Đột biến cytochrome b ty thể gây thiếu hụt enzyme chuỗi hô hấp nghiêm trọng ở người và nấm men: Báo cáo khoa học

A mitochondrial cytochrome bmutation causing severe

respiratory chain enzyme deficiency in humans and yeast

Emma L. Blakely

, Anna L. Mitchell

, Nicholas Fisher

, Brigitte Meunier

, Leo G. Nijtmans

Andrew M. Schaefer

, Margaret J. Jackson

, Douglass M. Turnbull

and Robert W. Taylor

1 Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, The Medical School, University of Newcastle upon

Tyne, UK

2 Wolfson Institute for Biomedical Research, University College London, UK

3 Nijmegen Center for Mitochondrial Disorders, Department of Pediatrics, Radboud University Nijmegen Medical Center, the Netherlands

4 Department of Neurology, Royal Victoria Infirmary, Newcastle upon Tyne, UK

The mitochondrial bc

complex (ubiquinol–cytochrome

coxidoreductase or complex III; EC 1.10.2.2) is a

membrane-bound enzyme that catalyses the transfer of

electrons from ubiquinol to cytochrome c, coupling

this process to the translocation of protons across the

inner mitochondrial membrane. In higher eukaryotes,

the enzyme complex is composed of 11 polypeptide

subunits. One subunit, cytochrome b, is encoded

by the mitochondrial genome (mtDNA) [1] whilst the

others are encoded by the nucleus and synthesized on

cytoplasmic ribosomes prior to being imported into

mitochondria and assembled into a functional com-

plex. As a hydrophobic, integral membrane protein

consisting of eight transmembrane helices, cyto-

chrome bis fundamental for the assembly and function

of complex III, and together with cytochrome c

and

the iron–sulfur protein (ISP) it forms the catalytic core

of the enzyme.

It has become apparent in recent years that dis-

orders of the mitochondrial respiratory chain due to

Keywords

Mitochondrial DNA (mtDNA); complex III;

complex I; cytochrome b; yeast mutants

Correspondence

R.W. Taylor, Mitochondrial Research Group,

School of Neurology, Neurobiology and

Psychiatry, The Medical School, Framlington

Place, University of Newcastle upon Tyne,

NE2 4HH, UK

Fax: +44 191 2228553

Tel: +44 191 2223685

E-mail: r.w.taylor@ncl.ac.uk

(Received 15 April 2005, accepted 19 May

2005)

doi:10.1111/j.1742-4658.2005.04779.x

Whereas the majority of disease-related mitochondrial DNA mutations

exhibit signiﬁcant biochemical and clinical heterogeneity, mutations within

the mitochondrially encoded human cytochrome bgene (MTCYB) are

almost exclusively associated with isolated complex III deﬁciency in muscle

and a clinical presentation involving exercise intolerance. Recent studies

have shown that a small number of MTCYB mutations are associated with

a combined enzyme complex defect involving both complexes I and III, on

account of the fact that an absence of assembled complex III results in a

dramatic loss of complex I, conﬁrming a structural dependence between

these two complexes. We present the biochemical and molecular genetic

studies of a patient with both muscle and brain involvement and a severe

reduction in the activities of both complexes I and III in skeletal muscle

due to a novel mutation in the MTCYB gene that predicts the substitution

(Arg318Pro) of a highly conserved amino acid. Consistent with the dra-

matic biochemical defect, Western blotting and BN-PAGE experiments

demonstrated loss of assembled complex I and III subunits. Biochemical

studies of the equivalent amino-acid substitution (Lys319Pro) in the yeast

enzyme showed a loss of enzyme activity and decrease in the steady-state

level of bc

complex in the mutant conﬁrming pathogenicity.

Abbreviations

BN-PAGE, Blue-native PAGE; ISP, iron–sulfur protein; MELAS, mitochondrial encephalopathy, myopathy, lactic acidosis and stroke-like

episodes; MERRF, myoclonic epilepsy and ragged-red fibres; MTCYB, mitochondrial cytochrome bgene; mtDNA, mitochondrial DNA;

MRI, magnetic resonance imaging; Q

, quinol oxidation site; Q

, quinol reduction site; RFLP, restriction fragment length polymorphism;

RRF, ragged-red fibre.

