Báo cáo khoa học: Disease-related mutations in cytochrome c oxidase studied in yeast and bacterial models pptx

While the stop mutation at the C-terminus of subunit 3 and the short deletion were highly deleterious and abolished the assembly of the mitochondrial enzyme, the four missense mutations

reported in association with a range of diseases In this work

we used yeast and bacterial mutants to assess the effect of

human mutations in subunit 1 (L196I) and subunit 3 (G78S,

A200T, DF94–F98, F251L and W249Stop) While the stop

mutation at the C-terminus of subunit 3 and the short

deletion were highly deleterious and abolished the assembly

of the mitochondrial enzyme, the four missense mutations

caused little or no effect on the respiratory function Detailed

analysis of G78S, A200T and DF94–F98 in Rhodobacter

tions in the catalytic core of cytochrome oxidase The yeast enzyme is highly similar to the human enzyme and provides

a good model to assess the deleterious effect of reported mutations The bacterial system allows detailed biochemical analysis of the effect of the mutations on the function and assembly of the catalytic core of the enzyme

Keywords: cytochrome oxidase; diseases; Rhodobacter; assembly

Cytochrome oxidase (complex IV), embedded in the inner

mitochondrial membrane, is the terminal enzyme complex

of the mitochondrial respiratory chain It catalyses the

reduction of oxygen to water and the translocation of

protons across the mitochondrial membrane This process

contributes to the electrochemical proton gradient, which is

then used to drive ATP synthesis by the ATP synthetase

Mitochondrial cytochrome oxidase is composed of up to 13

subunits Ten subunits in mammals (eight in yeast) are

encoded by the nuclear genome Three subunits (subunits 1,

2 and 3) are encoded by the mitochondrial genome They

form the catalytic core of the eukaryotic complex and are

homologous to the subunits of the aa3-type cytochrome c

oxidases of Rhodobacter sphaeroides and Paracoccus

deni-trificans Subunit 2 forms part of the docking site for

cytochrome c and binds CuA, the first electron acceptor

Subunit 1 binds heme a and the binuclear centre (heme

a3-CuB), the site of oxygen reduction Subunit 3 has no redox

centre; it has been shown to prevent suicide inactivation of

the Rhodobacter aa3-type oxidase via long range interactions

with the CuBcentre [1] The role of the nuclear encoded

subunits is unclear but many of them are likely to have an

assembly or stability function The assembly of the enzyme is

a complex process and requires a large number of nuclear factors, which have been identified by extensive studies of yeast respiratory mutants, deficient in cytochrome oxidase (reviewed in [2])

Cytochrome oxidase deficiency in humans is associated with a wide range of clinical phenotypes Most cytochrome oxidase deficiencies are autosomal recessive and usually show early onset and a fatal outcome Mutations have been found in four of the nuclear-encoded assembly factors, namely, Surf1p, Cox10p, Sco1p and Sco2p [3–7] Mutations in the nuclear-encoded structural subunits of the enzyme have not been observed but disease-associated mutations in the mitochondrially encoded subunits 1, 2 and 3 have been reported These mutations are relatively rare and the disease symptoms usually present during late childhood to adulthood They are associated with a variety

of clinical presentations including LHON (Leber’s here-ditary optical neuropathy), MELAS (mitochondrial ence-phalomyopathy, lactic acidosis and stroke like episodes), AISA (acquired idiopathic sideroblastic anaemia) and encephalomyopathy In addition to these mutations sus-pected to cause enzymatic dysfunctions, a number of nonpathogenic polymorphisms in the mitochondrial genes for subunits 1, 2, and 3 have been reported (www.mito map.org)

In this paper, we re-examine the disease mutations in the mitochondrially-encoded subunits 1 and 3 by using yeast and R sphaeroides mutants to explore their effect on the respiratory function We have previously produced five yeast strains, which carry mutations found in patients, A223S, M273T, I280T and G317S in subunit 1, and a short in-frame deletion DF94–F98 (human sequence) in subunit 3 In this work, another five human mutations were studied in yeast: L196I in subunit 1, and G78S,

