Subunit composition and biogenesis of mitochondrial cytochrome b

Biochimica et Biophysica Acta, 456 (1976) 291-313 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in The Netherlands BBA 86035

S U B U N I T C O M P O S I T I O N A N D BIOGENESIS OF M I T O C H O N D R I A L CYTOCHROME b H A N N S WEISS

European Molecular Biology Laboratory, 69 Heidelberg, Postfach 10 22 09 (G.F.R.) (Received May 26th, 1976)

CONTENTS 1.

Introduction

II.

Purification of cytochrome b and its solubility in detergents

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

292

I II.

Some properties of the heine centre of purified cytochrome b . . . . . . . . . . . .

295

IV.

Molecular weight of cytochrome b . . . . . . . . . . . . . A. M i n i m u m molecular weight per mol protoheme . . . . . . B. Molecular weight in denaturing detergents . . . . . . . . . C. Molecular weight in mild detergents . . . . . . . . . . .

. . . .

295 295 296 297

V.

Subunit composition of cytochrome b

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

299

VI.

A m i n o acid composition of cytochrome b . . . . . . . . . . . . . . . . . . . .

302

VII.

Have different cytochrome b species been isolated from the inner mitochondrial membrane? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

304

VIII.

Biogenesis of cytochrome b . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mitochondrial inheritance . . . . . . . . . . . . . . . . . . . . . . . . . B. Mitochondrial translation . . . . . . . . . . . . . . . . . . . . . . . . .

304 304 305

I X.

Conclusion

310

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Acknowledgements References

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291

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311

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311

I. I N T R O D U C T I O N

Two functionally different cytochrome b species form part of the respiratory chain of mitochondria. Their characteristic light absorption properties led to their discovery in situ, i.e. in intact mitochondria or even in intact organisms. However, the combined spectrophotometric, potentiometric and kinetic techniques [1-9] which have been applied to their investigations, allowed only the characterisation of properties of their heme centres, whereas the characterisation of their protein components has for a long time been given insufficient attention. This is due to the fact that cytochrome b is an integral protein [10] of the inner mitochondrial membrane and that its purification and in vitro handling are still technically difficult. By the use of mild detergents [1 I] the mitochondrial membrane could be dissected into distinct electron transfer complexes [12]. The smallest segment, however, which contains the two b-cytochromes (as well as cytochrome ct and an iron-sulfur protein) in catalytically active form, still consists of at least seven distinct protein components [13]. Any further subdivision of this so-called Complex III leads to strong modifications

292 of the b-cytochromes. Thus, it was not yet possible to decide conclusively whether or not the functional dimerity of cytochrome b results from two distinct heine proteins or from the different membrane environment of one single heme protein. In addition, cytochrome b has become of special interest in the field of mitochondrial genetics and biogenesis. In recent years, it has been established that the inner mitochondrial membrane consists of two types of polypeptides derived from two separate genetic systems [14,15]. The bulk of the membranous polypeptides is coded for by nuclear DNA and is synthesized on cytoplasmic ribosomes. A small number of polypeptides [16-25], however, is synthesized on mitochondrial ribosomes and most probably specified by genes on mitochondrial DNA. These polypeptides are the most hydrophobic and are deeply embedded in the membrane interior [23, 25-30]. it has recently been established that a cytochrome b apoprotein belongs to this group of polypeptides [22,24]. It is the aim of this article to summarize and discuss our knowledge about the structure and the biogenesis of mitochondrial cytochrome b. This subject had not been treated in earlier reviews [13,31-34] as most of the information has been made available only in recent years. II. PURIFICATION OF CYTOCHROME b AND ITS SOLUBILITY IN DETERGENTS Presuming that with cytochrome b in situ the protoheme is uniquely associated with a single polypeptide and is not relocated to a different polypeptide upon purification, a preparation can be referred to as purified cytochrome b when it displays a state of homogeneity such that the molecular weight of the heine-carrying polypeptide determined by physical methods agrees with the minimum molecular weight derived from the specific heme content. As purified cytochrome b has a strong tendency to form aggregates even in the presence of the detergents used for its isolation, this criterium of purity could not be examined with certainty for a long time. The introduction of dodecyl sulfate gel electrophoresis has to a large extent solved this problem [35,36]. From the numerous cytochrome b preparations which have been described in the literature [22,24,28,37-48], only those which displayed a degree of purity allowing some characterisation of the protein structure will be considered in this chapter [22, 24,28,4244,46-48]. Most authors [42-44,4648] started with the purification of Complex Ill, a catalytically active electron transport segment which could easily be isolated from beef heart mitochondria as a physically homogeneous particle, by solubilisation with bile acids and precipitation with ammonium sulfate. In this Complex IlI, two functionally different b-cytochromes could still be distinguished with regard to the position of their light absorption bands, their redox potentials, their redox kinetics and their response to the respiratory chain inhibitor antimycin [13]. They were always present in equimolar amounts. Dodecyl sulfate gel electrophoresis of this complex revealed seven distinct bands corresponding to polypeptides of the molecular

