Effects of mutations in mitochondrial cytochrome b in yeast and man : Deficiency, compensation and...

Eur. J. Biochem. 268, 1155±1162 (2001) q FEBS 2001

R E V I E W A R T I C L E

Effects of mutations in mitochondrial cytochrome b in yeast and manDeficiency, compensation and disease

Nicholas Fisher and Brigitte Meunier

Department of Biology, University College London, UK

The mitochondrial cytochrome bc1 complex is a key proton-

motive component of eukaryotic respiratory chains. The

mitochondrially encoded cytochrome b forms, with cyto-

chrome c1 and the iron±sulfur protein, the catalytic core of

this multimeric enzyme. Mutations of cytochrome b have

been reported in association with human diseases. In the

highly homologous yeast cytochrome b, several mutations

that impair the respiratory function, and reversions that

correct the defect, have been described. In this paper, we

re-examine the mutations in the light of the atomic structure

of the complex, and discuss the possible effect, at enzyme

level, of the human cytochrome b mutations and the

correcting effect of the reversions.

Keywords: bc1 complex; human disease; mutations; rever-

sions; yeast.

The mitochondrial bc1 complex is a membrane-boundenzyme, composed of up to 11 subunits, that catalyses thetransfer of electrons from ubiquinol to cytochrome c andcouples this electron-transfer process to the translocation ofprotons across the inner mitochondrial membrane. Itscatalytic mechanism, the Q-cycle, requires two distinctquinone-binding pockets, the quinol-oxidation (Qo) andquinone-reduction (Qi) sites, which are located on oppositesides of the membrane but linked by a transmembraneelectron-transfer pathway (Fig. 1). The latter is provided bycytochrome b, which, with cytochrome c1 and the iron±sulfur protein (ISP) forms the catalytic core of the enzyme.Cytochrome b is a hydrophobic integral membrane proteinencoded by the mitochondrial genome. In contrast, theother subunits of the bc1 complex are encoded by thenucleus and synthesized on cytosolic ribsomes beforeimport and assembly into the mitochondrial inner mem-brane. In humans, several point mutations of cytochrome bhave been associated with disease. In the highly similaryeast cytochrome b, point mutations have been reportedthat impair the respiratory competence of yeast, and alsointragenic reversions which correct the defect. The crystalstructure of the chicken and bovine cytochrome bc1

complexes have been determined and the co-ordinates areavailable (Brookhaven database: PDB 1 BCC and 3 BCCfor the avian enzyme; PDB 1BGY and 1BE3, for the bovineenzyme; for a review of the bc1 structure and function, see,for instance, http://www.life.uiuc.edu/crofts/bc-complex_site/index.html). The atomic structure of the yeast enzyme[1] has recently been solved but the co-ordinates have not

yet been released. We have re-examined the mutations inyeast and human cytochrome b and, from a structuralviewpoint, addressed two questions: first, how do thereversions correct the defect caused by the primarymutations in yeast; and secondly, what are the effects ofthe disease-related human mutations on the enzymaticfunction.

R E S P I R A T O R Y- D E F I C I E N C YM U T A T I O N S I N Y E A S T C Y T O C H R O M E bA N D T H E I R R E V E R S I O N S

A number of missense, nonsense or frameshift mutationsin yeast cytochrome b have been reported that impairrespiratory growth in yeast. Nonsense or frameshiftingmutations that result in truncated cytochrome b almostinvariably abolish complex assembly. The missense muta-tions may also impair the complex assembly or stability,leading to a dramatic decrease in the levels of assembledenzyme, or they may alter the catalytic activity of thecomplex with little effect on its assembly. Eleven missensemutations have been described that cause respiratorygrowth deficiency, i.e. the mutant cells are unable to growon medium containing glycerol as an aerobic respiratorysubstrate (Table 1, Fig. 3). Five mutations (G33D, G131S,L282F, G340E and G352V) affect the complex assembly orstability, as judged spectrophotometrically by a dramaticdecrease in the cytochrome b optical signal. Two mutations,W142R and M221K, inhibit the catalytic activity of thecomplex without impairing its assembly. Four mutations,C133Y, G137V, G137E and S206L, lead to an unusualphenotype, causing stringent respiratory growth deficiencyeven though the bc1 complex activity is partially active andenzyme content in cells is not, or only partly, decreased. Ithas been suggested that the mutations impede the proton-motive activity of the complex, although it retains a highelectron-transfer activity, giving rise to an uncoupled system.The decrease in proton transfer and subsequent impairmentof ATP synthesis may explain the growth impairment.

Correspondence to B. Meunier, Department of Biology, University

College London, London WC1E 6BT, UK.

E-mail: [email protected]

Abbreviations: ISP, iron±sulfur protein; LHON, Leber's hereditary

optic neuropathy.