Correspondence to B Meunier, Wolfson Institute for Biomedical

Research, University College London, Gower Street,

London WC1E 6BT, UK Fax: 44 20 79165994,

E-mail: b.meunier@ucl.ac.uk

Abbreviations: AISA, acquired idiopathic sideroblastic anaemia;

CCCP, carbonyl cyanide m-chlorophenylhydrazone; Cox, genes

encoding for cytochrome oxidase subunits; LHON, Leber’s hereditary

optical neuropathy; MELAS, mitochondrial encephalomyopathy

lactic acidosis and stroke like episodes.

(Received 20 November 2002, revised 23 January 2003,

accepted 27 January 2003)

A200T, F251L and W249Stop in subunit 3 (Fig 1,

Table 1) The subunit 3 mutations G78S, A200T and

DF94–F98 were also introduced in R sphaeroides This

allowed us to compare the effects of the mutations in two

different systems The yeast enzyme is highly similar to the

human enzyme (there is, for instance, 57% and 44% amino

acid identity between subunits 1 and 3, respectively) and

therefore provides a good model to assess the deleterious

effect of reported mutations The structure of the

R sphaeroides cytochrome oxidase is nearly identical to that of the catalytic core of the mammalian enzyme, as predicted by their sequence identities (54% and 49% between subunits 1 and 3, respectively) The bacterial system allows detailed biochemical analysis of the effect of the mutations on the function and assembly of the catalytic core of cytochrome oxidase

Table 1 Disease-related mutations in the mitochondrially encoded subunits of cytochrome oxidase.

Mutations Disease and references Yeast sequence Effect of the mutation in yeast References Subunit 1

McArdle’s disease [27]

Subunit2

Subunit 3

Fig 1 Location of disease-related mutations in

the catalytic core of cytochrome oxidase The

structure has been drawn from the

co-ordi-nates of the bovine enzyme [17] Subunits 1

and 3 are in light and dark blue, respectively.

The mutations are in yellow The hemes a and

a 3 are in red The numbering is according to

the human sequence, which is highly identical

to the bovine sequence (approximately 90%).

Spectra were generated by scanning cell suspensions reduced

by dithionite with a single beam instrument built in-house

The cells, grown on 1% yeast extract/2% peptone/3%

glucose plates for 48 h, were resuspended in 5% Ficoll at a

concentration of approximately 200 mg of cells per ml and

reduced by dithionite A quadratic baseline compensation

was carried out on the data as described in [10] to remove

the distortion of the baseline

Production of theRhodobacter sphaeroides mutants

In order to create the subunit 3 mutants G78S, A200T

(A205 in R sphaeroides) and DF94–F98 in the R

sphaero-ides aa3-type oxidase, plasmid pMB301 [11], containing only

coxIII (the gene for subunit 3) of R sphaeroides, was

mutagenized using the QuikChange site-directed

mutage-nesis system (Stratagene) Presence of the correct mutation

was verified by DNA sequencing of the altered coxIII genes

A 956-bp SmaI fragment was restricted from each of the

mutated pMB301 plasmids and cloned into pMB307 [11],

yielding pUC-based plasmids that contained coxI6Xhis on

one strand and the coxII-III operon (coxII, cox10, cox11,

coxIII) on the other An EcoR1–HindIII fragment that

contained all of these cox genes was restricted from the

pMB307 plasmids and cloned into pRK415 [12] in order to

create plasmids capable of replicating and expressing in

R sphaeroides These three pRK415-based plasmids,

pMAG78S, pMAA205T and pMADF94–F98, were each

conjugated into R sphaeroides YZ200, a strain with a

deletion of the genomic copy of the coxII-III operon [13], by

established methods [14]