293 weights in the range of 8000 to 50 000. The attribution of any one of these polypeptides to cytochrome b, however, was not possible at this stage of purification, because dodecyl sulfate led to the dissociation of the heine from the protein. (A review of the composition, structure and function of Complex III has been published separately in this journal by Rieske [13]). A further purification of cytochrome b was obtained by Goldberger and coworkers [43]. These authors subjected Complex III from beef heart mitochondria to heat treatment in a cholate solution. Thereby, cytochrome b formed a flocculent precipitate while most of the other polypeptides remained in solution. Solubilization of the precipitation with small amounts of sodium dodecyl sulfate (0.3 g/g protein) and subsequent ammonium sulfate precipitation resulted in a cytochrome b preparation which contained 35/~mol protoheme per g protein. This preparation, however, consisted of huge aggregates and only upon treatment with the cationic detergent cetyldimethyl ammonium bromide, could a monodisperse protein solution be obtained. Ohnishi [44] used a high concentration of cholate in the presence of ammonium sulfate to cleave Complex III and simultaneously precipitate cytochrome b. Following this, treatment with a bacterial protease in the presence of cholate led to a resolubilisation ofcytochrome b. The final purified cytochrome b contained 47 #tool protoheme per g protein. Although this preparation formed a clear solution in the presence of cholate and Emasol (polyoxyethylene-sorbitan-monolaureate), it also consisted of polydisperse aggregates. It was not soluble in cetyldimethyl ammonium bromide. Yu and co-workers [46] also cleaved Complex I11 from beef heart mitochondria by treatment with cholate and ammonium sulfate. They then resolubilized the precipitated cytochrome b with small amounts of dodecyl sulfate. By means of repeated gel filtration on Sephadex in a solution containing low concentrations of dodecyl sulfate (in the absence of mercaptoethanol), they reported having obtained a physically homogenous heme protein of molecular weight 37 000, which contained 26 /zmol protoheme per g protein and possessed the spectral characteristics of a b-type cytochrome. Katan and co-workers [47,48] purified Complex 1II from yeast mitochondria and precipitated cytochrome b with guanidine hydrochloride [45]. They obtained a preparation containing 20/~mol protoheme per g protein. This preparation, however, was insoluble in the presence of bile acids or Triton X-100. Dodecyl sulfate gel electrophoresis showed one main polypeptide of molecular weight 32 000 and two minor polypeptides with molecular weights 14 000 and 11 000. The protohemecarrying polypeptide could not be identified among them, as the heine was dissociated from the protein during dodecyl sulfate treatment. Weiss and co-workers [22,24,28] applied a completely different procedure for the purification of cytochrome b from Neurospora crassa and from Locusta migratoria [24]. First, they separated the mitochondrial membrane proteins by means of an amphipathic chromatographic system [49]. The stationary phase of this system was polymethacrylic acid resin, some of the carboxylic groups of which were linked to

294

A

556 1

1

I

559 I tn ,¢

Lf Wavetength (nm } Fig. 1. Low-temperature difference absorption spectra (reduced minus oxidized) of (A) intact Neurospora crassa mitochondria and (B) purified Neurospora crassa cytochrome b. The absorption spectra were obtained with a split beam spectrophotometer designed by M. Klingenberg.

oleylamine by means of an amide bond. The mobile phase was a solution of the detergents cholate and deoxycholate. Thus, the mobile phase and the resin phase both contained weakly acidic carboxylate groups and hydrophobic aliphatic chains. Mitochondrial membrane proteins solubilized with deoxycholate were bound to the resin, the stationary carboxylate and oleyl groups replacing the detergent. On elution with gradients of increasing detergent concentration and increasing salt concentration in the presence of detergent, cytochrome b appeared well separated from cytochrome aa3, cytochrome c, and from a number of proteins not containing heme. Secondly,

295 the crude cytochrome b preparation was submitted to gel filtration on a Sephadex G-100 column in a deoxycholate plus salt solution. By means of this procedure, cytochrome b preparations with specific heme contents of about 35/zmol per g protein were obtained. These preparations differed from those reported above in that they were soluble at a monodisperse low molecular weight state in a solution containing deoxycholate and salt. The preparations were not soluble in Tween 80, Triton X-100, or cetyldimethyl ammonium bromide. Their protein moiety was soluble in dodecyl sulfate or laureate. Treatment with the last four detergents led to the dissociation of the protoheme from the protein. ilI. SOME PROPERTIES OF THE HEME CENTRE OF PURIFIED CYTOCHROME b Whereas the functional characteristics of the two b-cytochromes present in Complex Iil closely resemble that of the intact mitochondria [13,51-53], further purification produced a strong modification. The fact that the two b-cytochromes thereby became indistinguishable is most striking [19,51,53]. For example, with Neurospora crassa, the low temperature absorption spectrum of intact mitochondria shows two distinct cytochrome b bands in the a-region, at 556 and 563 nm [54-56], whereas only one assymetric a-band with a maximum at 559 nm is seen in the low temperature absorption spectrum of purified cytochrome b [19,53] (Fig. 1). Titration of cytochrome b in intact Neurospora mitochondria, using a combined potentiometric and spectrophotometric technique [6], revealed two redox components with the midpoint-potentials Era, 7 ~ --40 mV and Em, 7 ~ -{-60 mV [53]. Titration of purified cytochrome b, however, revealed only one component with Em,7 ~ --56 mV [53] (Fig. 2). Further signs of conformational changes occurring during the isolation of cytochrome b from its proper surroundings are the reactivity with carbon monoxide and with oxygen of the reduced form of purified cytochrome b [43,44,50]. The replacement of the detergent by phospholipids does not restore the original properties of cytochrome b in situ [50]. IV. MOLECULAR WEIGHT OF CYTOCHROME b

