involvement of quinone in photosynthetic systems of green plants or photosynthetic bacteria is also established [5-8] as it has been for mitochondrial systems. The quinones and the length of isoprenyl side chains in photosynthetic systems are more diverse than those in mitochondria. For example, plastoquinone is involved in green plants [124-127], ubiquinone in purple photosynthetic bacteria, and menaquinone in green photosynthetic bacteria [5]. Detection of a semiquinone anion upon the illumination of isolated reaction centers, chromatophores, or chloroplasts indicates a specific protein-quinone inter-

action or binding. Quinones which participate in light energy harvesting and electron transfer are bound to specific proteins [111,113], analogous to the mitochrondrial Q-binding proteins.

IliA. Q-binding proteins in photosynthetic bacteria As in the mitochondrial inner membrane, bacterial chromatophores contain, on a molar basis, excess quinone [9]. Quinones in the chromatophores can be classified into four populations [5] : primary electron acceptor of the reaction center; secondary acceptor of the reaction center; a component, Z, active in the cytochrome b-c2 region of the electron-transport chain; and a large pool of unbound quinone. Several studies on primary and secondary electron acceptors have been done and Z is currently undergoing extensive study in many laboratories.

IliA-1. Q-binding proteins in the reaction center The reaction center of photosynthesis is a protein complex which exhibits a light-induced electrontransfer reaction used for energy conversion [6]. In this section, we will be concerned with the reaction center only as that complex performing the primary photochemical reaction. Many bacterial reaction center preparations are available, the best characterized of which are those obtained from Rhodopseudomonas sphaeroides mutant R-26 [129], or Rhodospirillure rubrum [130]. The reaction center preparation of Rps. sphaeroides R-26 as prepared with detergent (LDAO) has a molecular weight of 95 000. Three protein subunits with molecular weights of 21 000, 24 000 and 28 000 in a 1 : 1 : 1 stoichiometry have been detected in SDS-polyacrylamide gel electrophoresis [129,131]. These subunits were designated as L, M and H for light, medium and heavy, respective, according to their molecular weight. A reaction center preparation with only two subunits, L and M, has been obtained from Rps. sphaeroides as well as from R. rubrum [129]. The reaction center prepared from Rps. sphaeroides R-26 contains four bacteriochlorophylls, two bacteropheophytin, two ubiquinones and one iron per reaction center. The reaction center-bound Q or Fe can be selectively removed and photochemical activity assays for such Q- or Fe-depleted preparations are available [13]. Q was removed by a treatment with lauryl-

120 dimethylamine N-oxide and o-phenanthroline. The amount of Q removed can be altered by adjusting the concentration ofo-phenanthroline and detergent. The conditions required for the removal of Q demonstrate that one of the two Q molecules is bound more tightly than the other. Photochemical activity assays show that the tightly bound Q (designated QI) acts as a primary electron acceptor. QI is distinct from the second Q (QII), which is bound to the reaction center and serves as a secondary electron acceptor. Ql and Qn are believed to act in series with tire iron located between them. In the reaction center, Q showed only a broad EPR at g 1.8 [129,132] which was believed to be due to the magnetic coupling between both Ql and QII and iron (Fe2+). After removal of iron from the reaction center, a typical Q free radical was observed [ 133,134]. Evidence for the involvement of Q as a one-electron acceptor during the photochemical reaction in the reaction center came from lightinduced absorbance changes in the reaction center by illumination in the presence of a weak reductant observed by Slooten [135] as well as by the flash experiments of Vermeglio [136] and Wraight [132]. A typical differences spectrum of semiquinone versus quinone was observed. Although the semiquinone spectra for both QI and QH have been observed, binding behavior differs greatly between QI and QH. Binding for QI is less specific than that for QH, as various types of quinone and lengths of side chains can be used, whereas the binding site for Qll accepts only the native ubiquinone-50. The protein responsible for primary quinone (Q1) binding has been identified as the M subunit of the reaction center through photoaffinity labeling [137]. The Qi-depleted reaction center was first reconstituted with all-labeled 2-azidoanthraquinone and then illuminated with ultraviolet light to form a covalent attachment between the quinone and the protein at the quinone-binding site. The radioactivity was located specifically on the M subunit after the treated sample was subjected to SDS-polyacrylamide gel electrophoresis. Although the photoaffinity labeled quinone analogue used is quite different from Q, the capability of the analogue in the photochemical reaction and the specific distribution pattern of radioactivity among the three subunits confirmed that the M subunit is indeed responsible for the primary Q-binding site. No comparable information for Qu is