Preparation of bacterial cytochromec oxidase,

measurement of oxygen consumption and proton

pumping activity, and determination of subunit 3

content

R sphaeroidescells were grown in minimal medium to late

exponential phase and cytochrome c oxidase was purified

from cytoplasmic membranes solubilized in

N-dodecyl-b-D-maltoside by chromatography on Ni2+-nitrilotriacetic

acid agarose (Qiagen) as previously described [13] Oxygen

reduction assays were as described in [1] and proton

pumping was measured in a stopped-flow apparatus as

described in [15] The content of subunit 3 was determined

by densitometry of Coomassie-stained SDS-urea gels [16]

using a Personal Densitometer and ImageQuant software

(Molecular Dynamics) Subunit 3/subunit 2 density ratios

catalytic core of human enzyme is essentially identical (90%) to that of the bovine enzyme, the structure of the bovine oxidase can be used to predict some of the conse-quences of the human mutations Most of the mutations discussed below occur in residues that are completely conserved in humans, yeast and R sphaeroides; the remain-ing residues are conservative replacement within conserved regions Six human mutations reported in patients suffering from a range of disorders were studied here: G78S, DF94– F98, A200T, F251L and W249Stop in subunit 3, and L196I

in subunit 1 (Table 1) As shown in Fig 1, the mutations are located far from the redox centres of cytochrome oxidase They were therefore unlikely to directly affect the catalytic activity of the enzyme but they might alter its assembly L196I, G78S and DF94–F98 are located at the interface between subunits 1 and 3 and might weaken the assembly of these two subunits A200T is close to residue S195, which is required for enzyme assembly [9] F251L and W249Stop, located at the C-terminal end of subunit 3 might alter its folding In order to assess the effect of the mutations

on the respiratory function, and in particular on the assembly and/or stability of the complex, the mutations were introduced into yeast mitochondrial genome using biolistic transformation methods as described in [8,9] The yeast mutants were then used to monitor the effect of the mutations on the respiratory growth and on the cytochrome oxidase content Three mutations, G78S, A200T and DF94– F98 in subunit 3 were chosen for more detailed analysis and introduced in R sphaeroides (Materials and methods) The effect of the mutations on oxygen consumption, proton-pumping activity, and on the binding of subunit 3 were examined

W249Stop and DF94–F98 in subunit 3 alter the assembly of cytochrome oxidase The W249Stop mutation, which is predicted to result in the loss of the last 13 amino acids of subunit 3, had a dramatic effect on respiratory function in yeast The cells were unable

to grow on respiratory medium (Fig 2B) The mutation abolished the assembly of the complex as no cytochrome oxidase signal was detected by optical spectroscopy (Fig 2A) That seems to indicate that the well-conserved C-terminal end of subunit 3 is required for the correct folding and assembly of the subunit into the oxidase complex This severe effect on enzyme assembly was identical to that induced by the short deletion DF94–F98 The deletion of F94–F98 in helix 3 is likely to severely

compromise the ability of subunit 3 to bind to subunit 1,

particularly as one of the principal contacts between the two

subunits is an ion pair of H103 (subunit 3)-D227(subunit 1)

located one turn above F98 [17,18] Shortening of helix 3

should disrupt this interaction The W249Stop mutation

may cause a similar disruption as the region of helix 7 from

W249 onwards comes close to the F94–F98 region of

helix 3 So, like DF94–F98, loss of the C-terminus of

subunit 3 could weaken the H103 salt bridge at the top

of helix 3 and disrupt assembly Because in yeast, subunit 3

is required for the assembly or stability of the other subunits

and unassembled subunits are rapidly degraded by the

AAA proteases (ATPases associated with diverse cellular

activities) of mitochondria, no further analysis could be

performed in yeast

In order to study in more detail the assembly defect

caused by DF94–F98, the short-deletion was introduced

in R sphaeroides as described in Materials and methods

Contrary to the yeast enzyme, the bacterial

subcom-plex containing only subunits 1 and 2 is stable in the

absence of subunit 3, which allows further analysis

As expected, the mutant enzyme contained little subunit 3

(Fig 3), but the remaining subunits 1–2 oxidase was active

(Vmax¼ 900 s)1) However, as a result of the loss of

subunit 3, the mutant enzyme underwent rapid suicide

inactivation with catalytic turnover (Fig 4) and it pumped

protons with reduced efficiency (Fig 5)