A. Minimum molecular weight per mol protoheme On the basis of the specific heme or iron contents of the preparations, the following minimum molecular weights were evaluated for purified cytochrome b: 22 000 by Ohnishi [44], 27 000 by Weiss et al. [22,28], 28 000 by Goldgerger et al. [43] and 37 000 by Yu et al. [46]. The nonconformity among the first three data is small, taking into consideration that completely different techniques have been applied for heme and protein determination. Thus, when comparing the absolute absorption spectra of the cytochrome b preparation, the ratios of the heme absorbance at 416 nm to the protein absorbance at 278 nm were found to be more in agreement. It amoun-

296

10050A

o

O-50-

y,

~" 100 -

E

O- s0J

-100-1,5

-1

I

-0,s

i

0,5

1,5

Log (ox/red) Fig. 2. Redox titration curves of (A) the b-type cytochromes in intact Neurospora crassa mitochondria and (B) purified Neurospora crassa cytochrome b. The redox titration was performed by yon Jagow [53] with mitochondria at pH 7 and with purified cytochrome b at pH 8 at the wavelength pair 562-575 nm in the presence of redox mediators as described by Wilson and Dutton [6].

ted to 1.38 with cytochrome b from Neurospora crassa [22], 1.30 with cytochrome b from beef heart [44] and 1.28 with cytochrome b from Locusta migratoria [24]. Unfortunately, such data are not given for the 37 000 molecular weight cytochrome b described by Yu et al. [46], although the good solubility in dodecyl sulfate reported for this preparation should make it particularly suited for such measurements. B. Molecular weight in denaturing detergents

The most widely used method for molecular weight determination of polypeptides of oligomeric proteins is now dodecyl sulfate gel electrophoresis [35,36]. When applied to purified cytochrome b, a molecular weight of 27 000-32 000 was obtained by Weiss et al. [22,28], 32 000 by Lorenz et al. [24], 32 000 by Katan et al. [47,48] and 37 000 by Yu et al. [46]. The basis of this method is the assumption that the relative amount of dodecyl sulfate bound to the membrane protein cytochrome b is equal to the relative amount bound to the water-soluble proteins used for calibration [57]. This assumption, however, might be invalid. The high content of unpolar amino acids in cytochrome b (Table II) suggests that it binds more dodecyl sulfate. This would explain why the

297 molecular weights determined by this method were somewhat higher than the minimum molecular weights derived from the specific heme contents. In a very early paper, Bomstein et al. [42] reported on analytical ultracentrifugation studies of a cytochrome b preparation [43] in the presence of the cationic detergent cetyldimethyl ammonium bromide. From sedimentation velocity and sedimentation equilibrium experiments, these authors calculated an average molecular weight of 20 000. As the binding of detergent [58,59] to cytochrome b has not been considered in their studies, this molecular weight determination must be looked upon as a rather crude approximation. Later attempts by other authors [44, 48,50] to use this cationic detergent for the solubilisation of cytochrome b have been unsuccessful. C. Molecular weight in mild detergents In contrast to dodecyl sulfate, the mild detergent [l l] deoxycholate does not generally cause oligomeric (heine) proteins to dissociate into their polypeptides (and heine). In the presence of this detergent, however, most cytochrome b preparations were either insoluble [47,48] or were only soluble in a highly polymerized state [43, 44]. Only the purification procedure developed by Weiss et al. [22,24,28] yielded cytochrome b preparations which were soluble at a low molecular weight state in a solution containing deoxycholate and salt. During gel filtration on a Sephadex G-100 column in a solution containing deoxycholate, cholate and salt, the cytochrome b preparations from Neurospora crassa and from Locusta migratoria behaved like homogeneous 55 000-58 000 molecular weight heine proteins when compared with water-soluble proteins of known molecular weight [22,24,28]. From analytical ultracentrifugation experiments performed in a deoxycholate and salt solution, the following hydrodynamic parameters and molecular weights were obtained for the cytochrome b preparation from Neurospora crassa: sedimentation coefficient S2o,w = 4.2, diffusion coefficient D2o,w = 6.3, frictional coefficientf = 1.25, molecular weight calculated from s and D, Mr (s,D) = 63 000 and molecular weight from high speed sedimentation equilibrium Mr = 62 000 [28]. These values were corrected to standard conditions (viscosity and density of the buffer). The used partial specific volume of cytochrome b (0.754 ml/g) was estimated from the amino acid compositions (Table II). The values do not include corrections for the protein bound deoxycholate. In a multi-component system, however, such as the cytochrome b preparation in the deoxycholate solution, an interaction of the detergent with the protein has to be considered. In order to determine the amount of protein-bound deoxycholate, the cytochrome b preparation was subjected to gel filtration on a Sephadex column equilibrated with [14C]deoxycholate [28,60,61]. A quantity of 0.3 g deoxycholate bound per g cytochrome b protein resulted from the amount of radioactivity that coeluted with cytochrome b (Fig. 3). As pointed out by Tanford et al. [58] the molecular weight of a protein in a

298

200-3

E c

-2 ~ 100.<

E

E

0 ._>

o~

2OO-

LI

100-

ol o

V0tume (mL)

