Biochem. J. (2012) 444, 199–204 (Printed in Great Britain)
doi:10.1042/BJ20120116
199
Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase
for facile purification of mutant forms
Brigitte MEUNIER*, Amandine MARÉCHAL† and Peter R. RICH†1
*Centre de Génétique Moléculaire du CNRS, UPR 3404, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France, and †Glynn Laboratory of Bioenergetics, Institute of Structural and
Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
Yeast CcO (cytochrome c oxidase) has been developed as
a facile system for the production and analysis of mutants of a
mitochondrial form of CcO for mechanistic studies. First, a 6H
tag (His6 tag) was fused to the C-terminus of a nuclear-encoded
subunit of CcO from yeast Saccharomyces cerevisiae. This
allowed efficient purification of a WT (wild-type) mitochondrial
CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded
Cox13 subunit), with a recovery yield of 45 %. Its catalytic-centre
activity [≈180 e·s − 1 (electrons per s)], UV–visible signatures of
oxidized and reduced states and ability to form the PM [‘peroxy’
(but actually a ferryl/radical state)] and F (ferryl) intermediates
confirm normal functioning of the histidine-tagged protein. Point
mutations were introduced into subunit I of the 6H-WT strain. All
mutants were screened for their ability to assemble CcO and grow
on respiratory substrate. One such mutant [6H-E243DI (the 6HWT strain with an additional mutation of E243D in mitochondrial
DNA-encoded subunit I)] was purified and showed ∼ 50 % of the
6H-WT catalytic-centre activity, consistent with the effects
of the equivalent mutation in bacterial oxidases. Mutations in
both the D and the H channels affect respiratory growth and
these effects are discussed in terms of their putative roles in CcO
mechanism.
INTRODUCTION
bovine CcO [2] and is more weakly evident in bacterial CcOs. In
bovine CcO, this H channel, with breaks, spans the entire subunit
I and includes residues that interact directly with haem a ring
substituents.
A consensus view has yet to emerge on the roles of these
hydrophilic channels in coupled proton transfer and on whether
they might even have different roles in some types of CcOs [6,7].
Mutations in all three channel regions of bacterial CcOs have
confirmed that the D and K, but not the H, channels have crucial
proton-transfer roles [8,9]. Equivalent information in mammalian
CcO is sparse because site-directed mutation of the mtDNA
(mitochondrial DNA)-encoded core subunits is difficult. However,
results with a hybrid system in which bovine subunit I with
mutations in the H channel was incorporated into human CcO
in an immortal cell line [10] have suggested that the H channel
is the route for translocated protons. The yeast Saccharomyces
cerevisiae provides a flexible alternative system for studies of
mitochondrial forms of CcO since it is amenable to genetic
transformation of both nuclear and mitochondrial genomes.
Furthermore, all 11 subunits of yeast CcO are homologous with
mammalian CcO subunits and share extensive sequence identities.
A predicted model of its structure, on the basis of homology
modelling with bovine CcO, suggests that most structural
and functional features have been conserved between yeast and
mammalian forms [11]. Hence many structure/function aspects
of yeast CcO are likely to be similar in mammalian CcOs. Point
mutations have to date been successfully introduced in the D and
K channels of yeast CcO [12,13], but not in the H channel.
The present paper describes the construction of a modified
yeast CcO 6H-WT [yeast CcO with a 6H (His6 ) tag on the
nuclear-encoded Cox13 subunit; WT is wild-type], the 6H tag
allowing rapid and efficient purification. Further mutations were
CcO (cytochrome c oxidase, also called Complex IV) is the
terminal enzyme of many respiratory chains that catalyses transfer
of electrons from reduced cytochrome c to molecular oxygen [1].
It is a transmembrane complex embedded in the plasma membrane
of Gram-negative bacteria or the inner membrane of eukaryotic
mitochondria. In mammals it is a homodimer, with each monomer
composed of 13 protein subunits with a combined molecular mass
of 204 kDa [2]. In contrast, bacterial homologues are composed of
only three or four subunits [3,4]. All forms of CcO share conserved
subunits I, II and III that constitute the catalytic core and which,
in eukaryotes, are usually encoded by the mitochondrial genome.
