Crystallization and preliminary structure of beef heart mitochondrial cytochrome-bc1 complex

BB ELSEVIER

Biochimica et Biophysica Acta 1275 (1996) 47-53

Biochi~ic~a et BiophysicaA~ta

Crystallization and preliminary structure of beef heart mitochondrial cytochrome-bc I complex Chang-An Yu a,*, Jia-Zhi Xia a, Anatoly M. Kachurin a, Linda Yu a, Di Xia b, Hoeon Kim b, Johann Deisenhofer b a Deparmwnt of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3035, USA h Howard Hughes Medical Institute and Department of' Biochemistry, Unirersity qf Texas Southwestern Medical Center, Dallas, TX 75235-9050, USA Received 15 April 1996: accepted 16 April 1996

Abstract

The method reported for isolation of ubiquinol-cytochrome-c reductase complex from submitochondrial particles was modified to yield a preparation for crystallization. The cytochrome bq complex was first crystallized in large thin plate form and diffracts X-rays to 7 A resolution in the presence of mother liquor. This crystalline complex was enzymatically active and contains ten protein subunits. It had 33 tool phospholipid and 0.6 mol ubiquinone per tool protein. With slightly modified crystallization conditions, different crystal forms were obtained. Crystals grown in the presence of 20% glycerol diffracted X-rays up to 2.9 ,~ resolution using a synchrotron source. Four heavy atom derivatives have been obtained. The 3-D structure of the cytochrome bc~ complex was solved to 3.4 A resolution. Crystalline cytochrome bc~ complex is a dimer: most of the masses of core proteins I and II protrudes from the matrix side of the membrane, whereas the cytocbrome b protein is located mainly within the membrane. There are 13 transmembrane helices in each monomer. Most of the mass of cytochrome c~ and iron-sulfur protein including their redox centers are located on the cytoplasmic side of the membrane. The distances between these redox centers have been determined, and several electron transfer inhibitor binding sites in the complex have been located. Keywords: Mitochondrial cytochrome bc I complex; Electron transport: Protein crystallization; Co-crystallization of cytochrome bCl-Cytochrome c complex: Inhibitor complex

1. Introduction

Cytochrome bc I complex (commonly known as Complex III or ubiquinol-cytochrome-c reductase) is a segment of the mitochondrial respiratory chain which catalyzes antimycin-sensitive electron transfer from ubiquinol to cytochrome c [1,2]. The reaction is coupled to the translocation of protons across the mitochondrial inner membrane to generate a proton gradient and membrane potential for ATP synthesis. Bovine heart mitochondrial cytochrome bc~ complex was first isolated in 1962 [1]; since then several purification methods have been introduced [3-6]. The purified cytochrome bc~ complex contains 11 protein subunits, as revealed by high resolution sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) [7,8]. The amino acid sequences of all subunits are known,

* Corresponding author. Fax: +1 (405) 7447799: e-mail address: cayuq @okway.okstate.edu. 0005-2728/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 0 0 5 - 2 7 2 8 ( 9 6 ) 0 0 0 4 9 - 7

some of them determined by peptide sequencing [9-14] and others deduced from nucleotide sequences [15-17]. The essential redox components of the cytochrome bc~ complex are: two b cytochromes (b565 and b562) , o n e c-type cytochrome (cl), one high potential iron-sulfur cluster (2Fe-2S Rieske center), and a ubiquinone. As a result of recent intensive investigation into the electron transfer and proton translocation mechanisms, investigators in the field are now generally in favor of the proton motive Q-cycle hypothesis [18,19]. The key feature of the Q-cycle hypothesis is the involvement of two separate quinone/quinol binding sites: one at which quinol is oxidized (Qo) and another at which quinone is reduced (Qi). According to the Q-cycle, quinol is first oxidized by Rieske iron sulfur center to generate a reactive semiquinone which reduces the low-potential cytochrome b heme (b E). The reduced b L rapidly transfers an electron to the highpotential cytochrome b heme (b H) located on the opposite side of the membrane. The reduced b H is then oxidized by