Fig. 3. Deoxycholate-binding of cytochrome b. (A) 0.27 ml 112 #M cytochrome b from Neurospora crassa in 0.5% potassium deoxycholate, 1.5 M KC1, 0.05 M Tris-acetate pH 8 was subjected to gel filtration on a 0.5 × 21 cm Sephadex G-150 column equilibrated with 0.2570 potassium [~4C]deoxycholate(0.126/~Ci/ml), 0.2M KCI and 0.05 M Tris-acetate pH 9. (B) 0.25 ml, 0.5 ~ potassium deoxycholate, 1.5 M KCI, 0.05 M Tris-acetate pH 8 was applied to the same column. Elution rate 1 ml/h, temperature 22 °C; f-~-- -(3, cytochrome b absorbance; • • , radioactivity.

detergent solution can be determined by sedimentation equilibrium, if the experimental data is calculated using an equation for a multicomponent system. Applying a buoyant density factor calculated for the protein-detergent complex by means of the partial specific volume of the cytochrome b protein (0.754 ml/g) and of the deoxycholate (0.778 ml/g) [58], a molecular weight of 51 000 was obtained for the pure cytochrome b excluding the bound detergent [28,60]. Obviously, this molecular weight of cytochrome b in deoxycholate is about twice the apparent molecular weight of the cytochrome b polypeptide(s) in dodecyl sulfate and is also about twice the minimum molecular weight derived from the

299 specific heme content. This suggested that purified cytochrome b is a dimeric heme protein consisting of two heme-binding polypeptides of about equal size [22,24,28, 60] (Table I).

TABLE 1 MOLECULAR WEIGHT OF MITOCHONDR1AL CYTOCHROME b FROM AND FROM L O C U S T A M I G R A T O R I A

NEUROSPORA

CRASSA

Detergent used for solubilisation

Determination method

1. Deoxycholate and (or) cholate

Sedimentation and diffusion coefficient Sedimentation equilibrium Gel filtration Sedimentation equilibrium corrected for bound deoxycholate

51 000

Gel electrophoresis after intramolecular cross-linkage Gel electrophoresis Gel filtration

55 000 27 000--32 000 32 000 30 000 30 000

Minimum molecular weight per tool protoheme

26 000-30 000

2. Dodecyl sulfate

Cytochrome b purified from Neurospora crassa a

Locusta migratoria b

63 000 62 000 58 000

55 000

a Weiss et al. [22,28,60]. b Lorenz et al. [24].

v. SUBUNIT COMPOSITION OF CYTOCHROME b The dimeric character of the cytochrome b preparation was further confirmed by means of intramolecular cross-linking of the subunits prior to their dissociation by dodecyl sulfate [60]. Treatment with the bifunctional reagents dimethyl suberimidate [62] or glutaric dialdehyde [63] yielded a product which showed upon dodecyl sulfate gel electrophoresis the molecular weight 55 000 (Fig. 4B). This molecular weight was about twice that obl~ained by dodecyl sulfate gel electrophoresis of the cytochrome b polypeptides which were not cross-linked (Fig. 4A). This 55 000 molecular weight product was not obtained when cytochrome b was dissolved in dodecyl sulfate prior to treatment with the bifunctional reagents (Fig. 4A). The question as to whether the two cytochrome b subunits are identical or nonidentical polypeptide chains has not yet been definitely answered. During dodecyl sulfate gel electrophoresis, a splitting of the cytochrome b protein into two bands of about equal protein staining could sometimes be observed [28]. This might indicate a difference in molecular weight of 1000-2000.

300 10-3 . apparent motecutac

6,0 s,o 4,o 3,o

weight

2o,

10

A

t.n

L .o ,K

Distance migrated (cm]

Fig. 4. Dodecyl sulfate gel electrophoresis of purified Neurospora crassa cytochrome b treated (A) with dimethyl-suberimidate after the addition of dodecyl sulfate and (B) with dimethyl-suberimidate prior to the addition of dodecyl sulfate. 6 mg/ml dimethyl suberimidate were added to 30 #M cytochrome b in 0.5 % potassium deoxycholate, 0.5 % potassium cholate, 0.2 M KCI, 0.02 M triethanolamine HCI, pH 8.5. After incubation for 2 h at 25°C, the solution was dialysed against water for 5 h and then lyophilized. Thereafter dodecyl sulfate gel electrophoresis was performed as described [16] but modified by using 7.5 % polyacrylamide gels.

A higher reproducible separation of the cytochrome b protein was obtained by means of hydroxyapatite chromatography in the presence of dodecyl sulfate [28,60] a method which was first applied by Moss and Rosenblum [64] for the separation of virus membrane proteins: at a low sodium phosphate concentration in the presence of sodium dodecyl sulfate and dithioerytritol, the total cytochrome b protein is bound to hydroxyapatite. Using a linear gradient of sodium phosphate the cytochrome b protein was eluted as two distinct peaks (Fig. 5). Whereas the two latter results suggested non-identity of the two cytochrome b subunits, no difference could be established between them when comparing their peptide fragments: when each of the two subunits was cleaved with cyanogen bromide and then subjected to gel filtration on Bio Gel P-30 in 85 % formic acid, their elution patterns were found to be indistinguishable (Fig. 6). This suggests that both subunits might have the same or at least a very similar primary structure [60]. Unfortunately,

301

1000-

-0,5

A

-0/,.