Subunit III has no prosthetic group and may provide a channel
for substrate O2 to diffuse to the catalytic site. Subunit II houses a
dinuclear copper centre, CuA , which is the first electron acceptor
from cytochrome c. The subsequent electron acceptors, located
in subunit I, are haem a and a BNC (binuclear centre) consisting
of haem a3 and another copper atom, CuB , where the oxygen
reduction chemistry takes place.
A full catalytic cycle of CcO consumes eight protons from
the negative (matrix or cytoplasmic) side of the membrane. Four
are substrate protons used to produce two water molecules, and
four are translocated across the membrane, contributing to the
protonmotive force that drives ATP synthesis. All protons must
travel through the protein structure along channels composed
of protonatable residues and associated water molecules. Two
such possible channels were identified in the first bacterial CcO
structure from Paracoccus denitrificans [5]: a K channel leading
to the BNC and a D channel leading to a conserved buried glutamic
acid residue (Glu278 in P. denitrificans, Glu242 in bovine and Glu243
in yeast CcO). A third possible H channel was first identified in
Key words: cytochrome c oxidase, energy coupling, mutagenesis,
proton channel, subunit I, yeast.
Abbreviations used: BNC, binuclear centre; Cc O, cytochrome c oxidase; DDM, n-dodecyl β-D-maltoside; F, ferryl intermediate; FR, fully reduced; 6H
tag, His6 tag; 6H-WT, yeast Cc O with a 6H tag on the nuclear-encoded Cox13 subunit; 6H-E243DI , the 6H-WT strain with an additional mutation of E243D
in mtDNA-encoded subunit I; KPi, potassium phosphate; mtDNA, mitochondrial DNA; PM , ‘peroxy’ intermediate (but actually a ferryl/radical state); WT,
wild-type; YPGal, 1 % yeast extract, 2 % peptone and 2 % galactose.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
200
B. Meunier, A. Maréchal and P. R. Rich
introduced in the vicinity of the D or H channels, and the ability
of the resulting strains to assemble CcO and grow on respiratory
substrate was assessed.
EXPERIMENTAL
in 50 mM KPi, pH 7.4, 100 mM KCl and 5 mM potassium EDTA
and repelleted, followed by two washing cycles using 50 mM
KPi, pH 7.4, and 2 mM EDTA by resuspension/centrifugation at
40 000 rev./min for 30 min at 4 ◦ C using a T647.5 rotor. The final
pellet was homogenized with 50 ml of 50 mM KPi, pH 8.0, and
stored at − 80 ◦ C.
Materials
Yeast extract was purchased from Ohly and CO gas was from
BOC. All other reagents were purchased from Sigma–Aldrich
unless otherwise specified.
Introduction of 6H tags and point mutations in yeast Cc O
Modified yeast strains were derived from strain W303-1B [14].
The alleles of the COX4, COX5A, COX8 and COX13 nuclear genes
with a 6H tag sequence on their 3 ends at their chromosomal
loci were produced as described previously [15]. A short linker
encoding the sequence GARGS was also inserted before the
6H tag of COX13. The sequence of the genes was checked
and respiratory growth competence of the strains was monitored
to verify that the introduction of the 6H tag did not induce a
respiratory deficiency. Mutations in mtDNA-encoded subunit I
were then introduced into the 6H-WT by biolistic transformation
as described previously [13].
Respiratory growth competence
Respiratory growth competence was monitored from growth on
agar plates with respiratory medium (1 % yeast extract, 2 %
peptone and 2 % glycerol). For this test, a strain with a low
respiratory growth capacity was used: CKWT (mat a, leu1, kar11; derived from [16]). Because of the low respiratory growth
capacity, a moderate defect in respiratory function results in
a significant decrease in respiratory growth. The mitochondrial
genomes carrying the COX1 mutations were transferred into the
CKWT strain by cytoduction [16], resulting in isogenic strains
that differed only in the point mutation in COX1.
Batch growth for protein purification
Strains were first grown in 5 ml of YPGal (1 % yeast extract, 2 %
peptone and 2 % galactose) medium for 24 h at 28 ◦ C with shaking
at 150 rev./min. Cells were then transferred (1:100 dilution) into
50 ml of YPGal in a 1 litre flask and grown under the same
conditions. Finally, cells were transferred (1:100 dilution) into
10×500 ml of YPGal in 2 litre high-aeration shake flasks and
grown for 16 h at 28 ◦ C with shaking at 200 rev./min. Cells were
harvested in late exponential phase by centrifugation at 6500 g for
5 min at 4 ◦ C. Cells were washed by resuspension/centrifugation
cycles in 50 mM KPi (potassium phosphate), pH 7.0, until the
supernatant was clear (usually three times). Typically, 16 and
10 g of wet cells/litre of culture were obtained for the 6H-WT
and 6H-E243DI (the 6H-WT strain with an additional mutation
of E243D in mtDNA-encoded subunit I) strains respectively.