C.-A. Yu et al. / Biochimica et Biophysica Acta 1275 (1996) 47-53

48

a quinone or semiquinone at the Qi site. Proton translocation thus occurs as the result of deprotonation of QH 2 at the Qo site and protonation of the reduced Q at the Qi site. The Q-cycle mechanism is supported by several experimental observations such as the oxidant-induced reduction of cytochrome b [20,21], the detection of antimycin-sensitive and -insensitive transient Q radicals [22,23], and the 2H ÷ ejection per one electron transfer in cytochrome bc l complexes from many sources [25-27]. Photoaffinity studies [28,29] using photoactivatable azido-ubiquinone derivatives have located the two possible quinone binding domains in cytochrome b protein. The involvement of cytochrome b562 a t the Q i site is suggested by the different EPR characteristics of cytochrome b562 in the presence and absence of Q [30]. Chemical labeling and proteolytic enzyme digestion studies have provided substantial information concerning the spatial arrangement of this enzyme complex in the membrane [8]. Amino acid sequence analysis of cytochrome b [31,32] and the biophysical measurements [33-35] have predicted separate locations for the two b hemes. Mutagenesis studies [36] have contributed substantially to the structural study of the cytochrome bc I complex. However, the lack of knowledge of three-dimensional structure has long been an obstacle. The recent success in protein crystallization of the cytochrome bc~ complex [37-44] provides an opportunity for structural analysis of this large membrane protein complex. Over the past two decades, we have used multiple approaches to investigate the cytochrome bcl complex: protein components resolution and reconstitution, organic syntheses of ubiquinone derivatives and quinone-like inhibitors, molecular genetics manipulation and protein X-ray crystallography. In this review we summarize our work on the X-ray crystallographic studies of this complex, emphasizing protein crystallization and structure analysis. For

other work on this and related complexes, excellent reviews are available [45,46].

2. Preparation of cytochrome bc~ complex Bovine heart mitochondrial cytochrome bcj complex (or Complex III) was first isolated from NADH-cytochrome-c reductase in 1962 [1]. This method is still used by many investigators [38,40]. Trumpower and co-workers developed a method for isolation of bc~ complexes from various sources, which involves expensive laurylmaltoside and two ion exchange column chromatography [6]. The cytochrome bc l complex used in our laboratory is prepared from a highly purified succinate-cytochrome-c reductase preparation [4]. Succinate dehydrogenase in extensively dialyzed succinate-cytochrome-c reductase is solubilized and removed upon alkalization (to pH 10) under anaerobic conditions. The resulting residue is termed the cytochrome bc~ particle and contains both cytochrome bc~ complex and a protein fraction (QPs) that converts succinate dehydrogenase into succinate-Q reductase. The cytochrome bc~ particle is capable of reconstitution with pure succinate dehydrogenase to form succinate-cytochrome-c reductase. Highly purified cytochrome bc~ complex is obtained upon solubilization of the bc~ particle by deoxycholate and removal of QPs and other contaminants by a four-step ammonium acetate fractionation [4]. The purified complex contains 8.3 nmol cytochrome b, 4.7 nmol cytochrome c 1, 3.5 nmol ubiquinone and 250 nmol phospholipid per mg protein. For protein crystallization the number of steps of ammonium acetate fractionation was increased to 15. The specific activity of the purified complex is 11 ~mol cytochrome c reduced per min per nmol cytochrome b at 23°C. When the purified complex is subjected to high resolution SDS-PAGE [7,8] eleven protein bands are observed.

Table 1 C y t o c h r o m e b-cj c o m p l e x crystals obtained b y different investigators Investigators

Space g r o u p

Resolution

Unit cell dimension

Solvent content

Vm

Detergent used

Yue et al. (1991) K u b o t a et al. (1991)

P4122 P2j

(,~) low 7.5

(.~3) 4.3 4.4

in i s o l a t . / i n crystal. SC, D O C / D M G DOC/SML

P6122 orP6522 I4~22 P61(P65) or P41(P43) C2221 14122 14122

b = 159, 196, b = 253, 13 = b = 212,

(%) 69.4 -

Berry et al. (1992)

(,~) a = a = c = a =

65.7

4.96

LM/OG

a a a a a a c

b = 157, c = 590 b = 131, c = 7 2 0 b = 190, c = 445 384, b = 1 1 8 c = 177 b = 153, c = 597 b = 153.5, 597.7

70 4.4 69.4

4.0 3.9

SC, D O C / D M G DOC/SML

4.35 4.0

LM/OG LM/HECAMEG SC, D O C / S P C or D M G

Yu et al. (1994) K a w a m o t o et al. (1994) B e r r y et al. (1995) Lee et al. (1995) Xia et al. (1996)

4.7 4.5 6.5 3.8 3.3 2.9

= = = = = = =

c = 593 179 97 ° c = 352

D M G , d e c a n o y l - N - m e t h y l g l u c a m i d e ; D O C , d e o x y y cholate; H E C A M E G , 6-O-(N-heptyl-carbamoyl)-methyl-ct-D-glucopyranoside; LM, lauraryl maltoside; O G , octyl a - D - g l u c o p y r a n o s i d e ; PC, phosphotidylcholine; SC, sodium cholate; S M L , sucrose monolaurate; SPC, short side c h a i n e d - P C ; Vm, M a t t h e w s ' coefficient.