(Received 10 November 2000, revised 3 January 2001, accepted

11 January 2001)

As these missense mutations cause respiratory growthdeficiency, revertants can be selected easily on respiratorymedium. Reversions are secondary genetic events thatrestore, sometimes only partially, the respiratory com-petence impaired by a primary deficiency mutation. Theymay be located at the same codon, or at a second site, whichcan be close to or far apart from the primary lesions.

Reversions close to the faulty site

The correction of the bc1 defect by reversions close to thefaulty site can usually be explained by the removal orlessening of steric or electrostatic hindrance induced by theprimary mutations. The majority of the reversions reportedin cytochrome b fall into this category.

Some deficiency mutations are only corrected by replac-ing the faulty residue. This is the case for the deficiencymutations G33D, W142R and M221K. The Ca hydrogenatom of G33 is within a hydrophobic environment at theQi site, 4.0 AÊ from the porphyrin ring of haem bh. Intro-ducing the bulkier and anionic aspartyl side chain at thisposition would be sterically and thermodynamicallyunfavourable, explaining the failure of the bc1 complex toincorporate haem and assemble in the G33D mutant. Thereversion G33A re-introduces a smaller group, which doesnot disrupt side chain packing or interactions around thehaem [2,3]. The mutations W142R and M221K result infully assembled but catalytically inactive bc1 complex[3±5]. The highly conserved W142 is located withinsurface helix cd1 on the P side (positive side) of themembrane. In W142R, the bulky arginine side chain maysterically hinder entry of quinol into the Qo site or causesubtle structural changes at Qo, impairing the catalyticactivity of this region. This steric hindrance would beremoved or lessened by the reversions W142S, W142T andW142K. M221, located at the N-terminus of transmem-brane helix E, is typically replaced by phenylalanine inhigher eukaryotes and forms a major structural componentof the Qi site. The mutation M221K is likely to induceelectrostatic repulsion with nearby H222, which wouldresult in local structural perturbation at the Qi site andinhibition of the reaction with substrate quinone. Two

Fig. 1. The catalytic mechanism of bc1: a quick overview. Redox

prosthetic groups are located within three subunits: cytochrome c1 and

the ISP, which are membrane proteins with large hydrophilic domains,

and cytochrome b, a predominantly hydrophobic protein consisting of

eight transmembrane helices which contains two haems b of differing

redox potential (low potential, bl, and high potential, bh) and forms the

two quinol-binding sites, Qo (site of quinol oxidation) and Qi (site of

quinol reduction). A quinol molecule binds at the Qo site, is

deprotonated, transfers one electron through the ISP and cytochrome

c1 to cytochrome c and forms a highly unstable semiquinone species,

which immediately reduces the bl. The electron is then transferred to

bh, then to a quinol bound at the Qi site, forming a stable semiquinone

species. A second quinol-oxidation event at the Qo site completes the

Q-cycle, with the formation of fully reduced quinol at the Qi site.

Overall, two molecules of quinol are oxidized to quinone at the Qo site

and one molecule of quinone is reduced to quinol at Qi, with the

concerted transfer of two protons per quinol oxidized from the N

(negative) to the P (positive) side of the inner mitochondrial membrane

(IMM). For further details of the catalytic mechanism see http://

metallo.scripps.edu/PROMISE/CYTBC1.html and http://www.life.

uiuc.edu/crofts/bc-complex_site/index.html and references within.

Table 1. Yeast mutations in cytochrome b.

Primary

mutation

Same-site

reversion

Second-site

reversion (, 10 AÊ )

Long-distance

reversion (. 10 AÊ ) References

Mutations affecting the enzyme

W142R K, T, S ± ± [5,8]

M221K E, Q ± ± [2,3,8]

Mutations affecting the complex assembly

G33D A ± ± [2,3]

G131S ± ± G260A [6,8,25]

L282F I, C, T N256I, I281F D287H [8,9]

G340E A,V, D340 C342G, E345G K288N [8,9]

G352V ND ND ND [26]

Mutations with untypical effects

C133Y S, D, N, F L130M, I176S A126T, H343T [6±9]

G137V A ± ± [6±8,11]

G137E C133S, H141Y, N256K I125T, I147F, F151L, [6±8,11,27]

S206L T, V N208Y, K, W30C [8,14,15]

1156 N. Fisher and B. Meunier (Eur. J. Biochem. 268) q FEBS 2001

reversions have been reported, M221Q and M221E [2]. Theintroduction of the uncharged glutamine would ease thiselectrostatic hindrance. A glutamate is likely to be toleratedbecause it is not buried within the hydrophobic interior ofthe membrane and energetically favourable charge com-pensation by nearby H222 is also likely to occur.

