(Received for publication, April 19, 1995; and in revised form, August 6, 1995)
From the
To explore the nature of proposed ligands to the Cu center in cytochrome c oxidase, site-directed
mutagenesis has been initiated in subunit II of the enzyme. Mutations
were introduced into the mitochondrial gene from the yeast Saccharomyces cerevisiae by high velocity microprojectile
bombardment. A variety of single amino acid substitutions at each of
the proposed cysteine and histidine ligands (His-161, Cys-196, Cys-200,
and His-204 in the bovine numbering scheme), as well as at the
conserved Met-207, all result in yeast which fails to grow on
ethanol/glycerol medium. Similarly, all possible paired exchange
Cys,His and Cys,Met mutants show the same phenotype. Furthermore,
protein stability is severely reduced as evidenced by both the absence
of an absorbance maximum at 600 nm in the spectra of mutant cells and
the underaccumulation of subunit II, as observed by immunolabeling of
mitochondrial extracts. In the same area of the protein, a variety of
amino acid substitutions at one of the carboxylates previously
implicated in binding cytochrome c, Glu-198, allow (reduced)
growth on ethanol/glycerol medium, with normal intracellular levels of
protein. These results suggest that a precise folding environment of
the Cu
site within subunit II is essential for assembly or
stable accumulation of cytochrome c oxidase in yeast.
Cytochrome c oxidase accepts electrons from cytochrome c to reduce dioxygen to water in the final step of cellular respiration, according to:
Coupled to this electron transfer, the enzyme pumps protons against an electrochemical gradient, and the energy stored in this gradient is subsequently utilized for the production of ATP(1, 2) . Although eukaryotic cytochrome c oxidase contains up to 12 subunits(3, 4) , subunits I, II, and III (encoded by the mitochondrial genome) are thought to form the core of a functional enzyme. Within this core reside the redox active metal centers.
The site of dioxygen
reduction is a binuclear metal center comprised of cytochrome a and Cu
. In addition, two metal
centers serve in electron transfer from cytochrome c to the
oxygen binding site. Electrons from the one electron carrier cytochrome c enter cytochrome c oxidase through the
intermembrane (cytosolic) face of the protein, via Cu
(5) and/or cytochrome a(6, 7) . The
binuclear oxygen binding site then accepts electrons from Cu
and/or cytochrome a and transfers them to bound
dioxygen. Finally, coupled to one or more of these electron transfers,
the enzyme pumps protons across the inner mitochondrial
membrane(2) .
Recent mutagenesis studies in Escherichia
coli quinol oxidase and an aa-type cytochrome c oxidase from Rhodobacter sphaeroides have
identified six histidine residues in subunit I as ligands to cytochrome a, cytochrome a
, and
Cu
(8, 9, 10) . In contrast,
subunit II has long been thought to provide ligands to
Cu
(11, 12, 13, 14) .
Electron nuclear double resonance spectroscopic studies of isotopically
substituted enzyme have defined two histidines and one cysteine as
ligands to Cu
(14, 15, 16) .
Evolutionary constraints further require that subunit II contributes at
least one cysteine ligand to the Cu
center(14) .
Specifically, sequence alignment of each of subunits I, II, and III
against a diverse group of species reveals only two conserved
cysteines, located at positions 196 and 200 (numbering according to the
bovine enzyme) in a highly conserved region of subunit II, as shown in Fig. 1. The two conserved cysteines have been implicated as
ligands to Cu
by differential labeling studies (17) . Within the C-terminal end of subunit II, there are only
two conserved histidines, located at positions 161 and 204, and
cross-linking studies have implicated the region of subunit II near
position 160 (as well as near position 198) in the binding between
cytochrome c oxidase and cytochrome c(18) .
Recent mutagenesis studies on C-terminal fragments of subunit II from E. coli quinol oxidase (19, 20) and from the aa
-type cytochrome c oxidase from Paracoccus denitrificans(21, 22, 23) are consistent with these
assignments.
Figure 1: Sequence conservation in subunit II. Capitals indicate either invariant or conservative substitutions. Amino acids targeted for substitution are shown in bold. Residues which have been shown to be post-transcriptionally edited are indicated by an underline.