available although indirect evidence suggests that the H subunit may be responsible for the secondary Q-binding site [6]. The amino acid compositions of the Ql-binding protein and the possible QH-binding protein are available [6]. Recently, a protein with a molecular weight of 11 000 was isolated from the chromatophores of R. rubrum [138] by molecular sieve column chromatography in the presence of sodium cholate and deoxycholate. Tire isolated protein contains l m o t Q/tool protein and stimulates the photoactivity of the 'Q-deficient' reaction center.

ILIA-2. Q-binding proteins in the electron-transport chain of the cytoehrome b-c2 complex The photosynthetic bacterial electron-transport chain resembles the mitochondrial cytochrome b-Cl complex, containing cytochromes b, cytochrome c2, Rieske iron-sulfur protein [139], Z (Q) [140,141] and phospholipids. The electron-transfer reaction in the cytochrome b-c2 region is sensitive to antimycin A and to many other quinone analogues such as 2,5-dibrom o-3-methyl-6-hydroxy-4,7-dioxobenzothiazole or 5-{n-undecyl)-6-hydroxy-4,7-dioxoben zothiazole. Z is probably analogous to the Q-QPc entity of mitochondria. Extraction and reconstitution studies indicate that Z is a Q-protein complex, with a two-electron two-proton redox couple (/:'m,7 = 155 mV, n = 2). Component Z may act as an oxidant [142] or a reductant ofcytochrome b [141], or both [143]. Recent advances have shown that Z is a specific Q molecule which reduces Rieske iron-sulfur protein, and an interaction between Z and iron-sulfur protein has been implied [ 124,144]. ILIA-3. Free Q in the bacterial photosynthetic chromatophore There are 25 mol Q/tool reaction center in the isolated chromatophore [128]. Only 3 - 4 tool of Q were found in protein-bound form and were needed for fully active, complete electron transfer in single turnover flashes [128]. Most of Q in the chromatophore does not participate directly in electron transfer and light energy-harvesting activity. The unbound Q is easily extracted by organic solvents. This portion of Q, although not bound to a protein, is capable of a redox equilibrium with the bound Q such as Qn in the reaction center. It probably functions as a storage

121 reservoir in the light energy-harvesting event to ensure that the electron acceptors, QI and QII, are in a state of readiness. The Em, 7 value for this Q pool is 90 mV, which is considerably higher than that of Qt and QtI. No physical exchange occurs between the tightly bound Q and the Q pool [145], according to a 14Clabeled Q experiment. Exchange between the Q pool and bound Q involves physical detachment of the bound Q from its apoprotein, whereas a redox equilibrium between bound Q and free Q requires only physical contact between them. In this scheme, the Q-protein complex exists like other electron-transfer components in which the prosthetic groups are permanently associated with the apoprotein.

IIIB. Q-binding proteins in the photosynthetic apparatus of green plants The photosynthetic apparatus of green plants is much more complex than that of photosynthetic bacteria. It is composed of two photosystems; PS I, which generates NADPH for the reduction of CO2 to carbohydrates, and PS II, which oxidizes water and generates ATP. Each photosystem has its own reaction centers and electron-transport components [146]. While the involvement of quinone in PSI is not certain, its participation in PS II is well established [124,146]. The participation of quinone is analogous to the bacterial photosynthetic apparatus except that PQ is used instead of Q in PS II. Native PS II contains eight to ten polypeptides [7] whereas the reaction center preparation for PS II [146] is composed of only three polypeptides with molecular weights of 43 000, 27 000 and 6500. The small molecular weight polypeptide is believed to be associated with cytochrome b-559. It would be of interest to determine whether or not this particular protein is also associated with PQ. The evidence for involvement of PQ in PS II also comes from studies of the extraction of PS II with organic solvent (0.2% methanol in hexane) [147] followed by reconstitution. An entity called X-320 by Stiehl and Witt [ 148] is believed to be the primary electron acceptor in PS II, and was recently identified as the plastosemiquinone anion [149,150]. The slight shift in the peak position and the difference in the extinction coefficient between X-320 and that of PQ in methanol solution were believed to be due to the binding of