For the other mutations described, such as C133Y,G137E, S206L, L282F and G340E [6±9], the respiratory

defect is corrected by replacement of the altered residue orintroduction of another change in the vicinity (, 10 AÊ ) ofthe original mutation (Fig. 2A). C133 is located at theC-terminus of transmembrane helix C, and hence is also inclose proximity to residues forming the Qo site. Theintroduction of a bulky tyrosyl side chain into this regionmay be expected to cause local structural perturbation at theQo site, explaining the reduced level of haem b-565 bound

Fig. 2. Location of the yeast second-site reversions in the cytochrome b structure. The structure has been drawn with quanta from the

co-ordinates of the bovine enzyme. The numbering is according to the yeast sequence. The location of primary mutations causing the deficiency is

marked by circle. (A) Whole structure of cytochrome b and the location of deficiency mutations (positions 133, 137, 282, 340 at the P side and

position 206 at the N side) and `close to site' reversions. Each primary mutation and its set of reversions is indicated by a brace. (B) Enlargement of

the P side of the subunit showing three deficiency mutations and their long-distance reversions. The deficiency mutations in position 131, 133 and

137 and their reversions are marked in blue, green and white, respectively.

q FEBS 2001 Mutations in yeast and human cytochrome b (Eur. J. Biochem. 268) 1157

in the C133Y mutant [8]. The respiratory defect is correctedby the reversions C133S, D, N (all of which could poten-tially ease the steric problem) and F [6,7]. The latterchange, which restores the cytochrome b content to wild-type level, is surprising because the aromatic side chains oftyrosine and phenylalanine are of similar size. This wouldsuggest that the hydroxy moiety of tyrosine is apparentlyultimately responsible for impaired haem binding in theC133Y mutant. The hydroxy group of the tyrosine residuecould hydrogen bond with the side chain of S140 (animportant and highly conserved structural component of theQo site, located within helix cd1, which is within 5 AÊ ofresidue 133), possibly influencing the local fold around thisresidue. The deleterious effect of C133Y is also compen-sated for by the reversion I176S [6]. The side chain of I176is within 4.5 AÊ of residue 133 and so it is likely that I176Srelieves steric hindrance introduced by the bulky tyrosylgroup. Hydrogen-bonding between these two hydroxylatedresidues may also be a stabilizing factor. Respiratorycompetence is also restored to the C133Y mutant by thereversion L130M. The a-carbon atoms of these tworesidues are within 6 AÊ of each other, and mutation froma branched to non-branched aliphatic side chain at position130 is presumably favoured on steric grounds.

The conserved residue G137 is located on the P side ofthe membrane in a loop that links transmembrane helix C tosurface helix cd1. This region forms part of the Qo-bindingpocket and it is possible that the binding of quinol issterically hindered in G137E. In addition, the introductionof the charged side chain into the hydrophilic but pre-dominantly non-ionic environment of helix cd1 and associ-ated regions may cause local structural perturbation,although the overall assembly or stability of the bc1

complex in the mutant strain does not appear to be impaired[8,10]. Respiratory growth is partially restored by thesecond mutations C133S, H141Y and N256K [6]. C133S isa conservative structural change, and so it is not clear howthis reversion restores respiratory competence. It is con-ceivable that a deprotonated C133 thiol group (pK 8.2)could generate electrostatic conflicts with the glutamylgroup of G137E given the close proximity of these residues,which would be alleviated by the C133S reversion.Although N256 is located in the large surface loop con-necting transmembrane helix E to helix ef, the Ca atoms ofG137 and N256 are separated by only 5 AÊ . Therefore thelysyl side chain of the N256K reversion presumably charge-compensates for the glutamyl group of G137E. Thereversion H141Y is difficult to explain. Phenylalanine iscommonly found in this position in other organisms, butfrom a structural point of view it is not apparent why themutation to tyrosine should result in the restoration ofrespiratory competence to the G137E bc1 mutant. A secondmutation of G137 resulting in respiratory deficiency,G137V, has been described [11]. The introduction of abulky aliphatic side chain at this position is also likely tosterically hinder quinol binding. This suggestion issupported by the observation that reversion of this residueto alanine restores respiratory growth [6,7].