FEBS Journal 272 (2005) 3583–3592 ª2005 FEBS 3583

pathogenic mutations in mtDNA genes are amongst

the most common inherited human metabolic diseases

[2]. Many pathogenic mtDNA mutations are hetero-

plasmic, affecting a population of mitochondrial

genomes within the cell, and all cause disease by a

common mechanism, the impairment of cellular oxida-

tive phosphorylation [3]. Patients can present with a

wide spectrum of clinical phenotypes affecting predo-

minantly skeletal muscle, heart and the CNS, which is

dependent upon the abundance and segregation of

the causative mutation, the gene that is mutated and

the associated biochemical defect; point mutations in

mitochondrial tRNA genes and large-scale mtDNA

rearrangements cause multiple respiratory chain abnor-

malities through impairment of mitochondrial trans-

lation [4], whilst mutations in protein-coding genes

generally affect only a single enzyme complex [5,6].

A number of patients with respiratory complex III

deﬁciency due to nonsense, missense or frameshift

mutations in the cytochrome b(MTCYB) gene have

now been described [7]. Although MTCYB mutations

are associated with numerous clinical presentations

including mitochondrial encephalopathy [8,9], cardio-

myopathy [10,11], septo-optic dysplasia [12] and a

multisystem disorder [13], the overwhelming majority

have been reported in patients with severe exercise

intolerance and myopathy, often associated with mus-

cle weakness and ⁄or myoglobinuria and evidence of

mitochondrial proliferation as evidenced by ragged-red

ﬁbres (RRF) on muscle biopsy [14–20]. In many of

these patients the genetic defect arises sporadically,

does not appear to be transmitted to offspring and is

restricted to skeletal muscle with no evidence of the

mutation in mitotic tissues such as peripheral lympho-

cytes. Although this absence of pathogenic mutations

in ﬁbroblasts and platelets precludes their study in

transmitochondrial cybrid cell lines, signiﬁcant insight

into the underlying molecular mechanism of human

MTCYB mutations has been made by introducing

the equivalent amino-acid substitutions into the yeast

(Saccharomyces cerevisiae) enzyme [21,22].

A curious observation in some patients with

MTCYB mutations is that whilst the genetic defect

resides within a structural component of complex III,

the in vitro assay of respiratory chain enzyme activities

in muscle mitochondria reveals deﬁciency of both

complexes I (NADH–ubiquinone oxidoreductase) and

complex III [16,18,19]. Conversely, mutation of the

nuclear encoded complex I gene NDUFS4 also results

in a reduction of complex III activity in addition to

complex I deﬁciency [23]. Recent studies using both

human and mouse cell lines harbouring deleterious

MTCYB mutations have demonstrated a structural

dependence among complexes I and III, highlighting

the need for a fully assembled respiratory complex III

for the stability and activity of complex I [24]. Further-

more, these experiments corroborate previous two-

dimensional blue native electrophoresis data which

revealed that individual respiratory chain complexes

physically interact with each other to form supercom-

plexes [25].

Here we report our studies in a patient with mito-

chondrial encephalopathy and a previously unreported

15 699 GﬁCMTCYB mutation that predicts a highly

conserved amino-acid substitution (Arg318Pro) in the

C-terminal portion of the protein. In agreement with

recent ﬁndings, analysis of the patient’s muscle biopsy

reveals a severe biochemical deﬁciency in the activities

of both complex III and complex I which is reﬂected

by a dramatic loss in the steady state levels of both

complexes, whilst modelling of the equivalent mutation

in yeast (Lys319Pro) reveals a bc

defect, thus conﬁrm-

ing the pathogenicity of the human 15 699 GﬁC

mtDNA transversion.

Results

Biochemical analysis of patient’s muscle biopsy

reveals a combined deficiency of complexes I

and III

Enzyme histochemistry of the patient’s muscle revealed

normal activities of both cytochrome coxidase (COX)

and succinate dehydrogenase, although approximately

1% of total ﬁbres showed evidence of subsarcolemmal

accumulation of mitochondria, typical of ragged-red

ﬁbres. Measurements of the individual respiratory

chain complexes in patient muscle mitochondria

revealed speciﬁc defects involving complexes I and III

activities (Table 1), both of which were decreased to

approximately 5% of control values. Furthermore, the

activities of these enzymes and other respiratory chain

complexes were entirely normal in muscle from both

the patient’s clinically unaffected mother and her sister

(Table 1).