The loss of subunit 3 could be due to weaker binding to

subunit 1, as suggested above, and/or to structural

insta-bility and degradation of the mutant subunit in vivo When

DF94–F98 was isolated from R sphaeroides cells grown

only to mid-log phase, the purified enzyme contained 20–

25% of the normal amount of subunit 3 (Fig 3) However,

what seemed to be proteolytic fragments were apparent

below the subunit 3 band Indeed, when the mutant oxidase

was isolated from bacterial cells grown to late stationary

phase, where protein degradation is highly active, subunit 3

was completely absent in the purified product These data

suggest that in the bacterial cell, the short deletion in helix 3 affects the folding and stability of subunit 3 In contrast, the wild-type oxidase was purified from stationary phase cells with normal amounts of subunit 3 Bound subunit 3 was not removed from the DF94–F98 mutant enzyme by

Fig 3 Subunit 3 content of the R sphaeroides mutants Cytochrome oxidase samples were separated on SDS-polyacrylamide gels contain-ing urea as described in [16] and stained with Coomassie Blue The location of subunit 1 (M r ¼ 48), subunit 2 (M r ¼ 37 and 35) and subunit 3 (M r ¼ 20) are indicated by the arrows Subunit 2 runs as a doublet due to incomplete C-terminal processing [13] Lanes A and G contain wild-type oxidase; lane B, G78S; lane C, A200T; lane D, DF94– F98 isolated from cells grown to mid-log phase; lane E, as D but repurified on Ni2+-nitrilotriacetic acid agarose; lane F, DF94–F98 isolated from cells in stationary phase Based on densitometry meas-urements (see Materials and methods) G78S and A200T contain as much subunit 3 as the wild-type oxidase and DF94–F98 in lanes D and

E contains  25% of the normal amount of subunit 3 Note that subunit 2 rather than subunit 1 is used as the reference in order to determine the amount of subunit 3 In the absence of subunit 3, sig-nificant amounts of a free form of subunit 1 (termed subunit Ia, see [19]) accumulate in the membrane As subunit Ia contains a histidine tag, it

is isolated along with the subunit 1–2 oxidase and leads to an apparent overabundance of subunit I in the absence of subunit 3 (lane F).

Fig 2 Respiratory growth and optical spectra

of the yeast strain harbouring human mutations

in cytochrome oxidase subunits 1 and 3 (A)

Optical spectra of reduced cell suspensions

(see Materials and methods) (B) Respiratory

growth The cells were incubated on glycerol

medium for 4 days at 28 °C.

increasing the detergent concentration or by multiple

chromatographic separations on Ni2+-nitrilotriacetic acid

agarose (Fig 3) Thus, while weaker binding of the mutant

subunit 3 seems likely, it could not be demonstrated by this

method

The results indicate that the short deletion affects the

assembly of the oxidase at some point after the assembly of

subunits 1 and 2 This is consistent with previous studies

showing that subunit 3 is not required for assembly of the

redox centres in subunits 1 and 2, nor is subunit 3 required

for the association of subunits 1 and 2 [11,19] Assuming

that the assembly of the catalytic core is not largely

different in mitochondria and bacteria, the same effect

would be expected in yeast and human cells However,

while subunits 1 and 2 form a stable subcomplex in the

bacterial membrane, the analogous subcomplex fails to

accumulate in the inner mitochondrial membrane in the

absence of normal subunit 3 [20] If subunit 3 is not

directly necessary for the association of subunits 1 and 2 in

mitochondria, it seems likely that the binding of subunit 3

is a necessary prerequisite for the binding of critical,

nuclear-encoded subunits that stabilize the growing

com-plex Thus, while the binding of subunit 3 seems to be an

end-stage event in the assembly of the bacterial oxidase, it

may be an indispensable middle step in the assembly of the

mitochondrial enzyme

chrome oxidase were more affected at higher temperature [9], we monitored the respiratory growth and cytochrome oxidase content at 35°C Again, the mutations had no effect (data not shown) We have previously observed that