S206, located in the D-E loop on the N-side of themembrane, is a central structural component of the Qi site.The side chain of this serine residue has a 33 AÊ 2 contactwith the quinone headgroup, forming a hydrogen bond tothe methoxy oxygen atom [12]. This serine is highly

conserved in eukaryotes and has been proposed to form partof a quinone-binding motif in other respiratory andphotosynthetic systems [13]. Furthermore, the b-carbonatom of S206 in the wild-type bc1 complex is 5 AÊ from apropionate group of haem bh: the introduction of a branchedaliphatic side chain in the S206L mutant therefore may beexpected to generate some steric conflicts or generalstructual perturbation in this region. Indeed, there is adecrease in spectral cytochrome b content (45%) and aredshift of the cytochrome bh optical spectrum [8]. Thereversions S206T and S206V which restore respiratorygrowth to the wild-type rate [14] are likely to reduce thesesteric conflicts, although the side chains of valine andleucine differ by only a single methylene group. It isinteresting to note that valine is compatible with quinonebinding and turnover at the Qi site despite the inability ofits side chain to participate in hydrogen-bonding. Threesecond-site reversions have been described: W30C, N208Yand N208K [14,15]. The side chains of N208Y and K arebulkier than N208, but both could form hydrogen bonds toquinone bound at the Qi site, which may thus be stabilized.W30 is within 5 AÊ of S206 and the ring nitrogen atom ofthis aromatic residue is separated from a propionate groupof haem bh by a distance of less than 3 AÊ . A dipolar inter-action between these two groups is likely to occur. Mutationof this residue to the considerably smaller cysteine representsa major structural change and it is not clear why W30Cshould compensate for the original mutation.

The mutation L282F dramatically decreases the cyto-chrome b optical signal [8]. The invariant residue L282 islocated within the `PEWY' region (ef helix), forming closeatomic contacts with the Qo site antagonist stigmatellin[12]. The introduction of a bulky phenyalanine wouldgenerate steric problem and impair the assembly of theenzyme. This could be alleviated by the reported reversionsL282I, C and T [9]. L282I restores respiratory competenceto wild-type level, which would be expected becauseleucine and isoleucine are conservative structural isomers.The revertants L282C and T have poor growth rates, whichis not surprising because of loss of the leucyl structuralcomponent at the Qo site. The defect induced by L282Fis partially compensated for by N256I and I281F secondmutations. N256 is located within the surface loop con-nection helices E and ef, on the same side of the membraneas L282. The side chains of these two residues are < 7 AÊ

apart in the crystal structure of chicken bc1 complex, and soit is possible that the N256I reversion lessens somestructural steric hindrance in the L282F mutant complex,despite the similarity in size between the asparagine andisoleucine side chains. I281F may structurally stabilize theL282F mutant to a small degree by allowing favourablehydrophobic interactions between the aromatic moieties ofthe phenylalanine residues.

The G340D mutation affects the assembly or stability ofthe enzyme [8]. G340 is located at the C-terminus oftransmembrane helix G and is in close proximity to the Qo

site. This residue is not buried within the hydrophobicenvironment of the lipid bilayer, but is located within apredominantly hydrophobic pocket and so the introductionof the charged glutamic acid residue at the position wouldundoubtedly be a destabilizing event. Removal of thisanionic species by reversion to alanine (G340A), valine(G340V) or, remarkably, by the in-frame deletion of codon


340 restores the respiratory competence [9]. The respiratoryfunction is also, although only poorly, restored by thesecond-site reversions C342G and E345G [9]. The side chaincarboxylate moiety of E345 is <10 AÊ from the a-carbonatom of G340, and so replacing glutamate with glycine atposition 345 removes a possible source of destabilizingelectrostatic repulsion between the side chains in theG340E mutant. The reversion C342G introduces a moreconservative structural change, and may lessen some sidechain steric hindrance around E340.

The (partial) restoration of the respiratory function by thereversions located close to the primary mutations can beexplained by the lessening of the steric or electrostaticperturbations induced by the deficiency mutations. Incontrast, the beneficial effects of long-distance reversionare much more difficult to understand.

Reversions far from the faulty site. Second-site reversionwithin cytochrome b at positions far from the site of theoriginal mutation have been reported for the respiratorydeficient mutants G131S, C133Y, G137E, L282F andG340D. These long-distance reversions are situated atleast 10 AÊ away from the location of the original mutationwithin the native fold of the protein, ruling out a directinteraction between the side chains of the residues involved.Therefore, it is often very difficult to explain the effect ofthese reversions on the primary deficiency mutations forwhich they partly compensate. An exception is provided bythe long-distance second-site revertants for the respiratory-deficient mutant G137E. As described above, this glycineresidue is located in the vicinity of the Qo site. Respiratoryfunction is partially restored by the reversions I125T,I147F and F151L, situated 15±20 AÊ from G137 [6](Fig. 2B). These residues are structural components of theQo site, exhibiting close atomic contacts with stigmatellinin the inhibitor-bound avian crystal structure [12]. It ispossible that these long-distance reversions restore limitedrespiratory competence via subtle changes in the backbonefold at the Qo site, compensating for the hindranceintroduced by the G137E mutation.