Preparation of mitochondrial membranes
Wet cells were resuspended 1:1 (w/v) on ice in 650 mM Dmannitol, 50 mM KPi, pH 7.4, and 5 mM EDTA. For disruption,
glass beads (425–600 μm diameter) were added and 90–95 %
of cells were broken by mechanical lysis using a bead-beater
cell disruptor (BioSpec Products). Cell debris was removed by
centrifugation at 5600 g for 20 min at 4 ◦ C and mitochondrial
membranes were pelleted by centrifugation at 40 000 rev./min for
1 h at 4 ◦ C using a T647.5 rotor. The membranes were resuspended
c The Authors Journal compilation c 2012 Biochemical Society
Purification of Cc O
The membrane preparation was diluted to 1.8 mg of protein/ml
in 50 mM KPi, pH 8.0, and solubilized by incubation on ice
for 30 min with 2 % (w/v) DDM (n-dodecyl β-D-maltoside;
Melford Laboratories). The solubilized material was centrifuged
at 40 000 rev./min for 35 min at 4 ◦ C using a T647.5 rotor.
Imidazole at 5 mM was added to the supernatant before it was
incubated for 1 h with Ni2 + -iminodiacetic acid resin (His-bind®
resin, Novagen) previously equilibrated with 50 mM KPi, pH 8.0.
The resin was loaded into a column and washed with 5 column
vol. of 50 mM KPi, pH 8.0, 150 mM KCl, 5 mM imidazole
and 0.009 % DDM followed by 3 column vol. of 50 mM KPi,
pH 8.0, 10 mM imidazole and 0.009 % DDM. The protein was
eluted by increasing the imidazole to 100 mM and was directly
loaded on to a DEAE Sepharose CL-6B column previously
equilibrated with 20 column vol. of 50 mM KPi, pH 8.0, and
0.015 % DDM. The column was washed with 2 column vol.
of 50 mM KPi, pH 8.0, 50 mM NaCl and 0.01 % DDM. The
purified protein was eluted by increasing the NaCl to 250 mM
and concentrated in a pressure cell with a 100 kDa cut-off
membrane (YM100, Millipore) to 10–20 μM. CcO concentration
was estimated from a dithionite-reduced minus oxidized visible
absorption difference spectrum using an assumed ε603–621nm of
26 mM − 1 ·cm − 1 [17]. SDS/PAGE was performed in denaturating
conditions with a 16.5 % acrylamide gel containing 6 M urea as
described previously [18,19].
O2 -consumption rates and catalytic-centre activities
CcO was diluted to 10–20 nM in 67 mM KPi, pH 6.2, containing
40 μM TMPD (N,N,N ,N -tetramethyl-p-phenylenediamine),
50 μM horse heart cytochrome c and 0.05 % DDM in the chamber
of a Clark-type oxygen electrode at 24 ◦ C. The reaction was
initiated by addition of 20 mM sodium ascorbate. Catalytic-centre
activities [expressed in e·s − 1 (electrons per s)] were calculated
from the steady-state rates of oxygen consumption.
UV–visible absorption spectroscopy
UV–visible absorption spectra were recorded with a single
beam spectrometer constructed in-house and equipped with a
stepped dispersive monochromator and photomultiplier detector.
All spectra were recorded in a standard 1 cm-path-length quartz
cuvette in 50 mM KPi, pH 8.5, and containing 0.009 % DDM.
Starting from the oxidized (O) state, the following CcO species
were generated: FR (fully reduced), with excess of sodium
dithionite; FR-CO compound, by saturation of a FR sample with
CO gas; PM [‘peroxy’ intermediate (but actually a ferryl/radical
state)], by bubbling with CO under aerobic conditions; and F
(ferryl intermediate), by addition of 500 μM H2 O2 . The extent
of PM or F formation was assessed from A610–633 or A580–633
using assumed molar absorption coefficients of 10.1 and 4.3
mM − 1 · cm − 1 [17].