800-

J

600-

-0,3

400 -

L0,2

200-

-0,1

0

0 -0,5

A ,,.,,..

E

~ -4--

B

"~ 300-

¢U t" ¢-n

t-n

¢'0 0

-0,4

5 O

tJ~

-0,3

200 -

-0.2 I00-0.1 0

I

0

50

I

I

100 150 Votume (mr)

I

200

Fig. 5. Hydroxyapatite chromatography in dodecyl sulfate of (A) cytochrome b from [3H]leucinelabelled Neurospora crassa and (B) a mixture of subunit I from cytochrome b of [aHlleucine-labelled Neurospora crassa plus subunit 1I from cytochrome b of ['4C]leucine-labelled Neurospora crassa. 0.2~).5 mg cytochrome b in 2-5 ml 0 . 2 ~ sodium dodecyl sulfate, 0.0l M sodium phosphate, pH 6.4, and 2 m M dithioerytritol were applied on a 0.6 × 8.5 cm column of Bio-Gel H T (Bio-Rad, Richmond, California) equilibrated in the above-mentioned solution. They were eluted with a linear 200-ml gradient ranging from 0.25 to 0.55 M sodium phosphate containing in addition 0.2 ~ dodecyl sulfate and 2 m M dithioerytritol. The flow rate was 3.5 ml/h, the temperature 28°C. • •, 3H-radioactivity; O - - - - O , 14C-radioactivity; . . . . , sodium phosphate.

real fingerprints from the two cytochrome b subunits are not yet available as (a) the peptide fragments obtained by cyanogen bromide cleavage are insoluble in the solvents usually applied for two dimensional peptide mappings and (b) cytochrome b proved to be very resistent to digestion with trypsin or chymotrypsin [50].

302

10 `3 10

8

6

I

i

I

apparent molecular weight 4 2 I

I

800-

600-

× 400? E x

200c: z~

8 ~

0 J

600-

t,O0-

200-

0

|

I

I

I

20

25

30

35

I

40

45

Volume (mr)

Fig. 6. Gel filtration of the fragments resulting from cyanogen bromide cleavage of (A) [aH]leucine. labelled cytochrome b ( Q - - O ) and (B) a mixture of subunit I from [aH]leucine labelled cytochrome b (O O) plus subunit II from [~4C]leucine-labelled cytochrome b ( ~ - - - A ) . For cyanogen bromide cleavage, 30/~g cytoehrome b was dissolved in 100 Ftl 90% formic acid. 3 mg cyanogen bromide were added, the tube was flushed with nitrogen and sealed. The reaction medium was applied 27 h later on a 0.6 × 250 cm Bio-Gel P-30 column in 85 % formic acid. At a hydrostatic pressure of 120 cm the flow rate was 0.5 ml/h. The temperature was 25°C [60]. VI. AMINO ACID COMPOSITION OF CYTOCHROME b The a m i n o acid compositions of the various cytochrome b preparations discussed in the above chapters are c o m p a r e d in T a b l e II. The four preparations displayed a similarly high c o n t e n t of leucine, isoleucine and alanine a n d a low c o n t e n t of lysine, histidine a n d arginine. The resulting low polarity [65] of 33-38 is characteristic of integral m e m b r a n e proteins.

303 TABLE lI AMINO ACID COMPOSITION OF MITOCHONDRIAL CYTOCHROME b FROM BEEF HEART S A C C H A R O M Y C E S C E R E V 1 S I A E AND N E U R O S P O R A C R A S S A Results are expressed in mol ~. Amino acid

Asx Thr Ser Glx Pro Gly Ala Cys Val Met lle Leu Tyr Phe Lys His Arg

Cytochrome b from Beef heart a 6.4 7.0 5.8 5.8 4.5 9.6 10.2 1.9 5.8 3.8 7.7 12.8 4. 5 7.0 2.6 1.9 3.2

Polarity [65] 33

Beef heart s

Saccharomyces cerevisiae c

Neurospora crassa d

Subunit I

Subunit 1I

7.9 6.4 5.8 5.6 6.0 7.9 7.9 2.4 5.1 3.3 7.6 13.3 3.6 5.5 3.7 3.0 2.9

8.6 5.3 5.7 6.6 4,9 6,5 7.6 9.4 2.6 8.1 11.5 3.4 8.4 4.4 2.9 3.9

8.9 5.0 8.3 7.7 4.9 7.9 7.6 1.2 6.8 2.2 8.3 12.1 4.1 5.8 3.8 2.5 4.3

8.7 5.0 8.6 5.9 4.9 7.9 7.6 2.7 6.9 2.8 8.6 13.3 4.2 6.8 2.8 2.6 3.7

35

37

38

37

a Ohnishi [44]. b Yu et al. [46]. c Katan et al. [47,48]. d Weiss et al. [28,60].