49

C.-A. Yu et al. / Biochimica et Biophysica Acta 1275 (1996) 4 7 - 5 3

A

13

C

D

E

F

Fig. 1

Fig. 2 Fig. 1. Crystals of cytochrome bcj complex grown under different conditions. (A), Native cytochrome bc~ complex; (B), Cytochrome c:cytochrome bcj complex; (C), UHDBT inhibited cytochrome bc] complex; (D), Stigmatellin inhibited cytochrome bc I complex; (E), Stigmatellin/antimycin inhibited cytochrome b £ 1 complex; and (F) Antimycin/myxothaizole inhibited cytochrome bc I complex. Fig. 2. Molecular shape of crystalline cytochrome bc 1 complex, inter-twined dimer.

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C.-A. Yu et al. / Biochimica et Biophysica Acta 1275 (1996) 47-53

3. Crystallization of cytochrome

bc I

complex

Purified cytochrome bcl complex in 50 mM Tris-Cl buffer (pH 7.8), containing 0.67 M sucrose, was first crystallized in 6% polyethylene glycol-4000 (PEG) in the presence of 0.25 M sodium chloride, 1.8% heptanetriol, 0.04% decanoyl-N-methyl-glucamide. Good-sized crystals formed within 2 - 4 weeks. The largest crystals grown in a Pasteur pipette had dimensions of 4 X 2 X 1 ram. Although the crystals showed a high degree of birefringence under polarized light, they diffracted X-rays only at low resolution [37]. When crystals are grown in X-ray capillary tubes in 50 mM MES buffer (pH 7.0), instead of 50 mM Tris-Cl buffer (pH 7.8), they diffract X-rays to 7 ,~ resolution in the presence of mother liquor using a Xuong/Hamlin area detector. The crystals have the symmetry of the tetragonal space group 14122 with cell constants a = b = 153.5 A, c =597.7A. Assuming one cytochrome bc 1 complex molecule per asymmetric unit, the crystals would have a solvent content of 70%. Removal of the mother liquor from the crystals causes severe loss of diffraction quality. Moreover, the tendency of the crystals to move in a liquid-filled capillary tube makes data collection difficult. To circumvent these difficulties, we developed a method to crystallize cytochrome b q complex in the gel state [44]. Purified ubiquinol-cytochrome-c reductase, 20 mg/ml, in 50 mM MES buffer (pH 7.0), containing 0.67 M sucrose was mixed, at 18°C, with an equal volume of precipitating solution containing 0.08% decanoyl-N-methyl-glucamide, 3.6% heptanetriol, 0.5 M sodium chloride, 12% polyethylene glycol, and 0.8-0.4% low gelling temperature agarose. The mixture was placed in capillary tubes, cooled to 4°C, overlaid with equilibrating solution, and incubated in a shock-free environment at 4°C. Under these conditions, cytochrome-bq complex crystals formed within 2 - 4 weeks. The size, shape and diffraction quality of these crystals approached those obtained from the liquid state. When crystallization was carried out under the reduced pressure, the rate of crystal growth increased substantially at the expense of crystal size. Recently, conditions used for growing cytochrome bc 1 complex crystals were further modified to improve crystal diffraction quality. The cytochrome bcj complex was diluted with an equal volume of 0.5 M sucrose and precipitated with an equal volume of 50% saturated ammonium acetate. The precipitates were collected by centrifugation and redissolved in 20 mM ammonium acetate buffer (pH 7.2), containing 20% glycerol, to a protein concentration of 20 mg/ml. This cytochrome bc~ complex was then mixed with 0.5 M MOPS buffer (pH 7.2), and 2% detergent (DMG, decanoyl-N-methylglucamide or SPC, short side chained phosphotidyl choline) solution to a final concentration of 50 mM MOPS and 0.1% detergent. A 0.57 to one volume of 12% PEG-4000 in 50 mM MOPS buffer containing 0.5 M KC1, 20% glycerol, and 0.1% detergent