Photolysis of the FR-CO compound was achieved with a single
flash from a Xenon flashlamp (20 J/flash; 6 μs duration at half
maximum) that was filtered through a 520 nm-cut-off filter. The
photomultiplier was screened with a 570 nm (OG570) cut-on
Site-directed mutagenesis in subunit I of yeast cytochrome c oxidase
201
Figure 1 Steady-state levels of expression of Cc Os with 6H-tagged nuclearencoded subunits
Cells were cultivated in YPGal, and total proteins were extracted as described previously [35] and
analysed by SDS/PAGE. After blotting on to a nitrocellulose membrane, bands were hybridized
with the monoclonal antibodies anti-His6 (Qiagen) and anti-porin (Molecular Probes) as a control
for protein loading. Bands with bound antibodies were visualized by chemiluminescence.
filter. Photolysis and subsequent recombination kinetics at room
temperature (24◦ C) were monitored over 85 ms at 590 minus
610 nm, and 50 individual transients were averaged to improve
signal/noise. Averaged data were fitted to a single exponential
decay using OriginPro 8.1 (OriginLab Corporation).
RESULTS AND DISCUSSION
Construction of yeast Cc O with a 6H tag and mutations in subunit I
A 6H tag was fused to the C-terminus of the nuclear-encoded
subunits Cox4, Cox5A, Cox8 and Cox13 as described in the
Experimental section. The presence of the 6H tag on any of
these subunits had no significant effect on the respiratory growth
competence, indicating that it did not have a major impact on CcO
assembly and/or activity in vivo. Steady-state levels of expression
of the 6H-tagged subunits were assessed by Western blotting
(Figure 1). The levels of immunodetected 6H-tagged Cox4 and
Cox8 were significantly lower than those of 6H-tagged Cox5A
and Cox13. The lower levels could have resulted from faster
degradation of the tagged proteins or from proteolysis of the
6H tag from the subunits. Since the highest level of expression
was that of 6H-tagged Cox13, and because an alternative isoform,
Cox5B, can replace Cox5A under some conditions [20], this strain
(6H-WT) was selected for further work. Mutations in mtDNAencoded subunit I were then introduced into 6H-WT by biolistic
transformation as described previously [13]. The resulting strains
are isogenic to 6H-WT, differing only in the point mutation.
Purification of 6H-tagged Cc Os
The 6H-WT strain was grown in 5 litres of medium, yielding
approximately 80 g wet mass of cells containing 160 nmol
of CcO (assessed from a dithionite-reduced minus oxidized
spectrum of whole resuspended cells; results not shown).
Mitochondrial membranes were isolated after cell disruption,
solubilized with DDM and the CcO purified by Ni2 + chelating affinity and DEAE Sepharose CL-6B ion-exchange
chromatographies (see the Experimental section). Typically,
70 nmol of purified CcO was obtained, giving a purification
yield of 45 %. SDS/PAGE of the subunits of a representative
6H-WT CcO preparation is shown in Supplementary Figure
S1 at http://www.BiochemJ.org/bj/444/bj4440199add.htm, where
bands were tentatively assigned by comparison with previous
results [19]. The same procedure was used for the 6H-E243DI
Figure 2
UV–visible absorption spectra of purified 6H-WT yeast Cc O
(A) Absorption spectra of oxidized (O, continuous line), FR (dotted line) and FR with bound CO
(FR-CO, dashed line) forms of yeast 6H-WT Cc O. Spectra were recorded at room temperature in
50 mM KPi, pH 8.5, and 0.009 % DDM. The oxidized spectrum was obtained after incubation of
0.75 μM purified 6H-WT yeast Cc O with 5 μM K + -ferricyanide for 15 min. The FR state was
generated with a slight excess of sodium dithionite and was converted into the FR-CO form by
brief bubbling with CO gas. (B) FR minus O (lower trace) and FR-CO minus FR (upper trace)
difference spectra, derived from the absorption spectra in (A).
strain containing the E243D mutation in subunit I. In this case,
the same growth conditions yielded 50 g wet mass of cells (40 %
less than 6H-WT). However, these cells contained 100 nmol of
CcO, the same relative amount of CcO per g wet mass of cells.