The a m i n o acid compositions of the two subunits of the cytochrome b prep a r a t i o n f r o m N e u r o s p o r a c r a s s a [28] are very similar. Differences were f o u n d only in their lysine a n d cystein contents. The resulting difference in the lysine to leucine ratio between the two subunits was also observed when a cytochrome b p r e p a r a t i o n from cells simultaneously labelled with [aH]lysine a n d [~4C]leucine was subjected to hydroxyapatite c h r o m a t o g r a p h y : a n u n e q u a l distribution of all- artd 14C-radioactivity between the two polypeptides was o b t a i n e d c o r r e s p o n d i n g to the difference in their lysine to leucine ratios [60]. However, whether these differences between the two s u b u n i t s really exist or whether they are caused by lysine- a n d cystein-rich c o n t a m i n a t i o n s present in the p r e p a r a t i o n c a n n o t yet be decided with certainty. A t t e m p t s to determine the a m i n o e n d - g r o u p of cytochrome b from N e u r o s p o r a c r a s s a by m e a n s of the dansyl chloride m e t h o d have n o t given a n y result, n o r could

304 the protein be attacked by the solid-phase Edman degradation*. This suggests that the N-terminus of this protein is masked [50]. The C-terminal amino acids of the two cytochrome b subunits from Neurpspora crassa were determined by means of enzymatic hydrolysis. No amino acid was released by the action of carboxypeptidase A alone. Carboxypeptidase B released lysine from each of the two subunits. The combined action of carboxypeptidase A and B produced a release of lysine, tyrosine and leucine. These data suggest that both subunits have the C-terminal sequence Lys - Tyr - Leu [50].

vii. HAVE DIFFERENT CYTOCHROME b SPECIES BEEN ISOLATED FROM THE INNER MITOCHONDR[AL MEMBRANE? Several authors [66-68] have reported that a 15 000-17 000 molecular weight polypeptide of Complex IlI is a cytochrome b apoprotein. Their suggestion was based on the observation that this polypeptide seemed to be the main constituent of cytochrome b preparations analysed by dodecyl sulfate gel electrophoresis. The protein quantitation applied, however, presumed that all polypeptides present in the preparation displayed an equal specific Coomassie blue staining and that the protein which remained attached to the origin of the gel had a polypeptide composition identical to that which migrated into the gel. With membrane proteins, both presuppositions are seldom fulfilled. Peculiarly the 30 000 molecular weight cytochrome b polypeptide band shows an unusually low specific protein staining and has a strong tendency to form aggregates even in the presence of dodecyl sulfate [50]. Consequently, the relative amount of this polypeptide present in crude cytochrome b preparations might have easily been underestimated. Yu et al. [46] reported on the isolation of a cytochrome b with the molecular weight 17 000. Their experimental basis was the coelution of protoheme with a 17 000 molecular weight protein fraction upon Sephadex gel filtration in a dodecyl sulfate solution. The reported data did not prove conclusively that the protoheme is protein-bound. Free protoheme dissolved in dodecyl sulfate displays a light absorption spectrum and an apparent molecular weight upon gel filtration in dodecyl sulfate very similar to those reported by these authors for the 17 000 molecular weight preparation. viii. BIOGENESIS OF CYTOCHROME b A. Mitoehondrial inheritance

It is now well established that mitochondria contain DNA as well as all the enzymatic apparatus necessary for transcription and translation of the genetic information into proteins [14,15]. The mitochondrial DNA, however, is small and can

* The solid-phase Edman degradation was kindly assayed by Dr. E. Wachter, Mfinchen.

305 only accommodate genes for a rather limited number of proteins. Cytochrome b, as well as cytochrome oxidase and oligomycin-sensitive adenosine triphosphatase, have for a long time been considered possible candidates for intramitochondrial coded proteins, as mitochondrially inherited mutations in fungi [14,15,23] were found to cause respiratory deficiencies due to the loss of these enzymes. Most of these mutants, however, were pleiotropic with a general failure or loss of mitochondrial protein synthesis. The poky-mutation of Neurospora crassa [55, 56,69-71] is now considered to be a consequence of mitochondrial ribosome deficiency, which leads to a decrease of mitochondrial protein synthesis in general. The cytoplasmic petite or o--mutants of Saccharomyces cerevisiae [72-74] have deleted large regions of mitochondrial DNA that code for ribosomal and transfer RNA and thus showed no mitochondrial protein synthesis at all. Therefore, these mutations of mitochondiral DNA could only support evidence that an intact system of mitochondrial protein synthesis is necessary for the biogenesis of cytochrome b. Only recently, Tzagoloff et al. [75] succeeded in the selection of cytoplasmic mutants of Saccharomyces cerevisiae lacking the ability to grow on non-fermentable substrates, due to the specific lack of cytochrome b, but which have retained mitochondrial protein synthesis. Slonimski and Tzagoloff [76] called this new class of mutant mit--mutants. By means of genetic analysis, namely mapping of the mit-mutations relative to each other and to known genetic drug resistant markers, one distinct region on the mitochondrial DNA could be established in which mutations led to a selective loss of cytochrome b. It could not yet be decided, however, whether this part of mitochondrial DNA represents the structural gene of cytochrome b or a regulatory gene necessary for translation or assembly of cytochrome b. B. Mitochondrial translation The proteins of mitochondria can be labelled selectively according to their site of translation, namely the mitochondrial or the cytoplasmic ribosomes, by in vivo incorporation of radioactive amino acids in the presence of either cycloheximide, a specific inhibitor of cytoplasmic protein synthesis [77-80] or chloramphenicol, a specific inhibitor of mitochondrial protein synthesis [81-84]. For these labelling studies, the fungi Neuro~pora erassa and Saccharomyces ¢erevisiae have been widely used, due to the fact that the catabolism of radioactive amino acids added to cultures of these microorganisms is small compared to the incorporation into proteins. In order to define the site of biosynthesis of cytochrome b, Weiss and coworkers [22,28,60] used the following three procedures of double-labelling of Neurospora crassa proteins: [~H]leucine (1.5 mCi/g protein) was first added to an exponentially growing culture and was incorporated for 2 h. Then [l~C]leucine (0.2 mCi/g protein) was added, either in the absence of inhibitors (procedure l) or 2 min after the addition of cycloheximide (0.01 mg/ml, procedure ll) or 2 min after the addition of chloramphenicol (4 mg/ml, procedure 11I). The ceils were harvested 2 h later. Then cytochrome b was purified and analysed for 3H and 14C-radioactivity (a) after gel filtration in deoxycholate and salt solution, i.e. under conditions where the heine