Mitochondrial DNA analysis identifies a novel

MTCYB mutation

The patient’s clinical presentation, together with the

histochemical and biochemical ﬁndings in muscle,

prompted us to pursue a mitochondrial genetic abnor-

mality. Following a negative screen for common

pathogenic mtDNA mutations including the 3243

AﬁG mutation commonly associated with the

MELAS (mitochondrial myopathy, encephalopathy,

Novel cytochrome bmutation in human and yeast E. L. Blakely et al.

3584 FEBS Journal 272 (2005) 3583–3592 ª2005 FEBS

lactic acidosis and stroke-like episodes) phenotype,

we determined the sequence of the entire coding region

of the mitochondrial genome. This revealed several

common polymorphic sequence variants and a novel

GﬁC transversion at position 15 699 in the MTCYB

gene (Fig. 1A) that had not previously been reported

[7]. The 15 699 GﬁC mutation, which is predicted to

change an evolutionary conserved arginine to proline

at amino-acid position 318 of the human protein

(Fig. 1B), destroys an MwoI restriction site permitting

the assessment of heteroplasmy at this site by PCR-

RFLP analysis. This clearly demonstrated that the

mutation was heteroplasmic, with highest levels present

in the patient’s muscle (88% mutant load). Consistent

with a pathogenic role for this mutation, lower levels

were evident in mitotic tissues including urinary epithe-

lia (16% mutant load), circulating lymphocytes (13%

mutant load) and hair shafts (14% mutant load).

Moreover, the 15 699 GﬁC mutation was undetecta-

ble in skeletal muscle from the patient’s mother and

sister and urinary epithelial cells from her son

(Fig. 2A), suggesting that it had arisen de novo in the

patient and had not been transmitted to her child who

was clinically unaffected.

Distribution of 15 699 GﬁC mutation

in single fibres

Further supportive evidence to suggest that the 15 699

GﬁC mutation was responsible for the muscular

symptoms was provided by studies of individual skel-

etal muscle ﬁbres to determine whether the higher

amounts of mutant mtDNA were present in those

ﬁbres showing mitochondrial accumulation (COX-posi-

tive RRF) than those that did not (non-RRF). Using

PCR-RFLP analysis, we did detect higher levels of

the 15 699 GﬁC transversion in RRF [92.6 ± 1.25%

(n¼7)] than non-RRF [86.2 ± 2.43% (n¼9)],

although this just failed to reach statistical signiﬁcance

(P¼0.0513, Student’s t-test) (Fig. 2B).

The 15 699 GﬁC mutation is associated with

decreased steady–state levels of respiratory chain

complex I and III subunits

Western blotting studies were performed to investigate

the effect of the 15 699 GﬁC mutation on the expres-

Table 1. Respiratory chain complex activities in skeletal muscle mitochondria. The activities of complexes I and III are markedly decreased

in the patient’s muscle, whilst all respiratory chain complex activities were normal in muscle biopsies from the patient’s mother and sister.

Enzyme activities are expressed as nmol NADH oxidized per min per unit citrate synthase (CS) for complex I, nmol DCPIP reduced per min

per unit citrate synthase for complex II (succinate–ubiquinone-1 reductase) and the apparent first-order rate constant per s per unit citrate

synthase for complexes III and IV (x 10

). Control values are shown as mean ± SD. DCPIP, 2,6-dichlorophenol-indophenol; SD, standard devi-

ation.

Complex I ⁄CS Complex II ⁄CS Complex III ⁄CS Complex IV ⁄CS

Patient 0.042 0.369 0.094 0.941

Patient’s mother 0.196 0.350 2.020 0.907

Patient’s sister 0.338 0.413 2.133 0.961

Controls 0.240 ± 0.060 0.320 ± 0.088 2.10 ± 0.75 1.34 ± 0.39

Fig. 1. Identification of a novel 15 699 GﬁCMTCYB mutation.