The sequence of the C-terminal region of subunit II has
been compared to that of the copper binding region of blue copper
proteins (13) and, more recently, to nitrous oxide
reductase(20, 24) . While the former clearly
coordinate a single copper atom, the latter is thought to contain a
mixed valence binuclear copper site(25) . Recent multifrequency
EPR studies of cytochrome c oxidase suggest that the Cu center also contains a binuclear copper
site(25, 26, 27) . In any case, sequence
alignments between all three classes of sites suggest potential common
ligands to copper.
Modification of bovine cytochrome c oxidase with a water-soluble carbodiimide in the presence and absence of cytochrome c has identified specific negatively charged amino acids in subunit II which may be involved in the electrostatic interaction between cytochrome c oxidase and a positively charged face of cytochrome c(13) . Similarly, a monoclonal antibody to subunit II inhibits cytochrome c binding and protects regions of subunit II from reaction with the carbodiimide (28) . One of the protected carboxylates, Glu-198, is rigorously conserved and is located directly between the two conserved Cys residues at 196 and 200.
In the current study,
site-directed mutagenesis of subunit II in the eukaryotic enzyme has
been carried out in the yeast Saccharomyces cerevisiae to test
directly the involvement of the four most likely ligands to
Cu: His-161, Cys-196, Cys-200, and His-204, as well as the
potential ligand Met-207. Single substitutions have been introduced at
each site and include mutations intended either to retain or remove the
ability to coordinate copper. Additionally, double mutations have been
prepared which exchange proposed ligand functional groups (e.g. a mutation of Cys to His at one site combined with a mutation of
His to Cys at another). Finally, to complement studies of the closely
spaced Cys residues and to test the role of this region in the binding
of the physiological redox partner cytochrome c, the centrally
located Glu-198 has also been replaced by semiconservative, as well as
more dramatic substitutions.
Enzyme function was tested by
crossing TF189 rho with TF145 (cox2
); at the same time TF189 rho
was crossed with AB-4D/V25 to ensure that TF189 rho
cells did not loose their plasmid. A
control strain PT424, carrying a wild type cox2 plasmid, was
used as a control for AB-4D/V25 and TF145. Haploid mutants which grow
on ethanol/glycerol medium when crossed to TF145 were crossed with
AB-4D/V25, and the resulting diploids were screened by DNA
hybridization (Southern) analysis for the appropriate restriction site.
For nonrespiring mutations, diploids were selected from a cross to the
wild type haploid strain DL1 on minimal medium (SD, 0.7% bacto-yeast
nitrogen base without amino acids (Difco), 2% glucose, 2% bacto-agar
(Difco)); diploid colonies which failed to grow on an ethanol/glycerol
medium were picked for further characterization.
To monitor the stable accumulation of subunit II, an immunolabeling analysis was carried out on a mitochondrial extract prepared as described by Meunier et al.(33) . Mitochondrial protein extracts containing equal amounts of total protein (34) were loaded onto a 12.5% SDS-polyacrylamide gel and electrophoresed at a constant voltage of 200 V. Protein was electrotransferred to nitrocellulose (Schleicher & Schuell, BA83) in 80 mM Tris base, 12.8 mM glycine, 20% methanol, incubated with either the monoclonal antibody to cytochrome c oxidase subunit II, CCO6, or the polyclonal antibody YR6-T (provided by T. Mason), and detected using a horseradish peroxidase-conjugated secondary antibody (Amersham Corp.).
To assay for total heme content by
the pyridine hemochromogen assay (36) , spheroplasts were
prepared by lyticase (Sigma) treatment of yeast cells. Then 1.5 ml of
spheroplasts were collected by centrifugation and resuspended in 750
µl of 2% Triton X-100, 1% SDS, 100 mM NaCl, 1 mM EDTA, 10 mM Tris, pH 8. This suspension was centrifuged,
and the supernatant was retained. To 475 µl of supernatant, a pinch
(1 mg) of solid dithionite and 250 µl of pyridine were added,
and the solution was mixed by gentle inversion. The solution was
centrifuged again for clarification and 25 µl of 2 M NaOH,
2 mM EDTA were added to the supernatant. This process yields a
solution containing the pyridine adducts of all hemes in the sample and
allows for the quantitative distinction of hemes a, b, and c(37) .