The correcting effect of the other long-distance reversioncannot be explained from the structural informationcurrently available. The deficiency mutation C133Y, forexample, is partly compensated for by A126T and H343T[6,9] (Fig. 2B). Hydrogen-bonding between C133Y andthese new hydroxylated residues would be stabilizing.However, they are unlikely to be involved in directstructural interactions with Y133, however, because of the10±20 AÊ separation between the groups in the foldedcytochrome b. A limited respiratory growth is restored inL282F and G340D mutants by the introduction of D287Hand K288N, respectively, which are located 10±20 AÊ fromthe primary mutations [9]. No `close to site' reversions havebeen reported of the G131S mutations, but only a long-distance reversion G260A (Fig. 2B). G131 is located on theC-terminal side of transmembrane helix C, at the P sideof the membrane. The G131S mutation, which causes adramatic decrease in spectral cytochrome b, is likely tointroduce a major change in secondary or tertiary structureto cytochrome b, which either impairs the haem-bindingcompetence of the polypeptide or affects the stableassembly of the complex as a whole. This loss of cyto-chrome b signal is compensated for by a second-sitereversion, G260A. The introduction of G260A restores thecytochrome b content to the wild-type level whereas therespiratory-growth competence is only partially restored(4% of the wild-type level) [6]. G260 is located in the largeloop that links helices E and ef on the P side of themembrane which contains residues forming the `PEWY'motif of the Qo site. The a-carbon atoms of G131 and G260are separated by < 15 AÊ , and so it is not apparent why thesecond-site reversion should restore structural integrity(with albeit limited activity) to the complex.

The structural or mechanistic basis behind the beneficialeffect of long-distance reversions often cannot be readilyexplained. It is possible that long-distance second-sitereversion may result in subtle changes to the backbone foldor helical packing, which are mechanically transmittedthrough the protein structure, alleviating the effects of theoriginal defect to some extent. Changes to intersubunit

Table 2. Human mutations associated with diseases.

Mutation

Residues

in yeast Disease References

G34S G33 Exercise intolerance [18]

G166E G167 Cardiomyopathy [19]

D171N S172 LHON [24]

L236I M237 Cardiomyopathy [19,23]

D251±258 252±259 Myopathy [18]

G251D G252 Cardiomyopathy [21]

G290D G291 Exercise intolerance [22]

G339E G340 Myopathy [17]

V356M F357 LHON [24]

Fig. 3. Location of the yeast and human mutations in a model of

cytochrome b. Predicted secondary structure of yeast cytochrome b,

based on the avian cytochrome b structure of Zhang et al. [12]. Helices

are named ibid. The positions of the histidine ligands in haem bl (H82

and H183) and haem bh (H96 and H197) are underlined. The location

of the `PEWY' Qo site motif is indicated at the N-terminus of helix ef.

`PEWY' is an acronym for the highly conserved sequence Pro-Glu-

Trp-Tyr. The locations of the yeast respiratory-deficiency mutations (y)

and of residues associated with disease in humans (h) are indicated.


contacts with other components of the bc1 complex mayalso be a structurally stabilizing factor for some long-distance second-site revertants. It seems unlikely thatrespiratory deficiency induced by mutation of residuesdirectly involved in the chemistry at the quinone-bindingsites of the molecule could be ameliorated by beneficiallong-distance reversion. Such reversions are more likely toarise in strains where the assembly of the complex isimpaired. It is clear that more structural information isneeded to understand the effects of the mutations and theirreversions.

C Y T O C H R O M E b M U T A T I O N SA S S O C I A T E D W I T H D I S E A S E I NH U M A N S A N D T H E I R P R E D I C T E DE F F E C T O N T H E b c 1 C O M P L E X

Pathological mutations in human mtDNA result in respira-tory-chain dysfunction, with associated clinical manifesta-tions. Human diseases associated with mtDNA mutationinclude Leber's hereditary optic neuropathy (LHON),Kearns±Saye syndrome, stroke-like episodes (the MELASsyndrome), maternally inherited Leigh's syndrome, exer-cise intolerance and cardiomyopathy. Non-pathologicalpolymorphic variants have also been observed in humanmitochondrial genes such as the cytochrome b gene. Ithas been proposed that there are at least four differentcytochrome b genotypes in Caucasian individuals [16].

Unlike nuclear genes, mitochondrial genes are present inhundreds of copies in each somatic cell, and so hetero-plasmy is a common feature with pathological mtDNAmutations. The severity of the respiratory defect of the cells(and therefore the severity of the disease) is thus likely todepend on the proportion of mutant mtDNA.

Eight missense mutations and one in-frame short deletionin cytochrome b (Table 2, Figs 3 and 4) have been reportedin patients with various diseases, in addition to a fewnonsense mutations. The deleterious effect of nonsensemutations is easily explained because they result intruncated cytochrome b. The precise effects of the missensemutations on the enzyme activity have not been elucidated.However, their possible effect can be discussed withreference to their location in the structure and by com-parison with mutations in homologous cytochrome b.