PQ (X-320) to the lipoprotein membrane [151,152]. The relative amounts of primary acceptor and chlorophyll molecules ( 1 : 3 0 0 ) also suggest that the primary acceptor PQ is bound to the protein [150,151 ]. PQ acts as the primary, one-electron acceptor (PQH'/ PQ). Since its semiquinone form is stable enough to be detected, the binding between PQ and protein must be very tight. A specific Q-binding protein may be responsible. Although the semiquinone form of PQ is established in PS II, the search for a semiquinone EPR signal has not been successful. The same explanation as that given for the broadened EPR signal in the purple photosynthetic bacterial reaction center [13] was given for this lack of an EPR signal. More data to support the possible involvement of nonheme iron in the primary electron acceptor have been published [147]. Recently, a new light-induced EPR signal (g 2.004, AH = 8 G) was observed after removal of iron, similar to the phenomenon observed in the bacterial reaction center [133,134], suggesting that PQ does indeed complex with iron. In a role analogous to that in the bacterial reaction center, PQ may serve as the secondary electron acceptor. This belief is based on the light-induced binary oscillation of the fluorescence change in flash experiments [153] and the oscillatory behavior of the absorption change at 320 nm. The absorption peak at 320 nm was observed at odd flashes. Similar oscillatory behavior was observed in the bacterial reaction center [132]. The involvement of PQ as the secondary electron acceptor has been established by chemical analysis, and removal and reconstitution experiments. A specific protein With a molecular weight of 32000 has been determined indirectly through photoaffinity studies of quinone antagonists [154] to be involved in the binding of this secondary quinone. Participation of a PQ-binding protein in the electron-transport chain of the chloroplast is less clear, even though the electron-transfer sequence involving PQ after the primary and secondary acceptors has been reported. It is generally believed that the PQ involved belongs to pool PQ rather than to a specific PQ-protein complex. Since the electron-transport components cytochrome b-559, cytochrome f a n d PQ act as counterparts of the mitochondrial cytochrome b-cl complex, it is not difficult to predict that a QPctype protein may be involved. Further studies on the isolated chloroplast fragment enriched in the cyto-

122 chrome b-f-PQ complex could yield important information, especially on the possible detection of the semiquinone radical in the cytochrome b-f-PQ complex.

IV. Reaction mechanism of Q in the electron-transport reaction While there is no question that Q is essential to mitochondrial electron transport, or to photosynthetic reaction centers and subsequent electron transfer, opinions on the mechanism of Q in these reactions have been quite diverse. With the accumulating evidence on the existing Q-binding protein in mitochondrial electron-transport and photosynthetic systems, it is perhaps safe to say that the Q-protein complex is a true functional (redox) form of Q. Whether or not Q also participates directly in proton translocalion [19,155] is a question to be resolved experimentally. The role of Q in proton translocation in the energy-transducing membrane has been questioned recently [156,160]. Support for the direct 'loop' mechanism for proton translocation has weakened since the observation that cytochrome oxidase [156, 160] could function as a proton pump and since the challenge presented to investigators by the H+/e - ratio [42,55,158]. Excellent reviews both for and against the direct mechanism for proton translocation are available [3,19,45,46,159-161 ] and no duplication is necessary here. However, we would like to raise some points based on the data available to date, which are, in our opinion, important in the illustration of the reaction mechanism of Q in bioenergetic reactions. Detection or isolation of a Q-binding protein in the mitochondrial system suggests that only bound Q participates in electron transfer. A redox equilibrium between bound Q and free Q may exist, although the kinetic rate or turnover of the bulk Q pool is quite different from that of bound Q. The kinetic data available are mostly on free Q, and its oxidationreduction rate is quite sluggish as compared to the other redox components. Whether or not the lack of an EPR signal in isolated QPs, and the difficulty in detection of an EPR signal in the succinate-Q region can be explained by a phenomenon similar to that observed in the primary and secondary acceptors in the purple photosynthetic bacterial reaction center remains to be verified experi-