a relatively small decrease in cytochrome oxidase content

or activity strongly affects the respiratory growth compet-ence of yeast cells [9] It seems that there is limited buffering capacity in respiratory function in the strains used in our studies Monitoring the respiratory growth of the mutant strains is therefore a sensitive test to assess the deleterious effect of the mutations The results indicate that the missense mutations had little effect on cytochrome oxidase content or activity in yeast

For the L196I and F251L mutations, these results were not unexpected Leucine196 is located in transmembrane helix 5 of subunit 1, which is close to subunit 3 However the replacement of leucine by isoleucine should effect only

a minor steric change and the substitution does not modify the polarity of the residue It seems likely that the enzyme can accommodate a slightly larger sidechain at that position as the closest residues in subunit 3 are 5–6 A˚ away F251 is located in helix 7 at the C-terminal part of subunit 3 In the bovine enzyme, its sidechain extends into the lipid bilayer and has no obvious contact with other subunits It is likely that the replacement of a phenylalanine

by a leucine does not distort the helix and alter the folding

of the subunit

On the basis of the structure, it might have been expected that G78S and A200T could hinder, at least slightly, the folding or assembly of the enzyme G78 is located at the base of helix 3 of subunit 3 The alpha carbon of G78 comes close to the ring of F94 of subunit 1 (4 A˚), which makes hydrophobic contact with PE9, one of the two phospholipids specifically bound in the cleft of subunit 3 [18] Alteration of F94 to alanine appears to slightly weaken the interaction between subunits 3 and 1, as evidenced by

 20% reduction in the content of subunit 3 in the purified

R sphaeroidesoxidase (Hosler, unpublished results) There-fore, a polar or bulky group at position 78 could potentially disrupt the contact between F94 of subunit 1 and PE9 of subunit 3 A200 is located toward the top of the helix 6 of subunit 3 Its methyl group extends into the centre of the five helix bundle, into a locally hydrophobic area containing the sidechains of F94, L252 and I256 of subunit 3 [17,18] The introduction of the longer and more polar sidechain of threonine into this region could potentially destabilize the five-helix bundle and hinder the subunit folding A200 is also close to residue S195, which is involved in assembly or

Fig 4 O 2 reduction activity of cytochrome c oxidases of R sphaeroides

mutants A200T (5 pmol), DF94–F98 (9 pmol) and Subunit 3 (–), the

oxidase containing only subunits 1 and 2 (6 pmol) were assayed for O 2

reduction (as O 2 uptake) as in [1] O 2 uptake was initiated by the

addition of 40 l M horse heart cytochrome c The inactivation shown

by Subunit 3 (–) and DF94–F98 is irreversible The O 2 reduction

activity of G78S and the wild-type oxidase was essentially identical to

that shown for A200T.

stability of the enzyme complex in yeast [9] In addition, it

has been suggested that A200T affected the proton transfer

activity of the enzyme [21] in Paracoccus denitrificans

In order to study further their effects, G78S and

A200T were introduced in R sphaeroides Both mutants

showed wild-type levels of O2reduction activity (Vmaxof

G78S¼ 1600 s)1; Vmax of A200T¼ 1860 s)1) with no

indication of suicide inactivation (Fig 4), normal proton

pumping (Fig 5) and no loss of subunit 3 (Fig 3) These

results show that any disruption of the F94–PE9 interaction

caused by G78S is not sufficient to weaken the interaction

between subunits 1 and 3 to the point where subunit 3 fails

to bind, and that the introduction of a threonine in position

200 does not compromise folding and binding of subunit 3

In addition, the normal O2reduction and proton pumping

activity of the A200T mutant argues against a proposed

role for this region of subunit 3 as an exit pathway for

protons [21]