The purity of each CcO preparation was routinely assessed by
UV–visible spectroscopy from the absorbance ratio of protein at
280 nm/haem Soret band at 425 nm; this was between 3 and 4,
depending on the quality of the preparation. Catalytic activities
were assessed using a Clark-type oxygen electrode in 67 mM KPi
at pH 6.2, conditions previously reported to give optimal activities
for WT yeast CcO [21]. The 6H-WT protein had a catalytic-centre
activity under these conditions of 180 e·s − 1 , similar to the value
reported by Taanman and Capaldi [22] for purified WT yeast
CcO. This confirmed that addition of the 6H tag to the Cox13 Cterminus did not alter the catalytic-centre activity. However, the
additional introduction of the E243D mutation in subunit I reduced
the catalytic-centre activity to 93 e·s − 1 , representing 52 % of the
6H-WT value under the same conditions. Similarly decreased
activities have been reported for equivalent glutamate/aspartate
mutants in CcO of P. denitrificans (60 % activity relative to
WT [9]), Rhodobacter sphaeroides (50 % [23]) and the bo-type
oxidase of Escherichia coli (31 % [24]).
c The Authors Journal compilation c 2012 Biochemical Society
202
B. Meunier, A. Maréchal and P. R. Rich
O difference spectrum at pH 8.5 (Figure 3A) is characterized
by a trough/peak in the Soret region at 414/438 nm, a broad
band at 567 nm and an α-band at 610 nm (which is red-shifted
in comparison with the 607 nm peak of PM of bovine CcO).
As observed in bovine CcO [29], the extent of PM formation
was pH-dependent (results not shown) with approximately 26 %
(A610–633 = 0.008) formed at pH 8.5. The kinetics of reaction with
CO to form PM were also markedly biphasic, with the slow phase
most probably caused by a significant fraction of the oxidized
enzyme being in the chloride-ligated form [30].
Figure 3(B) shows the difference spectrum induced by addition
of 500 μM H2 O2 to oxidized 6H-WT at pH 8.5, a reaction that in
bovine CcO results in transient formation of PM followed by full
conversion into F [29]. In the case of the yeast 6H-WT, although
the same O→PM →F sequence occurs, a significant amount of PM
remains in the steady state. Nevertheless, the spectrum has a Soret
band that is slightly blue-shifted in comparison with PM , together
with a broad band centred at 580 nm that can be attributed to the
F form. The extent of F formation from O was estimated to be
12 % from the A580–633 .
Minor traces of an additional haem could be present to a variable
extent, appearing as peaks at 422 and 554 nm in FR minus O difference spectra. In Figure 2(B) (lower trace), this species is marked
with ‘*’. The same species can be seen in previously reported yeast
CcO preparations [21]. It might be a contaminating haemoprotein
or, possibly, a partially assembled/disassembled form of CcO.
Effect of mutations in the D and H channels on respiratory growth
Figure 3
Generation of the PM and F intermediates of 6H-WT yeast Cc O
(A) PM minus oxidized difference spectrum. The PM intermediate was formed by reaction of
3 μM oxidized Cc O with CO in aerobic 50 mM KPi, 0.009 % DDM at pH 8.5. (B) PM + F
minus oxidized difference spectrum. The F intermediate was partially formed by reaction of
2.3 μM oxidized Cc O with 500 μM H2 O2 in 50 mM KPi, pH 8.5, and 0.009 % DDM. See the
text for details.
UV–visible absorption spectroscopy
Figure 2(A) shows absorption spectra of purified 6H-WT CcO.
The O form (Figure 2A, continuous line) has a Soret band
maximum at 425 nm, consistent with that of WT yeast CcO
[19,25]. Addition of dithionite led to the characteristic 444 nm
band of the FR state (Figure 2A, dotted line). Subsequent
bubbling with CO induced the shift characteristic of formation
of the FR-CO compound (Figure 2A, dashed line); assuming a
molar absorption coefficient of 10.5 mM − 1 · cm − 1 at 590–610 nm
[26], its extent indicated essentially full formation of the CO
compound. The 6H-E243DI mutant displayed equivalent spectra
(results not shown), confirming that the UV–visible properties
of the two haem centres were not affected. The kinetics of CO
recombination after flash-photolysis of the FR-CO compound
of the 6H-WT could be fitted to a single exponential with a
rate constant of 82 s − 1 at 24 ◦ C (Supplementary Figure S2 at
http://www.BiochemJ.org/bj/444/bj4440199add.htm), consistent
with previous measurements on the WT enzyme [27]. A similar
rate constant for CO recombination was measured for the 6HE243DI mutant, also consistent with previous measurements on
isolated mitochondria [13].