(A) Sequencing electropherogram showing the 15 699 GﬁC trans-

version in the patient (arrow) which is predicted to result in an

arginine to proline change at position 318. (B) Multiple sequence

alignment of the MTCYB gene highlighting the evolutionary conser-

vation of this region of the protein, including the positively charged

arginine at position 318 (shown in bold) and the two flanking amino

acids (boxed). Interestingly, the equivalent residue in yeast

(Lys319) is similarly positively charged.

E. L. Blakely et al.Novel cytochrome bmutation in human and yeast

FEBS Journal 272 (2005) 3583–3592 ª2005 FEBS 3585

sion of mitochondrial respiratory chain subunit pro-

teins. In agreement with enzyme activity measure-

ments, immunoblotting with a panel of monoclonal

antibodies demonstrated a severe and almost complete

reduction in the amount of complex I (NDUFA9) and

complex III (Core 2 protein) subunits in patient mus-

cle, whereas the steady-state levels of other respiratory

chain subunits were unaffected (Fig. 3A). BN-PAGE

and in-gel activity assays further conﬁrmed the

observed decrease in both complex I and III activities

(Fig. 3B).

Modelling the human Arg318Pro mutation

in yeast

Arg318 is very highly conserved in the vertebrate,

plant and bacterial sequence data (Fig. 1B), but is

replaced by another positively charged residue (lysine)

in yeast, in which the equivalent residue is Lys319. To

investigate further the mechanism by which the 15 699

G > C mutation disrupts complex III activity, we

used biolistic transformation to generate a yeast

mutant harbouring the Lys319Pro mutation. As with

all yeast mitochondrial mutants, the strain was homo-

plasmic with every copy of the mitochondrial genome

containing the mutation. First, we investigated the

effect of the mutation on the level of cytochromes in

whole cells. Cytochrome bcontent (based on the dithio-

nite reduced spectra in the visible region (Fig. 4) was

decreased by 50% of the wild type strain, while the

cytochromes cand oxidase contents were not affected

(Table 2). It seems therefore that the Lys319Pro muta-

tion hinders the assembly of the complex, an obser-

vation which might have been predicted from the

structural analysis. Lys319 is located at the C-terminal

region of a b-turn linking helices F2 and G (Fig. 5)

and probably acts as a backbone H-bond donor to

Asn316. Mutation of Lys319 to a proline would

remove this H-bond and probably be disruptive for

the geometry of the turn. Lys319 approaches with

4.5 A

˚of the 14 kDa subunit (although it is not

involved in strong interactions with this subunit) and

as such, any minor alteration of the architecture of the

associated turn may disrupt this interaction.

Mitochondrial membranes were prepared from the

wild type and mutant strains and the cytochrome c

reductase activity monitored spectrophotometrically as

a function of decylubiquinol (QH

) concentration. The

apparent V

and K

for QH

were calculated from

initial rate measurements using derived Eadie–Hofstee

plots (Table 2). The catalytic activity of the mutant

was decreased compared to wild type (V

value:

40 s

, 50% of the wild-type rate). The mutation

decreased the K

for quinol from 18 to 5 lm. The k

min

(an apparent second-order rate constant equal to

⁄K

) value for Lys319Pro was 8 lm

Æs

(com-

pared to 4.4 lm

Æs

for the wild type). The lower K

value indicates that the Q

site becomes saturated with

quinol at a lower concentration than the wild type,

suggesting slower rates of electron transfer from Q

.It

could be suggested that the mutation had long distance

effect and distorted the architecture of the Q

site.