Mutations in mitochondrially encoded subunits

of cytochrome oxidase in humans: correlating pathogenicity to the biochemistry elucidated in yeast and bacterial models

Mitochondrial genes are present in hundreds of copies in human cells Heteroplasmy is a common feature for mitochondrial genome mutations The severity of the respiratory defects and of the disease depends on the load

of mutated genes, which varies between tissues A few nonsense and frameshift mutations in the mitochondrially-encoded subunits of cytochrome oxidase have been found These mutations should result in truncated subunits, which abolish complex assembly and thereby cause a respiratory defect in the patients Similarly, the mutation M1T in subunit 2 causes a severe decrease of the level of subunit 2 and a low enzyme content [22] Several other mutations whose deleterious effects are more difficult to predict have

Fig 5 Proton pumping activity of the R sphaeroides mutants Cytochrome c oxidase was reconstituted into asolectin vesicles as previously described [15] Measurements of the absorbance of phenol red dye (100 l M ) were made in an Olis-RSM stopped-flow spectrophotometer and kinetic traces (average of at least three data sets) were taken at the isosbestic point for horse-heart cytochrome c (557 nm) Wild-type and G78S vesicles were measured using 0.1 l M enzyme and 6.5 l M cytochrome c2+, A200T with 0.15 l M enzyme and 5.5 l M cytochrome c2+, and DF94–F98 with 0.08 l M enzyme and 3 l M cytochrome c 2+ , all in 50 l M Hepes/KOH pH 7.4, 45 m M KCl, 44 m M sucrose and 2 l M valinomycin to relieve the membrane potential The bottom panel in each figure depicts the decrease in absorbance (acidification of the outside) showing the extent of proton pumping The top panel in each figure is the alkalinization seen in the presence of 5 l M carbonyl cyanide m-chlorophenylhydrazone (CCCP) In the presence of this concentration of uncoupler pumped protons are not observed and the alkalinization is due to the net consumption of protons in the synthesis of H 2 O In these experiments the H + /e – value for the wild-type oxidase averages 0.9 ± 0.2 The H + /e – values for G78S and A200T are, within error, the same as that of the wild-type enzyme, while the H+/e–value for DF94–F98 is significantly lower.

through the germline, and is present at high levels in

asymptomatic relatives of the patient, it is likely that the

mutation has only very mild effects In yeast, the same

mutation has no effect as shown in this work A223S has

been observed in a family with diverse clinical features

ranging from myopathy to a multisystem disorder [24]