was slowly added to the cytochrome bcj complex solution with constant stirring. After 2 h incubation at 0°C, the mixture was centrifuged for 10 rain at 40000 X g to remove precipitates formed, if any. The clear solution was used for crystallization. For a quick test, 4-1xl aliquots were used for hanging drop set-up, equilibrated with 200 txl of equilibrating solution, containing 18% PEG, 0.5 M KCI 20 mM Tris-Cl buffer (pH 7.0). Under these conditions, crystals formed overnight. To grow crystals for X-ray diffraction. 30- to 50-1M aliquots of centrifuged mixture were placed in small test tubes (6 X 50 mm) and incubated with equilibrating solution at 0 to 4°C. Crystals were mature in 3-4 weeks. Crystals are rectangular in shape, ranging in size from 0.5 to 1 mm. They can be frozen at high concentration glycerol. Under cryogenic conditions (100 ° K) these crystals diffract X-rays up to 2.9 resolution using a synchrotron source. These crystals were used for data collection. These crystals have the symmetry of body-centered tetragonal sl~ace group of I4122 and cell dimensions of a = b = 153.5 A and ¢ = 597.7 A. There are eight b¢ 1 dimers in a unit cell. Fig. 1 shows crystals of cytochrome b¢ 1 complex grown in 20% glycerol. Following our first report of crystallization of cytochrome bc~ complex in 1991 [37], several other investigators have also reported crystallization of this complex in various forms and with varying difractability. Table 1 summarizes the crystals obtained by different investigators.

4. Properties of cytochrome

bc 1

complex crystals

Crystalline cytochrome bc 1 complex is in the oxidized state and is composed of ten protein subunits. The complex contains 2.5 nmol ubiquinone, 8.4 nmol cytochrome b, 4.2 nmol cytochrome q , and 140 nmol phospholipid per mg protein. About 36% of the phospholipid associated with crystalline cytochrome bc 1 complex is diphosphatidylglycerol. These crystals are very stable in cold and show full enzymatic activity when redissolved in aqueous solution. Absorption spectra of the redissolved crystals show a Soret to UV ratio of 0.88 and 1.01 in the oxidized and the reduced forms, respectively. Cytochrome bc~ complex crystals are not very stable under X-ray beam at 4°C, but are very stable in the frozen state. At 100°K the crystal suffers no noticeable damage even after 24 h of bombardment with under X-ray beam from a synchrotron source. A single crystal is sufficient for a complete set of data collection.

5. Co-crystallization of cytochrome hibitor complexes

bc~

complex-in-

The cytochrome bc~ complex was precipitated by ammonium acetate as described in crystallization of native

C.-A. Yu et al. / Biochimica et Biophysica Acta 1275 (1996) 47-53

complex and redissolved in 50 mM MOPS buffer (pH 7.2), containing 20 mM ammonium acetate, 20% glycerol, and 0.14% SPC (or 0.11% DMG). This solution was incubated with a slightly molar excess of electron transport inhibitors [24], individually or in combination at 4°C for 30 min before being subjected to crystallization using the precipitating solution (0.57 volume) described for the native complex. The molar ratios used were 1.3, 2.1, 1.3, and 1.3 for antimycin, UHDBT, myxothaizole, and stigmatellin, respectively. The inhibitor solutions were made in 95% ethanol to a concentration of approx. 10 mM. Crystals of inhibitor complexes appeared in 3-4 weeks. The shapes and sizes of crystals of inhibitor-complexes were very similar to those of the native cytochrome bcl complex and also diffracting X-rays to a resolution comparable to native crystals.

6. Co-crystallization of cytochrome bcl complex-cytochrome c complex The cytochrome bc I complex was precipitated by ammonium acetate as described in crystallization of native complex and redissolved in 50 mM MOPS buffer (pH 7.2), containing 20 mM ammonium acetate, 20% glycerol, 0.14% SPC and incubated with a slightly molar excess (1.1 to 1) of beef heart cytochrome c. After incubation for 1 h at 0-4°C the mixture was subjected to crystallization using 0.46 volume of precipitating buffer solution or 0.51 volume of precipitating buffer solution containing no KCI. The crystals of cytochrome bc I complex/cytochrome c complex formed within 4 weeks. The presence of cytochrome c in the crystals was confirmed by spectral analysis. The crystals were washed thoroughly and redissolved in Tris-C1 buffer containing 0.1% deoxycholate. The molar ratio of cytochrome c:cytochrome bc I complex was found to be 0.75 and 1.0 in the crystals grown in the presence and absence of KC1, respectively. Preliminary results indicate that crystals of the cytochrome c/cytochrome bc I complex diffract X-rays to a resolution similar native crystals.