The G339E (G340 in yeast) mutation observed in apatient with myopathy (exercise intolerance) has a directcounterpart in yeast, where it abolishes bc1 complexassembly [8,17]. The mutation is most likely to have thesame effect in human cells because of the similaritybetween yeast and human cytochrome b. The mutationG34S (G33 in yeast) has also been observed in a patientwith exercise intolerance [18]. As mentioned above, twomutations of G33 have been characterized in yeast. G33Dimpairs the assembly of the complex whereas G33Arestores the respiratory function [2]. The serine side chainof the human G34S mutation is non-ionic and less bulkythan aspartate, but could still perturb the local environment(and perhaps redox potential) of haem bh, affecting thecatalytic behaviour at Qi.

The mutation G166E has been found in a patient withsevere cardiomyopathy [19]. The equivalent residue inRhodobacter sphaeroides cytochrome b (G182) has beenmutated to serine, which impaired the quinol oxidation

activity of the complex [20]. G166 is located in theextramembranous cd2 helix of cytochrome b, a region notthought to be directly associated with quinol binding at theQo site. However, G166 is within 5 AÊ of residues formingthe flexible `hinge' of the ISP, a region of the moleculeessential for catalytic turnover at the quinol-oxidation site.This flexible `hinge' facilitates macroscopic movement ofthe hydrophilic domain of the ISP, allowing the (2Fe22S)redox centre to shuttle electrons from ubiquinol bound atQo to cytochrome c1. Replacement of G166 by a bulkier orcharged residue could interfere with the movement of theISP, given the close proximity of the two subunits in thisregion.

The mutation G251D has been observed in a patientsuffering from cardiomyopathy [21]. The residue is locatedin the E±ef loop on the P side of the membrane. G251 iswell (but not totally) conserved across phyla, and is presentin yeast. Mutation to Asp will probably lead to electrostaticconflicts with neighbouring D252 (present in man, butreplaced by His in yeast) and more importantly, E272(PEWY residue). The delta oxygen atom of E272 is 5 AÊ

from the a-carbon of G251 in the chicken structure. Qo-sitechemistry may be impaired by the mutation, either directlyor because of assembly/misfolding problems.

An in-frame deletion of eight amino acids (residues251±258) has been detected in the muscle tissue of apatient also suffering from exercise intolerance [18]. Such alarge deletion is likely to severely disrupt complex

Fig. 4. Location of the mutations associated with human diseases.

The structure was drawn with quanta from the co-ordinates of the

bovine enzyme. The numbering is according to the human sequence.


assembly and activity. The deletion is located in a largesurface loop connecting helices E and ef on the P side ofthe membrane. The deleted region does not containany residues thought to be involved in quinone chemistryor binding, but the surface loop involved does connectwith a key structural component of the Qo site, the efhelix. Assuming that the bc1 complex is still capable ofassembly, it is likely that the structure of the Qo site isimpaired by this deletion, and bc1 activity inhibitedaccordingly.

The mutation G290D is associated with this exerciseintolerance [22]. G290 is very well conserved and locatedin transmembrane helix F1, a region of the molecule in theclose vicinity of the Qo site. Exchanging glycine foraspartate in the hydrophobic interior of the lipid bilayer isthermodynamically unfavourable and likely to severelyimpair the folding or assembly of cytochrome b. If theprotein were able to fold into a native-like structure despitethe presence of the introduced aspartate, it seems reason-able to suggest that quinone binding or the chemistry at Qo

may be diminished.L236I has been observed in a patient suffering from

cardiomyopathy [23]. This is a very conservativestructural change, and indeed isoleucine is found at thisposition in many other organisms. L236 is located withintransmembrane E, a region of the protein not associatedwith haem or quinone binding, nor is this residue involvedin intersubunit contacts with other subunits of the complex.This mutation is unlikely to be deleterious to the bc1

complex.Mutations D171N and V356M have been discovered in

individuals suffering from LHON [24]. The patients alsoharboured mutations in the mitochondrially encodedND5 subunit of complex I, which are likely to be theprimary cause of the disease. V356 is not conservedbetween phyla and is not likely to be involved inbinding substrate ubiquinone or involved in structuralinteractions with other components of the complex.D171 is located in a hydrophilic region of the protein atthe N-terminus of helix D. The residue is in the nearproximity of a surface b-turn at the P-side domain ofthe ISP. This b-turn contains three highly conservedbasic residues (K90, R92 and K94) within < 10 AÊ ofD172. If the docking of the ISP on to the P-side surfaceof cytochrome b during the bc1 complex catalytic cycleis stabilized by favourable electrostatic interactionsbetween D171 and this basic region of the ISP, themutations D172N could weaken this putative associationand impair the rate of quinol oxidation at Qo.