mentally. Observation of a ubisemiquinone radical upon addition of Q to Complex II [163] suggested that the Q radical EPR signal in succinate-cytochrome c reductase at the succinate-Q region may have been masked by the metal ions present. Although the results of Ohnishi and Trumpower [51] disprove this possible explanation, nevertheless, more experimental data are needed before the above possibility can be ruled out completely. Perhaps, a search for ubisemiquinone radical absorption spectra or a spectral shift resulting from the Q-protein interaction [164] will be fruitful in future investigations [86, 165,166]. The elegant fluorescence probe studies of Chance and Erecinska [167] indicate that the benzoquinone ring of Q is deeply buried, which would disprove the theory of Q as a proton translocator unless Q is guided by protein. Although an undirected movement of free Q and proton translocation has been reported [168] in the artificial membrane system, its significance in the native system is not clear. If the pathway of Q during the shuttle o f H + is dependent on the protein conformation change [169] then the proton pump mechanism is already working. It would be informative if the fluorescence probe experiment were repeated in a system in which only bound Q is involved. 111 other words, the phenomenon observed by fluorescence quenching studies must be made specific to the constituents involved. Since more free Q is present in mitochondria than bound Q, presumably the phenomenon observed is mostly due to tile free form of Q. Although the semiquinone anion spectra of QPc, QPs or QPn have not yet been established experimentally, based on the pK a measurements of the ubisemiquinone radical in organic solvents [122] and the results obtained in photosynthetic systems, it is highly likely that the Q radical in the mitochondrial system is indeed in the anionic form. The stability of QPc also indicates that Q: is probably located in that part of the protein where H + is not available. The increase in hydrophilicity of Q due to the reduction would cause the hydrophobic part of the protein, where Q is bound, to become hydrophilic in nature to accommodate Q-. Therefore, a significant rearrangement of protein molecules or rearrangement of Qmay occur during electron transfer and it is this change that would provide the energy for proton

123 translocation - through the proton pump [ 159,160, 162]. The kinetic inconsistency of proton release and electron transfer in mitochondria has provided a reason for questioning the direct mechanism of proton translocation [170]. A similar approach, using purified electron-transfer complexes [171], may provide valuable information, especially with chemically modified complexes. V. Other possible functions of Q Since the functionally active, protein-bound Q makes up only a small portion of the total Q present in the inner membrane of mitochondria or photosynthetic systems of bacterial chromatophores or chloroplasts of green plants, the role of most of the free Q is of interest. There are several possible functions for free Q in the mitochondrial system which need to be explored, such as (i) connecting the other dehydrogenases to the respiratory chain, (ii) serving as a phosphate carrier [172], and (iii) performing a regulatory function in energy metabolism. The possible regulatory effect of Q has been suggested by several investigators [173-175]. The following are some additional speculative bioenergetic regulatory effects of Q. Although the detailed molecular configuration of Q in the membrane is not yet known, it is perhaps reasonable to assume that oxidized Q and reduced Q exist in different configurations in the membrane. The effect on the membrane structure, and especially on the membrane fluidity [176] upon the change of redox state of Q should be quite marked even though no significant change was observed in the artificial membrane system. The direct correlation between electron-transport activity and membrane fluidity has been shown [ 164,177,178]. If the membrane is overenergized, the level of reduced Q is increased, and the membrane may become more rigid, assuming that reduced Q has an effect on the membrane similar to that of cholesterol [179,180]. This effect would decrease the electron-transfer activity, and thus slow down the energy input to the membrane. An increase in the reduced form of Q in the membrane could be achieved through increased contact between bound and free Q in the energized membrane [181]. Once the electron on bound ubisemiquinone is transferred to free Q it will disproportionate to Q and reduced Q.