Sequence conservation and spectroscopic analyses point to
specific cysteine and histidine residues within subunit II as potential
ligands to the Cu site. In order to directly probe the role
of these conserved amino acids, 14 single amino acid substitutions were
introduced into subunit II, targeting His-161, Cys-196, Cys-200, and
His-204 as described in Table 1and summarized in Fig. 2.
Substitutions for His include Cys and Asn, both polar amino acids and
potential replacement ligands to copper. Substitutions for Cys include
the polar amino acids His, Met, Ser, and Asp, as well as Ala, which
simply removes the cysteine sulfhydryl group. In subsequent studies,
four double mutant constructs were prepared which exchange individual
Cys and His residues, as shown in Fig. 2. In addition, sequence
comparisons and extended x-ray absorption fine structure spectroscopy
studies have prompted the proposal that Met-207 may coordinate
copper(38, 39) . To test this proposal, Met-207 was
replaced by Cys and double mutants were constructed to exchange Met and
Cys residues. Finally, Glu-198, which lies in the primary sequence of
subunit II between the two targeted cysteines and has been implicated
in the binding of cytochrome c, was substituted with single
amino acid mutations, as indicated in Table 1and Fig. 2.
Two types of mutations were intended, those that would be relatively
conservative (Gln and Asp) and those expected to be disruptive to
cytochrome c binding (His and Arg).
Figure 2:
Summary of mutagenesis results. All
mutations at His, Cys
, Cys
,
His
, and Met
, including mutations which
exchange Cys and His or which exchange Cys and Met, result in cells
which are unable to respire. Respiration is detectable only in cells
containing the mutations of Glu-198 to Asp, Gln, His, and to a lesser
extent Arg (underlined above). Immune detection using a
monoclonal antibody to subunit II shows an underaccumulation of subunit
II in all Cys, His, and Met mutants. Cys
Met and
Cys
Met showed similar results using a polyclonal
antibody. Pyridine hemochromogen extract analyses were performed on the
following: Cys
Ser, Met, Asp, His; Cys
Ser
, Met, His; His
Cys;
and His
Cys; and the C196H/H204C double mutant. In
all cases, accumulation of heme a was not observed.
Translation products from [
S]Met labeling are
not impaired. Examined only for mutants: Cys
Ser,
Met, Ala; Cys
Ser
, Met, Ala, Asp;
His
Cys; and His
Cys; and
C196H/H204C.
As described under
``Experimental Procedures,'' mutations in the gene encoding
subunit II of yeast cytochrome c oxidase were constructed
according to standard procedures in a bacterial mutagenesis system.
When possible, restriction sites have been either introduced or deleted
at or near the mutation to facilitate subsequent analyses, and mutants
were constructed so as to require a minimum of a 2-base reversion to
restore the wild type amino acid. Verified mutant constructs were then
introduced into rho yeast mitochondria via high
velocity microprojectile bombardment(30) . Haploid strains
containing transformed mitochondrial DNA were identified initially by
marker rescue and, in some cases, by DNA hybridization analysis of
restriction patterns and by DNA sequencing. To simply verify the
presence of the cox2 gene (mutant or wild type) by marker rescue,
candidate cells were crossed to strain AB-4D/V25, possessing a complete
mitochondrial genome, but containing a nonsense mutation near the
N-terminal end of the gene for subunit II. Since all mutant constructs
in the current study have modifications only near the C terminus,
homologous recombination in the diploid can result in a wild type
phenotype, verified by ability to grow on ethanol/glycerol media.
Finally, haploids containing the modified gene were then crossed to
either wild type strains or to strain AB-4D/V25, as appropriate, to
construct the diploid strain expressing the subunit II mutant in a
complete mitochondrial genomic environment.