On a structural basis, some `human' mutations such asG34S, G166E, G251D, D251-258, G290D and G339Eare likely to have deleterious effects on bc1 complexassembly or activity. Other mutations (D171N, L236I,V356M) are expected to have no or little effect. Thesepredictions need to be tested. A readily available methodis to rely on yeast mutants which harbour the samemutations (such as G339D) and use these models tocharacterize the effect of the mutations on therespiratory function. As yeast is amenable to mitochon-drial transformation, we are now introducing the humanmutations into yeast cytochrome b to study their effectsand, when the mutation is deleterious, to select and identifyreversions.

A C K N O W L E D G E M E N T S

This work is supported by a BBSRC Research Project Grant and a

MRC Career Development Award to B. M. We thank Elinor Thompson

for help with preparation of the manuscript and Dr GaeÈl Brasseur for

critical reading of the manuscript.

R E F E R E N C E S

1. Hunte, C., Koepke, J., Lange, C., Rossmanith, T. & Michel, H.

(2000) Structure at 2.3 AÊ resolution of the cytochrome bc1

complex from the yeast Saccharomyces cerevisiae co-crystallized

with an antibody Fv fragment. Struct. Fold. Des. 8, 669±684.

2. CoppeÂe, J.-Y., Tokutake, N., Marc, D., di Rago, J.-P., Miyoshi, H.

& Colson, A.-M. (1994) Analysis of revertants from respiratory

deficient mutants within the center N of cytochrome b in

Saccharomyces cerevisiae. FEBS Lett. 339, 1±6.

3. Brasseur, G. & Brivet-Chevillotte, P. (1995) Characterization of

mutations in the mitochondrial cytochrome b gene of Sacchar-

omyces cerevisiae affecting the quinone reductase site (QN). Eur.

J.Biochem. 230, 1118±1124.

4. Lemesle-Meunier, D., Brivet-Chevillotte, P., di Rago, J.-P.,

Slonimski, P.P., Bruel, C., Tron, T. & Forget, N. (1993) Cyto-

chrome b-deficient mutants of the ubiquinol±cytochrome c

oxidoreductase in Saccharomyces cerevisiae. Consequence for

the functional and structural characteristics of the complex. J. Biol.

Chem. 268, 15626±15632.

5. Bruel, C., di Rago, J.-P., Slonimski, P.P. & Lemesle-Meunier, D.

(1995) Role of the evolutionarily conserved cytochrome b

tryptophan 142 in the ubiquinol oxidation catalyzed by the bc1

complex in the yeast Saccharomyces cerevisiae. J. Biol. Chem.

270, 22321±22328.

6. di Rago, J.-P., Netter, P. & Slonimski, P.P. (1990) Intragenic

suppressors reveal long distance interactions between inactivating

and reactivating amino acid replacements generating three-

dimensional constraints in the structure of mitochondrial cyto-

chrome b. J. Biol. Chem. 265, 15750±15757.

7. di Rago, J.-P., Netter, P. & Slonimski, P.P. (1990) Pseudo-wild type

revertants from inactive apocytochrome b mutants as a tool for the

analysis of the structure/function relationships of the mitochon-

drial ubiquinol-cytochrome c reductase of Saccharomyces cere-

visiae. J. Biol. Chem. 265, 3332±3339.

8. Lemesle-Meunier, D., Brivet-Chevillotte, P., di Rago, J.-P.,

Slonimski, P.P., Bruel, C., Tron, T. & Forget, N. (1993)

Cytochrome b-deficient mutants of the ubiquinol-cytochrome c

oxidoreductase in Saccharomyces cerevisiae. J. Biol. Chem. 268,

15626±15632.

9. di Rago, J.-P., Hermann-Le Denmat, S., Paques, F., Risler, J.,

Netter, P. & Slonimski, P.P. (1995) Genetic analysis of the folded

structure of yeast mitochondrial cytochrome b by selection of

intragenic second-site revertants. J. Mol. Biol. 248, 804±811.

10. Giessler, A., Geier, B.M., di Rago, J.-P., Slonimski, P.P. & von

Jagow, G. (1994) Analysis of cytochrome b amino acid residues

forming the contact face with the iron±sulfur subunit of ubiquinol:

cytochrome c reductase in Saccharmoyces cerevisiae. Eur. J.

Biochem. 222, 147±154.

11. Tron, T. & Meunier-Lemesle, D. (1990) Two substitutions at

the same position in the mitochondrial cytochrome b gene of

Saccharmoyces cerevisiae induce a mitochondrial myxothiazol

resistance and impair the respiratory growth of the mutated strains

albeit maintaining a good electron transfer activity. Curr. Genet.