51

The electron density map for the bc 1 complex clearly shows the close interaction between two crystallographic symmetry related monomers (Fig. 2). The dimer can be divided into three regions, the trans-membrane helix region, the matrix region, and the inter-membrane space region. The majority of the molecular mass is located in the matrix region of the molecule, extruding from the trans-membrane helix region by 75 A. The inter-membrane space region extrudes 41 A into the cytoplasmic surface from the trans-membrane helix re~ion. The trans-membrane helix region is about 35 A thick with thirteen transmembrane helices in each monomer.

8. ~ i z a t i o n

of redox centers

Four high peaks in maps calculated using anomalous scattering data were interpreted as the redox-centers of the bc~ complex. Two of these sites are 20 A apart in the transmembrane region and are assigned to the heine irons of cytochromes b562 and b565, with the b562 close to the middle of the membrane and the b565 near the surface of the membrane. Another site near the cytoplasmic surface of the membrane and 27 A away from the nearest b-heme is the iron-sulfur center. The fourth site is the cytochrome c~ heme, which is 31 A apart from the iron-sulfur center. The distance between the b565s of the two monomers is less than 21 A, making the interaction between these two cytochromes very interesting. The relative locations of redox centers are indicated in Fig. 3. Some of the locations of redox centers and the distances between them determined here are very close to those obtained from the biochemical and biophysical studies [47-49].

7. Structural analysis Crystals grown in the ~presence of glycerol diffract X-rays to better than 3 A resolution under cryogenic conditions. The diffraction quality of heavy metal derivatized crystals is comparable to that of native crystals. Initial MIR phases were determined with four heavy metal derivatives. Electron density maps obtained with the MIR phases were subsequently improved with cyclic density modification procedures. Better phases were then used to improve further the initial MIR phases.

Fig. 3. The relative locations of redox centers of crystalline dimeric cytochrome bc I complex.

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C.-A. Yu et aL / Biochimica et Biophysica Acta 1275 (1996) 47-53

9. Localization of inhibitor binding domains Even without three-dimensional structural information, studies of interactions between inhibitors and electron transfer complexes have made significant contributions in the electron transfer pathway. For instance, antimycin inhibition of the cytochrome bc~ complex has been extensively studied. Antimycin blocks electron exit from cytochrome b562 and causes red spectral shift. Binding of antimycin to the cytochrome bcj complex also destabilizes the ubisemiquinone radical at the Qi site. It is generally believed that antimycin binds near the b562. Inhibitor-resistant mutant analysis also suggests that antimycin binds on the matrix side of the inner membrane [36,50]. Differences between the electron density maps calculated from X-rays diffraction data of antimycin-bound and native bc I complex reveal a strong area of density whose shape resembles that of antimycin. This density is located extremely close to the b562, causing local conformational changes both to the heme and to nearby residues. Another highly potent and specific inhibitor of electron transfer in the cytochrome bc 1 segment of the respiratory chain is UHDBT (5-undeceyl-6-hydroxy-4,7-dioxobenzothiazoi). UHDBT blocks electron transfer between the Rieske iron-sulfur and cytochrome c I [51] and inhibits oxidant-induced cytochrome b reduction [52]. It is generally believed that this inhibitor binds near the iron-sulfur cluster, causing modification of EPR signals from the iron-sulfur cluster. Preliminary analysis of the difference electron density maps between the UHDBT-cytochrome bc~ complex and the native complex also indicates that the dioxobenzothiazol part of UHDBT is likely to bind near the iron-sulfur cluster. More detailed analysis is needed before the precise binding site of this inhibitor can be established. Structural analyses of crystals of other inhibitor complexes and cytochrome c:cytochrome bc~ complex are currently in progress in our laboratories.

Acknowledgements The work described in this review was supported in part by a grant from the National Institutes of Health (GM 3072 to C.A.Y.) and from Welch Foundation (to J.D.). J.D. is an investigator in the Howard Hughes Medical Institute. The X-ray diffraction data were mainly collected at beamlines X12B and X4A of NSLS at Brookhaven National Laboratory and at beamline 7A of Stanford Synchroton Radiation Laboratory.

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