Comparison of Diverse Protein Sequences of the Nuclear-Encoded
Subunits of Cytochrome C Oxidase Suggests Conservation of Structure
Underlies Evolving Functional Sites
Jayatri Das,* Stephen T. Miller, and David L. Stern*
*Department of Ecology and Evolutionary Biology, and Department of Molecular Biology, Princeton University,
Princeton, New Jersey
Interspecific comparisons of protein sequences can reveal regions of evolutionary conservation that are under purifying
selection because of functional constraints. Interpreting these constraints requires combining evolutionary information
with structural, biochemical, and physiological data to understand the biological function of conserved regions. We take
this integrative approach to investigate the evolution and function of the nuclear-encoded subunits of cytochrome c
oxidase (COX). We find that the nuclear-encoded subunits evolved subsequent to the origin of mitochondria and the
subunit composition of the holoenzyme varies across diverse taxa that include animals, yeasts, and plants. By mapping
conserved amino acids onto the crystal structure of bovine COX, we show that conserved residues are structurally
organized into functional domains. These domains correspond to some known functional sites as well as to other
uncharacterized regions. We find that amino acids that are important for structural stability are conserved at frequencies
higher than expected within each taxon, and groups of conserved residues cluster together at distances of less than 5 Å
more frequently than do randomly selected residues. We, therefore, suggest that selection is acting to maintain the
structural foundation of COX across taxa, whereas active sites vary or coevolve within lineages.
Introduction
Rates of amino acid substitution are dependent upon
both structural and functional constraints acting at specific
sites. Amino acid replacements that disrupt protein folding
or functional interactions are eliminated by selection,
leading to lower rates of substitution at those sites. Thus,
phylogenetic comparisons can reveal conserved sites that
are likely to be critical for protein function. Integrating such
phylogenetic analyses with structural, biochemical, and
physiological information provides a meaningful context
for studying adaptive evolution and for generating new
hypotheses about function (Golding and Dean 1998). Here,
we apply this integrative approach to cytochrome c oxidase
(COX), the terminal enzyme in the electron transport chain
of oxidative phosphorylation.
COX, which catalyzes the transfer of electrons from
reduced cytochrome c to molecular oxygen, is the primary determinant of cellular oxygen consumption and is
thought to play a key role in regulating energy production
(Poyton and McEwen 1996). COX is a multisubunit
transmembrane protein located in the inner membrane
of the mitochondrion. In mammals, it is composed of
13 subunits, three of which are encoded by mitochondrial
DNA, and the remaining 10 are encoded in the nucleus.
The three large mitochondrial-encoded subunits compose
the catalytic core of the enzyme, which contains the
reaction centers. The smaller nuclear-encoded subunits are
arranged around the perimeter of the core enzyme
(Tsukihara et al. 1996). The mitochondrial-encoded
subunits are necessary and sufficient to carry out both
electron transfer and proton-pumping functions (Capaldi
1990), although numerous studies of bacterial and bovine
COX (Ruitenberg et al. 2002; Tsukihara et al. 2003) have
Key words: cytochrome c oxidase, nuclear-encoded, evolution,
structure, mitochondria.
E-mail: [email protected].
Mol. Biol. Evol. 21(8):1572–1582. 2004
doi:10.1093/molbev/msh161
Advance Access publication May 21, 2004
yet to fully determine the mechanisms by which these
processes occur.
The roles of the nuclear-encoded subunits have only
recently begun to be elucidated. Experimental studies
suggest that the primary function of the nuclear-encoded
subunits is to regulate COX activity. For example, paralogous isoforms of subunit VII in Dictyostelium discoideum
and subunit V in Saccharomyces cerevisiae are differentially expressed in response to oxygen concentration
(Schiavo and Bisson 1989; Burke et al. 1997). In vertebrates,
allosteric interactions that involve subunits IV and VIa are
thought to mediate a phosphorylation-dependent mechanism of COX regulation (Ludwig et al. 2001). Some of the
nuclear-encoded subunits are important for development
and physiology of multicellular organisms, as shown by
lethality and pleiotropic phenotypes caused by mutations
in subunit VIc in Drosophila (Szuplewski and Terracol
2001).
There is also evidence for coevolution of the
mitochondrial-encoded and nuclear-encoded subunits.
When mitochondrial DNA is placed in a foreign nuclear
background, the resulting disruption in mitochondrial
activity suggests that functional interactions between the
different subunits have coevolved. This finding has been
shown in backcrosses between different intraspecific populations of the copepod Tigriopus californicus (Edmands and
Burton 1998), in backcrosses between sister species of
Drosophila (Sackton, Haney, and Rand 2003), and in
xenomitochondrial cybrids of primate cell lines (Kenyon
and Moraes 1997). In support of these experimental studies,
analysis of evolutionary rates of mammalian COX subunits
has shown that residues on nuclear-encoded subunits evolve
more slowly when in close proximity to mitochondrialencoded subunits, whereas the rapid evolution of mitochondrial DNA allows optimizing interactions with residues on
nuclear-encoded subunits (Schmidt et al. 2001).