Alternatively, the regulatory effect could be exerted through interaction between ubisemiquinone and oxygen. The electron on semiquinone may be shuttled to 02 via free Q to form a superoxide anion, which would in turn become peroxide [182] with the help of superoxide dismutase [183]. The peroxide would then diffuse to the cytosol to be acted upon by catalase to become water and oxygen. In this sequence the participation of superoxide dismutase is important, because it prevents the accumulation of toxic superoxide anions and damage to the membrane structure as well as electron-transfer complexes. In this way, the energy from NADH or succinate is disposed of as heat with the return of NAD ÷, which is needed in the Krebs cycle and other essential NAD*linked reactions. The supply of the carbon source from the Krebs cycle to biosynthetic pathways will thus be continued and the cell will have normal function. In the light energy-harvesting system of chromatophores or chloroplasts, free Q may serve as an energystorage pool or act as a redox buffer system in order to help maintain electron acceptors, primary or secondary, in a state of readiness and to ensure maximal light energy and chemical energy conversion efficiency. Concerning the formation of ATP in the photosynthetic electron-transport system, the regulatory function of free Q as described for the mitochondrial system may be applied but is much less important. VI. Concluding remarks It is clear from the information described in the previous sections that the active species of Q in the electron-transfer reaction of the mitochondrial respiratory chain or in the photosynthetic apparatus is the Q-protein complex, rather than the free form of Q. There are three Q-binding proteins in the mitochondrial system, one of which has been isolated and characterized. The other two have been studied in the complex form and the specific proteins responsible for one of these have been identified through the photoaffinity labeling technique. The Q-binding proteins in the photosynthetic apparatus are comparable to those in the mitochondrial system. A Q-binding protein from the chromatophores of R. rubrum has also been isolated and characterized.

124 Although free Q in the system is in redox equilibrium with bound Q, the possibility of physical exchange between these forms has been ruled out. It should be emphasized that the Q-protein complex is a component of a given enzyme system, not an independent, separable redox component. QPs is an integral part or subunit of succinate-Q reductase, and the electron transfer (from succinate to Q) within this enzyme complex is uninterrupted. Similarly, QPn and QPc are parts of NADH-Q and ubiquinol-cytochrome c reductases, respectively. The native electron donor of ubiquinol-cytochrome c reductase is succinate-Q reductase or NADH-Q reductase rather than the free form o f ubiquinol. The physical separation of these enzyme complexes in the native membrane system is questionable. Even though exogenous ubiquinol is satisfactorily oxidized by ubiquinol-cytochrome c reductase, one should consider the Q-protein complex as a point where the redox equilibrium between bound and free Q occurs, but not as a point o f convergence o f electron-transfer complexes of the respiratory chain. However, the electron transfer between free and bound Q may serve as a mechanism for interaction between the electron-transport chain and other Q-linked reductases. If one extends the idea o f electron transfer between free and bound Q 1o other mitochondrial electron-transfer complexes, then the Q pool function o f Kr6ger and Klingenberg [18] can conceivably coexist with the Q-binding protein concept. More investigation is needed before the true electron-transfer mechanism involving Q is understood. Since the Q-protein complex-containing enzymes o f the respiratory chain are also involved in proton translocation, the topological arrangement of Q-binding proteins within the enzyme complexes as well as in the membrane will be very important in the elucidation o f the reaction mechanism of energy-conserving systems. Investigation in this area leading to clarification of the proton-translocation mechanism will presumably be very fruitful in the future.

Acknowledgements We are indebted to many colleagues, Drs. B.A. Ackrell, G.J. Arntsen, J.A. Berden, A. Crofts, G. Feher, C.R. Hackenbrock, G. Lenaz, P.A. Loacl~ S. Nagaoka, T. Ohnishi, M.Y. Okamura, S. Papa, J.S.

Rieske, H. Schneider, E.C. Slater, A.J. Swallow, B.L. Trumpower and B.R. Velthuys for supplying their preprints and unpublished information. The preparation o f this article and research from our laboratory are supported by research grants from NIH (GM 26292) and NSF (PCM78 01394).

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