18, 413±419.

12. Zhang, Z., Huang, L., Shulmeister, V.M., Chi, Y.-I., Kim, K.K.,

Hung, L.-W., Crofts, A.R., Berry, E.A. & Kim, S.-H. (1998)

Electron transfer by domain movement in cytochrome bc1. Nature

(London) 392, 677±684.


13. Fisher, N. & Rich, P.R. (2000) A motif for quinone binding sites

in respiratory and photosynthetic systems. J. Mol. Biol. 296,

1153±1162.

14. Coppee, J., Brasseur, G., Brivet-Chevillotte, P. & Colson, A.

(1994) Non-native intragenic reversions selected from Saccharo-

myces cerevisiae cytochrome b-deficient mutants. J. Biol. Chem.

269, 4221±4226.

15. Brasseur, G., Coppee, J., Colson, A. & Brivet-Chevillotte, P.

(1995) Structure-function relationships of the mitochondrial bc1

complex in temperature-sensitive mutants of the cytochrome

b gene, impaired in the catalytic center N. J. Biol. Chem. 270,

29356±29364.

16. Andreu, A.L., Bruno, C., Hadjigeorgiou, G.M., Shanske, S. &

DiMauro, S. (1999) Polymorphic variants in human mitochondrial

cytochrome b gene. Mol. Genet. Metab. 67, 49±52.

17. Andreu, A.L., Bruno, C., Shanske, S., Shtilbans, A., Hirano, M.,

Krishna, S., Hayward, L., Systrom, D.S., Brown, R.H.J. &

DiMauro, S. (1998) Missense mutation in the mtDNA cytochrome

b gene in a patient with myopathy. Neurology 51, 1444±1447.

18. Andreu, A.L., Hanna, M.G., Reichmann, H., Bruno, C., Penn,

A.S., Tanji, K., Pallotti, F., Iwata, S., Bonilla, E., Lach, B.,

Morgan-Hughes, J. & DiMauro, S. (1999) Exercise intolerance

due to mutations in the cytochrome b gene of mitochondrial DNA.

N. Engl. J. Med. 341, 1037±1044.

19. Valnot, I., Kassis, J., De Chretien, D., Lonlay, P., Parfait, B.,

Munnich, A., Kachaner, J., Rustin, P. & RoÈtig, A. (1999) A

mitochondrial cytochrome b mutation but no mutation of nuclearly

encoded subunits in ubiquinol cytochrome c reductase (complex

III) deficiency. Hum. Genet. 104, 460±466.

20. Brasseur, G., Saribas, A.S. & Daldal, F. (1996) A compilation of

mutations located in the cytochrome b subunit of the bacterial

and mitochondrial bc1 complex. Biochim. Biophys. Acta 1275,

61±69.

21. Andreu, A.L., Checcarelli, N., Iwata, S., Shanske, S. & DiMauro,

S. (2000) A missense mutation in the mitochondrial cytochrome b

gene in a revisited case with histiocytoid cardiomyopathy. Pediatr.

Res. 48, 311±314.

22. Dumoulin, R., Sagnol, I., Ferlin, T., Bozon, D., Stepien, G. &

Mousson, B. (1996) A novel gly290asp mitochondrial cytochrome

b mutation linked to a complex III deficiency in progressive

exercise intolerance. Mol. Cell. Probes 10, 389±391.

23. Marin-Garcia, J., Liu, Y.P., Ananthakrishnan, R., Pierpont, M.E.,

Pierpont, G.L. & Goldenthal, M.J. (1996) A point mutation in the

cytb gene of cardiac mtDNA associated with complex III

deficiency in ischemic cardiomyopathy. Biochem. Mol. Biol. Int.

40, 487±495.

24. Johns, D.R. & Neufeld, M.J. (1991) Cytochrome b mutations in

Leber hereditary optic neuropathy. Biochem. Biophys. Res.

Commun. 181, 1358±1364.

25. Brivet-Chevillotte, P. & di Rago, J.-P. (1989) Electron-transfer

restoration by vitamin K3 in a complex III-deficient mutant of

Saccharmoyces cerevisiae and sequence of the corresponding

cytochrome b mutation. FEBS Lett. 255, 5±9.

26. Edderkaoui, B., Meunier, B. & Colson-Corbisier, A.-M. (1997)

Functional mapping reveals the importance of yeast cytochrome b

C-terminal region in assembly and function of the bc1 complex.

FEBS Lett. 404, 51±55.

27. Bruel, C., Manon, S., GueÂrin, M. & Lemesle-Meunier, D. (1995)

Decoupling of the bc1 complex in S. cerevisiae: point mutations

affecting the cytochrome b gene bring new information about the

structural aspect of the proton translocation. J. Bioenerg.

Biomemb. 27, 527±539.


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