The absolutely conserved glutamic acid residue at position 198 lies immediately between the two Cys residues, within a proposed peptide loop. Modifications of Glu-198 to Asp, Gln, or His allow respiratory growth at 30 °C, indicating that a negative charge at this position is not essential for function. However, modification of Glu-198 to Arg allows only very weak respiratory growth, suggesting that the introduction of a large side chain and/or a positive charge interferes with function. Examining these effects more closely, in growth on ethanol/glycerol plates, the colony sizes of the His mutant are substantially reduced from those of wild type (although much larger than those of the Arg mutant), while the colony sizes of the Asp and Gln mutants were slightly reduced from wild type. To further characterize the effects of these mutations, the effect of growth temperature was examined. Mutations to Asp and Gln allow respiration at 15, 30, and 37 °C, while mutation to the potentially charged amino acids His and Arg allows respiration at 30 and 37 °C, but not at 15 °C.
The visible absorption spectrum of wild type whole cells,
shown in Fig. 3A, reveals three features corresponding
to the reduced alpha bands of cytochromes a, b, and c, centered near 600, 560, and 550 nm, respectively. In order
to more clearly distinguish changes in these features in mutant
strains, reduced minus oxidized difference spectra were obtained. To
obtain the oxidized spectrum, electron transfer from the cytochrome bc complex was first inhibited by the addition of
antimycin A, and cells were then oxidized by addition of
H
O
. To obtain the spectrum of the reduced
enzyme, dithionite was then added to cells, and a second spectrum was
measured (the order of treatment was not found to be critical). Whole
cell spectra of all yeast strains containing mutations to proposed His
and Cys ligands to Cu
show the absence of an absorbance at
600 nm and the presence of a new peak (see below) around 580 nm (e.g.Fig. 3B). The difference spectra of
reduced minus oxidized whole cells shown in Fig. 3D clearly identify the presence of the 600-nm absorbance in wild
type cells and the lack of such an absorbance at 600 nm in the mutants
targeting Cu
ligands. In contrast, in mutants substituting
Glu-198 by Asp, Gln, His, and Arg, the 600-nm absorbance is present (an
example is shown in Fig. 3C). The obvious lack of a
600-nm band for the nonfunctional mutants demonstrates that cytochrome c oxidase is not assembling properly.
Figure 3:
In vivo visible absorption
spectroscopy. Absorption spectra of concentrated suspensions of yeast
cells were recorded at room temperature, in order to determine the
presence of assembled cytochrome a. A, spectra of reduced and
oxidized cell suspensions. To fully oxidize the samples, antimycin A
was added prior to the addition of the oxidant HO
(dashed line); an excess of the reductant dithionite was
then added to provide spectra of the reduced samples (solid
line). B, similar spectra for a representative mutant,
C196D. Other nonrespiring mutants showed similar spectra. C,
spectra of a representative mutant, E198D, which shows reduced growth
on ethanol/glycerol medium. D, reduced minus oxidized
difference spectra of wild type and mutant cells clearly demonstrate
the absence of an absorbance for cytochrome a in the
mutant.
The absorption band
at 580 nm (denoted Hb in Fig. 3A) observed in
the spectra of the oxidized mutants also occurs in
HO
-oxidized wild type cells and has been
attributed to yeast hemoglobin(40, 41) . It appears in
the spectra of all respiration-deficient strains, including those that
lack subunit II completely (data not shown), those containing a nuclear
mutation which disrupts respiration(42) , and in strains with a
mutation in the cytochrome bc
complex(43) .
To assess the stable accumulation of hemes (specifically heme a), pyridine hemochromogen assays were performed on whole cell extracts from 10 of the mutants. The pyridine hemochromogen assay extracts the porphyrins and replaces the in vivo axial ligands of the hemes with the strong field ligand pyridine, resulting in a well defined environment independent of the original protein environment. In this assay, the absorption maxima for cytochromes b and c coincide near 550 nm, while heme a is characterized by an absorption at 587 nm. None of the respiration-deficient mutants examined has a detectable absorption maximum at 587 nm (data not shown).
Figure 4:
Immunolabeling and
[S]Met labeling of mitochondrial translation
products. A, for wild type and various mutant constructs, a
monoclonal antibody was used to detect accumulation of subunit II of
cytochrome c oxidase in mitochondrial extracts (see text) of
yeast grown to saturation in galactose medium. To verify that the
mutation did not disrupt the monoclonal epitope, a polyclonal antibody
was also used as a probe (last four lanes). E198Q*
represents purified enzyme from the E198Q mutant. C200S
and DL1 represent wild type yeast strains. B,
to assess the level of synthesis of subunit II, mitochondrial
translation products were selectively labeled with
[
S]Met in the presence of the cytoplasmic
translation inhibitor, cycloheximide. In each case, mitochondrial
extracts were run on a 12.5% SDS-polyacrylamide
gel.