Spectra of the PM and F reaction intermediates [28] of the
6H-WT CcO were also recorded (Figure 3). PM was formed by
reaction of CO with the O state in aerobic buffer. The PM minus
c The Authors Journal compilation c 2012 Biochemical Society
A range of mutations were introduced into subunit I of the 6HWT strain. These mutations are located in the vicinity of either
the D or H channels (Figure 4). Figure 5 shows the growth
profiles of all mutant strains. All three mutations in the D
channel (E243D, N99D and I67N) affected respiratory growth.
In the case of the E243D mutation, this correlates with a 50 %
lower O2 -consumption rate of the purified CcOs (see above).
I67N had previously been generated by random mutagenesis. It
severely inhibited CcO turnover [12] and it was proposed that
this might be caused by an interaction with Glu243 that prevented
it from changing configuration. N99D was chosen to mimic the
equivalent mutations of N131D in P. denitrificans and N139D
in R. sphaeroides which result in 62 and 270 % of WT activity
respectively [9,31]. Figure 5 shows that this mutation in yeast
greatly diminishes the ability of the strain to grow on respiratory
medium, suggesting an inhibitory effect. Taken together, these
results confirm the importance of the D channel in the catalytic
cycle of yeast CcO.
Of the strains with mutations in the vicinity of the H channel,
two (R37M and A446D) failed to assemble CcO. X-ray data of
bovine CcO showed that Arg38 is in hydrogen-bond interaction
with the formyl group of haem a. Mutation of the equivalent
residue in R. sphaeroides led to either loss of activity (R52A/Q)
or a decrease in catalytic-centre activity to 65 % of the WT level
(R52K) [7]. In all cases the redox spectra of assembled CcOs
with Arg52 mutations showed a shift of the α-band maximum,
confirming its interaction with haem a. Its replacement with a
methionine residue in yeast could destabilize binding of haem a
and preclude enzyme assembly. A446D, which had been identified
as causing respiratory deficiency by random mutagenesis, is
predicted to be located at the interface between subunits I and
II (Figure 4). Hence it is likely that the subunit I–II interaction is
destabilized by the replacement of a small apolar alanine residue
with a negatively charged aspartic acid. Replacement of Arg37 and
Ala446 with alternative residues that may result in assembled CcO
will be investigated in future work.
Site-directed mutagenesis in subunit I of yeast cytochrome c oxidase
Figure 4
203
Mutations and their predicted locations in yeast Cc O
Locations of mutated residues are shown on the predicted structural model of yeast Cc O [11]. Residues forming the three putative proton channels are coloured in purple (K channel), orange
(D channel) and green (H channel). Other residues that have been mutated are displayed in yellow. One strain (4Hmut in Figure 5) has four point mutations Q411L/Q413L/S455A/S458A.
Figure 5
Respiratory growth of Cox1 mutants
Serial dilutions in water of cells pre-grown on glucose plates (1 % yeast extract, 2 % peptone
and 2 % glucose) were spotted on to plates containing either glucose (fermentative medium) or
glycerol (respiratory medium) and incubated for 2 days at 28 ◦ C.