Fig. 2. Quantitation of the relative amounts of mutant and wild-type

mtDNA in patient tissues by PCR-RFLP analysis. (A) In wild-type

mtDNA, MwoI cuts the amplified 169 bp product into three frag-

ments of 84 bp, 54 bp and 31 bp (band not shown). The 15 699

GﬁC mutation abolishes a restriction site for MwoI, yielding frag-

ments of 138 bp and 31 bp and permitting the detection of mtDNA

heteroplasmy at this site. Lane 1, uncut PCR product; lane 2, con-

trol DNA; lane 3, patient’s blood; lane 4, patient’s muscle; lane 5,

patient’s urinary epithelial cells; lane 6, patient’s hair shafts; lane 7,

muscle from patient’s mother; lane 8, muscle from patient’s sister;

lane 9, urinary epithelial cells from patient’s son. (B) Segregation of

the 15 699 GﬁC mutation in COX-positive RRF and COX-positive

non-RRF. The mutation load was determined in individual muscle

fibres by PCR-RFLP analysis, and clearly demonstrates higher mean

levels of the mutation (horizontal bars) segregating with fibres that

show mitochondrial accumulation (RRF, black circles) than non-RRF

(open circles).

Novel cytochrome bmutation in human and yeast E. L. Blakely et al.

3586 FEBS Journal 272 (2005) 3583–3592 ª2005 FEBS

However the sensitivity to Q

site inhibitors such as

stigmatellin, myxothiazol and azoxystrobin was not

altered (Table 2). Therefore it is unlikely that the

mutation causes major structural change at the Q

site,

which would have explained the change in K

Additionally, it appeared that the overall stability of

mutant bc

complex in crude membrane preparations

was not noticeably different from that of the wild type

strain (based on loss of activity over the course of the

day and sensitivity to increased detergent concentra-

tion). This is in contrast to the previously studied

Gly167Glu mutation, located in an extramembranous

helix close to the hinge region of the ISP. The Gly167-

Glu mutation, which was ﬁrst documented in a patient

with cardiomyopathy [10], affected the stability of the

enzyme, possibly due to an altered binding of the

ISP on the complex [22]. In the Lys319Pro mutant,

although the steady-state level of bc

complex in cells

and membrane samples was decreased, the assembled

complex was stable.

Discussion

On account of the clinical and genetic heterogeneity

exhibited by mitochondrial disorders, the investigation

and diagnosis of patients suspected of a respiratory

chain abnormality remain a considerable challenge.

Although some patients present with a well-recognized

clinical phenotype due to a speciﬁc mutation (in either

Fig. 3. Western blotting and Blue native gel electrophoresis. (A) Immunoblotting of human skeletal muscle homogenates. Equal amounts

(15 lg) of protein from a control (lane 1) and the patient (lane 2) were subjected to SDS ⁄PAGE and blotted onto a PVDF membrane prior

to incubation with a cocktail of monoclonal antibodies as described in the Experimental procedures. The patient’s muscle homogenate

demonstrated a remarkable loss of complex I and complex III subunits, but normal steady-state levels of complexes II, IV and V subunits.

(B) BN-PAGE and in-gel activity assays were performed exactly as described [35], with 40 lg muscle mitochondrial protein loaded onto the

gel for both a control subject (lane 1) and the patient (lane 2).

Table 2. Properties of the yeast Lys319Pro mutant.

Parameters Wild type Lys319Pro

Cytochrome content (nmolÆg

)

Cytochrome b4.10 2.10

Cytochrome c7.60 7.50

Cytochrome oxidase 0.88 0.84

activity

max

)8040

(lMdecylubiquinol) 18 5

(nM)

Antimycin 3.0 3.0

Stigmatellin 2.4 2.4

Myxothiazol 2.7 2.7

Azoxystrobin 21.0 17.0

Cytochrome content in intact cells estimated from the dithionite-

reduced spectra.

Calculated from the decylubiquinol cytochrome c

reductase assay as described in [21].

Concentration in inhibitor

required to obtain 50% bc1 activity as measured as above at 40 lM

decylubiquinol.

Fig. 4. Yeast mitochondrial cytochrome content in intact cells Cells

were grown on YPD plates for 24 h, resuspended in 5% ficoll at a

concentration of around 200 mL of cells per ml and optical spectra

of reduced cell suspensions were obtained as described in [21].

Standard line, wild type spectra; bold line, Lys319Pro mutant spec-

tra. C, cytochrome c; B, cytochrome bc

; A, cytochrome oxidase.

E. L. Blakely et al.Novel cytochrome bmutation in human and yeast

FEBS Journal 272 (2005) 3583–3592 ª2005 FEBS 3587

Báo cáo khoa học: A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deﬁciency in humans and yeast

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