To determine whether these expressed subunits are stably
accumulating in the cell, crude mitochondrial extracts were probed with
antibodies specific to subunit II. Using a monoclonal antibody, all
mutant constructs which do not respire show a substantial
underaccumulation of subunit II compared to wild type. As shown in Fig. 4A, following a 1-h digestion of the cell wall
with lyticase in the absence of the protease inhibitor
phenylmethylsulfonyl fluoride, levels of subunit II are greatly reduced
for the mutant cells compared to wild type. A shorter lyticase
treatment (10 min) in the presence or absence of phenylmethylsulfonyl
fluoride (data not shown) yields higher levels of subunit II (but still
reduced 10-fold relative to wild type), suggesting that the mutant
proteins are less stable. Similar analyses of C196M and C200M using polyclonal antibodies to subunit II confirm this lack of
accumulation and further verify that the lack of immunolabeling is not
the result of direct modification of the antigenic determinant for the
monoclonal antibody to subunit II. These results further support a
model in which the precise structure of the Cu site is
required for the stability of the overall enzyme complex.
An understanding of the structural environment around the
Cu center in cytochrome c oxidase has remained
elusive, despite the availability of a wealth of spectroscopic and
sequence conservation data. Spectroscopic evidence has unambiguously
identified at least one cysteine sulfur ligand to the copper ion(s)
within the site, with strong arguments for a second cysteine
ligand(14) . Sequence analyses place the cysteines within a
highly conserved region near the C terminus of subunit II, shown below
(numbering in this manuscript follows that of the bovine enzyme).
Similarly, coordination by at least one His imidazole has been demonstrated(14, 15) , and spectroscopic arguments for two are very compelling(16) . In the current work, site-directed mutagenesis in yeast (the same system used in the spectroscopic studies of isotopically substituted protein) has been used to probe these four proposed ligand residues within subunit II. In addition, the importance of the conserved Glu-198, which lies directly between the two Cys residues, has been directly probed. The results provide constraints on the protein structure at this critical metal site.
The Cu center has been compared to the blue copper class of isolated
copper sites (13, 50) and, more recently, to a
binuclear center found in nitrous oxide
reductase(25, 51, 52) . In similar studies of
blue copper proteins, primary ligands to the copper have been
independently mutated(53, 54, 55) . Many
substitutions which are not tolerated in the Cu
center (e.g. Cys
Asp) nevertheless allow stable folding of the
blue copper proteins. The sensitivity of the Cu
center to
such semiconservative substitutions may be expected for a more complex
binuclear structure. Finally, mutagenesis has been carried out in a
peptide fragment engineered to restore a Cu
fold into a
homologous protein(19, 20) . As for azurin, most of
these mutations result in a folded structure which binds copper,
although typically more weakly than wild type. The mutations of
proposed ligands to the native eukaryotic Cu
site presented
here do not allow accumulation of folded protein. This most likely
results from effects on cytochrome c oxidase assembly or
stability in the cell.
Note Added in Proof-The crystal structures of
cytochrome c oxidase from bovine heart (Tsukihara, T., Aoyama,
H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K.,
Nakashima, R., Yaono, R., and Yoshikawa, S.(1995) Science269, 1069-1074) and from the bacterium P.
denitrificans (Iwata, S., Ostermeier, C., Ludwig, B., and Michel,
H.(1995) Nature376, 660-669) have recently been
solved at 2.8 Å resolution. The structures confirm that the
Cu center is binuclear and that the coppers are coordinated
by Cys-196, Cys-200, His-161, His-204, Met-207, and the backbone
carbonyl of Glu-198 (bovine numbering). The side chain carboxylate of
Glu-198 lies buried at the interface between subunits I and II, far
from the surface of the protein, and coordinates a Mg(II) ion at the
subunit interface. The Mg(II) ion is located along a potential electron
transfer path from Cu
to the heme edge of cytochrome a
.