306 protein remained intact, and (b) after gel electrophoresis and hydroxyapatite chrom a t o g r a p h y in dodecyl sulfate solution, i.e. under conditions where the heme protein is dissociated into subunits and heine (Table lII, Figs. 7, 8 and 9).

TABLE Ill THE EFFECT OF CYCLOHEXIMIDE AND CHLORAMPHENICOL ON THE INCORPORATION OF [14C]LEUCINE INTO VARIOUS PROTEIN PREPARATIONS AND CYTOCHROME b FROM NEUROSPORA CRASSA [3H]leucine was incorporated one generation before the addition of the inhibitors and of [t4C]leucine. For labelling procedures, see text. Labelling procedure

Preparation

Specific 3Hradioactivity of protein (cpm "/~g-~)

'4C/3H ratio of protein

I.

Absence ofinhibitors

Cytoplasma Mitochondrial matrix b Mitochondrial membrane c Cytochrome b

640 590 595

0.34 0.33 0.35 0.33

1I. Presence of cycloheximide

Cytoplasm Mitochondrial matrix Mitochondrial membrane Cytochrome b

630 610 615

0.01 0.01 0.08 0.31

111. Presence of chloramphenicol

Cytoplasm Mitochondrial matrix Mitochondrial membrane Cytochrome b

595 610 580

0.33 0.30 0.28 0.12

• 100 000 x g supernatant of disrupted cells [54]. b 100 000 × g supernatant of sonicated mitochondria [49]. c 100 000 × g sediment of sonicated mitochondria [49].

Under conditions of control labelling, the 14C/3H ratio, which represents the relative specific 3H content of protein, was fairly constant in all cell proteins, including the c y t o c h r o m e b peaks obtained u p o n gel filtration and gel electrophoresis (Table III, Figs. 7A and 8A). In cells treated with cycloheximide, the proteins of the cytoplasm and the mitochondrial matrix and 80-90 ~,, of the proteins of the mitochondrial m e m b r a n e were no longer synthesized. Their 14C/3H ratios were only a few of the percent of the corresponding ratios in untreated cells. In contrast, a large a m o u n t of [14C]leucine was incorporated into cytochrome b (Table III). The peak of 14C-radioactivity obtained u p o n gel filtration in deoxycholate coincided with the peak of heine absorbance and 3H-radioactivity (Fig. 7B). However, the peak of ~4C-radioactivity obtained u p o n gel electrophoresis in dodecyl sulfate was assymetrically distributed

307

10-3. apparent moLecuLar weight

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0,03-

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r-

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//~A~17nm / ~.

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-15

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III

I

1

25

30

0

35

VoLume (mr) Fig. 7. Gel filtration in deoxycholate of cytochrome b from Neurospora crassa having incorporated [3H]leucine in the absence of inhibitors and [14C]leucine (A) in the absence of inhibitors, (B) in the presence of cycloheximide, or (C) in the presence of chloramphenicol. Column: 0.7 x 150 cm, Sephadex G-100 in 0.25% potassium deoxycholate, 0.25% potassium cholate, 0.2 M KCI and 0.05 M Tris-acetate pH 8.

308

I0-3x -

apparent mol.ecuLar weight L,O 30 20 10 I

I

I

I

-~00

1ooiA 3H

-200

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0 -600

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-400

.-~ 100,,~

-200

~S

~S 0

~150-

0 I

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-t,00

100-200

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20 t+O Number of gel. slice

60

Fig. 8. Gel electrophoresis in dodecyl sulfate of cytochrome b from Neurospora crassa having incorporated [~H]leucine in the absence of inhibitors and [14C]leucine (A) in the absence of inhibitors, (B) in the presence of cycloheximide or (C) in the presence of chloramphenicol.