However this same change has also been listed as

Ôpoly-morphismÕ (www.mitomap.org) In addition, the mutation

in yeast has no effect [9], which seems to indicate that the

A223S is indeed a silent mutation Another silent mutation,

G317S, has been found in fibroblasts from a patient

presenting with lactic acidaemia and cytochrome oxidase

deficiency Residue G317 is a highly conserved residue

located next to T316, which is part of the K-channel Thus,

it might have been expected that the replacement of G317 by

serine could affect the catalytic activity of the enzyme

However, it was shown that the mutation had no effect on

human enzyme and that the disease was caused by a

mutation in the nuclear SURF1 gene [25] Consistent with

this, G317S had no effect in yeast [8] Two other mutations

in the region of the K-channel, I280T and M273T have been

observed in hematopoietic cells of patients suffering from

acquired idiopathic sideroblastic anaemia (AISA) In yeast

the mutations caused identical effects to those reported

in human cells They were mildly deleterious, showing a

twofold decrease in cytochrome oxidase activity and

perturbed binuclear centre properties [8] These changes

are likely due to altered K-channel function as both M273

and I280 are closely associated with two key residues of the

K-channel, K319 and T316 [17,18] The sulfur and carbonyl

oxygen of M273 are within 3.5 A˚ and 3.8 A˚, respectively, of

the terminal nitrogen of K319, while the sidechain of I280 is

within 4.1 A˚ of the sidechain hydroxyl group of T316

Substitution of threonine at these positions is likely to force

some rearrangement of K319 or T316 In addition, I280T

will place another hydroxyl group close to that of T316 As

these mutations do not eliminate cytochrome oxidase

activity in yeast their pathogenic significance is not clear

It is possible, however, that the high energy demands of

hematopoietic cells could not be fully met by mitochondria

having even mildly decreased respiratory function

Two other mutations have been reported but were not

studied in yeast: Y260H and Ter514K,Q,K,Ter Residue 260

is not conserved between species A histidine is found in some

sequences The replacement of the stop codon by lysine

extends the polypeptide by three residues This mutation has

been reported in patients with LHON [26] but also in a

patient suffering from McArdle’s disease, caused by a

Three mutations in subunit 3 have been associated with LHON: G78S [28], A178T [29] and A200T [28] The pathogenicity of G78S is controversial [30,31], as some reports list the mutation as primary while others suggest that the change is not pathologic but accidentally present in patients When introduced into yeast or R sphaeroides this mutation has no effect on respiratory competence or cytochrome oxidase function Therefore, it seems unlikely that G78S is a primary disease mutation Residue 78 is located in the helix 3 of subunit 3 at the interface with subunit 1, as discussed above The introduction of a polar residue can probably be compensated by re-arrangement of the solvent Likewise, the mutation A200T, located in the upper region of the five-helix bundle, has no effect on oxidase activity or assembly in yeast or R sphaeroides Thus, it is also unlikely to be a primary disease mutation Residue A178 is not conserved between species and is replaced by a tyrosine in yeast Therefore the mutation was not studied in yeast

F251L has been observed in a patient with MELAS [32]

As described above, on the basis of the structure, it was not expected that the mutation could severely hinder the assembly of the enzyme Indeed the mutation did not cause any respiratory dysfunction in yeast No decrease in cytochrome oxidase content could be detected

In contrast, the stop mutation at codon 249 (W249stop), which has been found in a patient with encephalopathy [33], inhibited the respiratory growth of yeast and prevented the assembly of cytochrome oxidase Similarly, cytochrome oxidase activity was severely decreased in the patient The deletion of five residues (DF94–F98) in a conserved region

of subunit 3, observed in a patient with myoglobinuria [20,34], severely affects oxidase assembly both in human and yeast cells and leads to the loss of subunit 3 in R sphaero-idesdue to instability and protein degradation

In conclusion, on the basis of the yeast and bacterial models, the human mutations could be placed into three classes (a) Two mutations in subunit 3, DF94–F98, W249stop, are highly deleterious and abolish enzyme assembly The consequences on energy production by the cells must be dramatic, depending on the load of mutations

in the cells These are clearly pathogenic mutations (b) Two mutations in subunit 1, I280T and M273T, have a signifi-cant but lesser effect on cytochrome oxidase activity that is likely to partly compromise cellular energy production Cells with a high demand in energy may be affected by these mutations, leading to disease (c) Several mutations, L196I, A223S and G317S in subunit 1, and G78S, A200T and

F251L in subunit 3, have no effect on respiratory

compet-ence in yeast G78S and A200T also have no effect on

cytochrome oxidase activity or assembly in R sphaeroides

If any of these mutations were the primary source of disease

in humans, it would indicate that the human enzyme is more

constrained in its structure than its yeast or bacterial

counterparts However, a large number of nonpathogenic

residue replacements have already been described,

suggest-ing that the human enzyme has significant flexibility in its

structure and can accommodate changes We cannot

exclude that the mutations have very mild effects in human

cells that are below detection with the yeast or bacterial

models Their pathogenicity is difficult to understand It

may be that some, or all, of this latter group of mutations

are also silent in humans and should more properly be listed

as polymorphisms

Acknowledgements

This work has been supported by a Medical Research Fellowship and a

BBSRC grant to BM, and by NIH Grant R01-GM56824 to J.P.H.;

D.M is supported by NIH Grant R37-GM26916 to S

Ferguson-Miller.

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