All other H channel mutant strains successfully assembled
CcO. Replacement of Gln413 (equivalent to bovine His413 and after
which the channel was named) with a leucine residue did not
affect respiratory growth, in contrast with the dramatic effect
of the nearby mutation Q411L. Mutation S382A, a residue in
hydrogen-bond interaction with haem a farnesyl, had a similar
effect on respiratory growth as E243D. Its replacement with an
alanine residue in R. sphaeroides decreased the CcO catalyticcentre activity to 67 % of the WT level [7]. S458A had an even
more pronounced effect on respiratory growth. This residue is also
present in bovine CcO, but is an alanine in P. denitrificans and
R. sphaeroides CcOs. In bovine CcO it is in hydrogen-bonding
distance with two crystallographically resolved water molecules
and might also interact with the farnesyl OH of haem a in a
redox-dependent manner [32]. In contrast, mutation S455A is
not expected to interact with haem a (Figure 4) and had little
effect on respiratory growth, in agreement with effects of the
equivalent mutation in P. denitrificans [9]. A multiple mutant
of Q411L/Q413L/S458A/S455A was also constructed (4Hmut,
Figure 5). Growth on respiratory medium was barely detectable,
suggesting that effects of some individual single point mutations
could be somewhat additive. Mutation S52D was also introduced
at the ‘top’ of the H channel to mimic the equivalent bovine CcO
Asp51 , a residue whose conformation is redox-linked and proposed
to provide the proton exit route, or valve, for proton translocation
through the H channel. Its mutagenesis to an asparagine residue
in bovine subunit I results in loss of coupled proton translocation
without inhibiting electron transfer [32]. In yeast CcO this
mutation had no effect on respiratory growth, and in bacteria
a glycine is present in the equivalent position. Mutation D445E,
first identified by random mutagenesis to inhibit respiration [33],
c The Authors Journal compilation c 2012 Biochemical Society
204
B. Meunier, A. Maréchal and P. R. Rich
was also introduced in the 6H-WT strain. The homology model
suggests that it is at the interface with Cox5A, a potential
regulatory supernumerary subunit [11]. Finally, the mutation
E39Q, equivalent to bovine Glu40 that forms the Ca2 + /Na + binding site near the ‘top’ of the H channel, also markedly
affected respiratory growth. Overall, these H channel mutants
indicate that this structure is playing a function of some kind in
yeast CcO. Whether this is one of coupled proton translocation
[10], of providing a dielectric channel [34] or, possibly, other
control functions can now be addressed through purification and
mechanistic studies of these and related mutations.
AUTHOR CONTRIBUTION
Brigitte Meunier, Amandine Maréchal and Peter Rich designed the research and wrote the
paper. Brigitte Meunier produced and characterized all histidine-tagged and mutant Cc O
strains. Amandine Maréchal grew the yeast cells, purified the Cc Os and carried out the
biochemical and biophysical analyses.
ACKNOWLEDGEMENTS
We thank Thomas Warelow and Talha Arooz for technical assistance.
FUNDING
This work was supported by the Agence Nationale de la Recherche [grant number ANR07-BLAN-0360-02 (to B.M.)] and the Biotechnology and Biological Sciences Research
Council U.K. [grant number BB/H000097/1 (to P.R.R.)].
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Biochem. J. (2012) 444, 199–204 (Printed in Great Britain)
doi:10.1042/BJ20120116
SUPPLEMENTARY ONLINE DATA
Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase
for facile purification of mutant forms
Brigitte MEUNIER*, Amandine MARÉCHAL† and Peter R. RICH†1
*Centre de Génétique Moléculaire du CNRS, UPR 3404, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France, and †Glynn Laboratory of Bioenergetics, Institute of Structural and
Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
Figure S2
Kinetics of CO recombination with FR 6H-WT Cc O
The FR-CO form of purified 6H-WT Cc O yeast was prepared by dithionite reduction of the oxidized
form in CO-saturated 50 mM KPi, pH 8.5, and 0.009 % DDM. CO recombination after flash
photolysis was followed at 590 minus 610 nm. Data were simulated with a mono-exponential
decay (in red) with a rate constant of 82 s − 1 at 24 ◦ C. Results are the average of 50 transients.
Figure S1
SDS/PAGE gel of purified 6H-WT Cc O
SDS/PAGE was performed with a 16.5 % acrylamide gel containing 6 M urea as described
previously [1]. A 2.8 μg portion of 6H-WT Cc O was incubated for 30 min at 40 ◦ C in a
denaturating buffer containing 2 % 2-mercaptoethanol. Protein subunits were separated by
electrophoresis at 50 mA over 15 h and revealed with either Coomassie Blue (left-hand lane)
or silver (right-hand lane) staining. Bands were tentatively assigned to specific subunits by
comparison with the migration profile obtained by Geier et al. [2] under similar conditions.
REFERENCES
1 Schägger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel
electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal.
Biochem. 166, 368–379
2 Geier, B. M., Schägger, H., Ortwein, C., Link, T. A., Hagen, W. R., Brandt, U. and von Jagow,
G. (1995) Kinetic properties and ligand binding of the eleven subunit cytochrome c
oxidase from Saccharomyces cerevisiae isolated with a novel large scale purification
method. Eur. J. Biochem. 227, 296–302
Received 18 January 2012/5 March 2012; accepted 7 March 2012
Published as BJ Immediate Publication 7 March 2012, doi:10.1042/BJ20120116
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society