309 A

.~_~1000

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Fig. 9. Hydroxyapatite chromatography in dodecyl sulfate of cytochrome b from Neurospora crassa having incorporated [aH]leucine in the absence of inhibitors and [t4C]leucine in the presence of cycloheximide. • • , 3H-radioactivity; ©- - -C), z4C-radioactivity;------, sodium phosphate. over the 3H-radioactivity peak (Fig. 8B). This suggested that the 3H-radioactivity peak resulted from the overlapping of two different polypeptides with different 14C/3H ratios, a suggestion which was confirmed upon hydroxyapatite chromatography of this double labelled cytochrome b preparation (Fig. 9). In cells treated with chloramphenicol, the synthesis of the proteins of cytoplasm and 80-90~ of the proteins of mitochondrial membrane and mitochondrial matrix remained unaffected, but no distinct [14C]leucine incorporation into cytochrome b took place (Table III, Fig. 7C, Fig. 8C). These labelling experiments provided direct evidence that the two subunits of Neurospora crassa cytochrome b were translated on mitochondrial ribosomes. A result consistent with that obtained by Weiss and co-workers [22,28,60] for Neurospora crassa was reported for Saccharomyces cerevisiae by Katan and coworkers [47]. These authors purified Complex III from cells in which the mitochondrially synthesized proteins have been selectively labelled by in vivo incorporation of [3H]leucine in the presence of cycloheximide. Subsequent separation of the polypeptides of Complex III by dodecyl sulfate gel electrophoresis and analysis of radioactivity among them displayed labelling of only the polypeptide at the molecular weight 32 000. This polypeptide was the main constituent in a further purified cytochrome b preparation. It showed an amino acid composition very similar to that of the cytochrome b preparation described by other authors (Table II). The mitochondrial translation of cytochrome b has also been established for Locusta migratoria [24]. The flight muscle of this insect proved to be an appropriate tissue for these studies. After the imaginal moult it rapidly develops from a precursor

310 muscle, showing a 50-fold increase of mitochondrial mass within a few days [86]. During this time, the catabolism of injected radioactive leucine is small compared to its incorporation into the muscle protein. The cytochrome b preparation from this insect showed strong similarity to the cytochrome b preparation from Neurospora crassa: during gel filtration in a deoxycholate and salt solution, it behaved like a homogeneous heme protein of molecular weight 55 000. Upon gel electrophoresis in a dodecyt sulfate solution, it migrated essentially as one protein band corresponding to a polypeptide with a molecular weight of 32 000 (Table I). When this cytochrome b was prepared from insects which were previously injected with a solution containing [3H]leucine plus cycloheximide, it displayed a specific 3H-radioactivity 40 times higher than the specific 3H-radioactivity of the cytoplasmic proteins. In the control experiment, injection of [3H]ieucine in the absence of cycloheximide, the specific radioactivity of cytoplasmic proteins and of cytochrome b were found to be of the same order of magnitude. The reason for carrying out these experiments with Loeusta migratoria was the fact that the length of mitochondrial DNA in insects and vertebrates is only 5 #m, whereas the length in fungi and higher plants is 20-30 #m [14]. Consequently, the question arose whether genes present on the large mitochondrial DNA are missing on the small mitochondrial DNA. The conformity in mitochondrial translation of cytochrome b among Neurospora crassa and Saccharomyces cerevisiae, on one hand, and Locusta migratoria, on the other, is in agreement with results on the mitochondrial translation of cytochrome oxidase subunits in these two types of organisms [15-24], suggesting that the mitochondrial translation of these polypeptides is universal. This might be considered as a hint that the regions of mitochondrial DNA missing in higher animals but present in eucariotic microorganisms, contain no further information for proteins of the inner mitochondrial membrane. IX. CONCLUSION In the opinion of the author, only one type of cytochrome b has so far been purified from the inner mitochondrial membrane. This cytochrome b is a dimeric heine protein with a molecular weight in the range of 50 000-55 000. It consists of two polypeptides of about equal size each carrying one protoheme. The dimeric character of this cytochrome b has long been overlooked due to the fact that purified preparations could for a long time be obtained only in highly polymerized states. The author would like to suggest that this dimeric heme protein is the molecular basis for the two functionally different cytochrome b species, which have been found to form part of the ubiquinone cytochrome c reductase. This assumption, however, has yet to be confirmed by determination of the molar relationship between the two cytochrome b polypeptides, on one hand, and the other polypeptides of this electron transfer complex, on the other. In the polypeptide conglomerate of this Complex III, the two cytochrome b subunits might interact each with a different polypeptide leading to different con-

311 formations and thus to the different properties of the two heme centres. During isolation from their proper surroundings, these conformational differences might be equalized and the two heme centres would no longer be distinguishable. Such a different membrane environment of the two cytochrome b subunits, however, has yet to be experimentally established. The two cytochrome b polypeptides are translated on mitochondrial ribosomes and, although this has not yet been proved formally, they are believed to be specified by the mitochondrial DNA. Most probably, both polypeptides are coded for by only one gene, but are modified in different ways after translation. The biological importance of the fact that cytochrome b is a mitochondrial translation product, whereas the other polypeptides of Complex III are cytoplasmic translation products is not yet understood. It could be speculated that by means of this biosynthesis, the correct arrangement of the Complex III polypeptides in the membrane is guaranteed.

ACKNOWLEDGEMENTS The studies on the structure and the biogenesis of Neurospora crassa cytochrome b were started in the Institut ftir Physiologische Chemie and Physikalische Biochemie der Universitat Mfinchen. I am very pleased to acknowledge the fruitful collaboration and help that I experienced in this institute. I particularly wish to thank Professor Dr. Dr. h.c. Theodor Bficher, Dr. Walter Sebald and Dr. Walter Neupert for their advice and discussions. I also thank Dr. Gebhard von Jagow for discussion and for the contribution of unpublished results to this article. The more recent data on Neurospora crassa cyotchrome b have been obtained in collaboration with Dr. Barbara Ziganke at the European Molecular Biology Laboratory, Heidelberg. I am greatly indebted to Sir John Kendrew, the Director-General of EMBL, for his support and interest in this project. It was supported by the Deutsche Forschungsgemeinschaft.

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