Because organisms have evolved to allocate their
energetic resources differently based on their specific
physiology and environment, understanding the regulation
Molecular Biology and Evolution vol. 21 no. 8 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
Evolution of Cytochrome C Oxidase 1573
Table 1
Nomenclature of Nuclear-Encoded COX Subunits IV, Va, and Vb Across Different Taxa
Vertebrates
H. sapiens
D. melanogaster
IV
Va
agCG46717 (XP_309490)
Y37D8A.14 (NP_499681)
COX6 (P00427)
S. pombe
A. thaliana
IV.1 (P13073), IV.2 (Q96KJ9)
CG10664 (NP_610013),
CG10396 (NP_610168)
agCG48672 (XP_314839)
W09C5.8 (NP_493394)
COX5a (P00424),
COX5b (P00425)
COX5 (O74988)
—
D. discoideum
IV (P30815)a
—
A. gambiae
C. elegans
S. cerevisiae
Va (P20674)
CG14724 (Q94514)
COX6 (Q9UTF6)
—
Vb
Vb (P10606)
CG11015 (NP_609046),
CG11043 (NP_609047
agCG50153 (XP_314835)
F26E4.9 (NP_492601)
COX4 (P04037)
COX4 (P79010)
AT3G15640 (NP_188185),
AT1G80230 (NP_178140)
V (P29505)
NOTE.—The gene ID according to the organism-specific databases is followed in parentheses by the NCBI accession number of the protein sequences used for
alignments in this study. Multiple sequences indicate the presence of multiple paralogs. Dashes indicate that no significant homologs were found in an organism’s genome
database or trace archive.
a
These subunits are poorly conserved in D. discoideum and were not included in further sequence alignment.
of COX will shed light on how this protein complex may
have evolved to accommodate metabolic adaptation. Here,
we describe the variation in COX subunit composition
across diverse taxa and show that the nuclear-encoded
subunits likely evolved subsequent to the origin of
mitochondria. We use multiple sequence alignments to
predict essential functional sites in these subunits and map
these conserved sites onto the crystal structure of bovine
COX. We conclude that these sites are structurally
organized into functional regions, some of which correlate
with known sites of interaction, whereas others are novel
regions that warrant future experimental study.
the PyMOL molecular graphics system (DeLano 2002).
PyMOL scripts for real-time visualization of identical and
conserved residues on the COX structure are also included
in Supplementary Material online.
A program was written to evaluate the spatial
distribution of conserved and identical residues. Distances
were calculated between all pairs of residues on the list of
conserved and identical amino acids described above.
Here, distance is defined as the smallest interatomic
distance between any two atoms in a pair of amino acids.
For a null set, a list of randomly selected residues (and
their dimer symmetry-related equivalents) of equal length
was generated and distances calculated as above.
Materials and Methods
Protein sequences for bovine COX nuclear-encoded
subunits were obtained from the Entrez Protein sequence
database (http://www.ncbi.nlm.nih.gov/Entrez/). Sequences for homologs in other species were obtained by use
of either PSI-Blast to search the unrestricted NCBI
database (Altschul et al. 1997) or protein-protein Blast
programs to search organism-specific databases (Drosophila melanogaster, http://www.fruitfly.org/blast/index.
html; Anopheles gambiae, http://www.ensembl.org/
Anopheles_gambiae/blastview; Caenorhabditis elegans,
http://www.wormbase.org/db/searches/blast; Arabidopsis
thaliana, http://www.arabidopsis.org/Blast/; Saccharomyces cerevisiae, http://genome-www2.stanford.edu/
cgi-bin/SGD/nph-blast2sgd; Schizosaccharomyces pombe,
http://www.genedb.org/genedb/pombe/blast.sp). Wholegenome sequence trace archives were also searched when
available (http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?).
Default parameters were used for all database searches.
The crystal structure of bovine cytochrome c oxidase was
obtained from the Protein Data Bank (PDB ID 1OCC;
http://www.rcsb.org/pdb/, Berman et al. 2000).
Homologous sequences were aligned by application
of the ClustalW multiple sequence alignment program
(Thompson, Higgins, and Gibson 1994) with default
parameters. All alignments are included in Supplementary
Material online. Residues identified in each alignment as
being identical or physicochemically conserved across at
least two taxa were mapped onto the crystal structure by
Results
Nomenclature of COX Nuclear-Encoded Subunits
The subunit composition of COX in vertebrates,
plants, and yeasts has been determined primarily by
resolution of the purified protein by polyacrylamide gel
electrophoresis (Capaldi 1990; Geier et al. 1995; Jansch et
al. 1996). Nomenclature of the individual subunits in each
taxon is derived from their order of molecular mass, rather
than from any sequence similarity between subunits.
Variation in the size of homologous subunits has,
therefore, resulted occasionally in conflicting nomenclature between taxa. Tables 1, 2, and 3 provide compilations
of the parallel nomenclature of homologous subunits. We
also list database accession numbers for homologous
sequences found in other organisms with completed
genomes. Because we have mapped conserved sites onto
the bovine crystal structure, we refer to the nuclearencoded subunits after the vertebrate nomenclature. There
is no detectable sequence similarity between different
nuclear-encoded subunits, nor do they appear to share any
significant structural similarity beyond transmembrane
helices. Although subunits IV, VIa, VIc, VIIa, VIIb, VIIc,
and VIII of bovine COX are all classified under a folding
pattern of a single transmembrane helix in the SCOP
database (http://scop.mrc-lmb.cam.ac.uk/scop/), each subunit is subcategorized as a unique structural superfamily.
The globular subunits, Va, Vb, and VIb, also do not share
1574 Das et al.
Table 2
Nomenclature of Nuclear-Encoded COX Subunits VIa, VIb, and VIc Across Different Taxa
Vertebrates
VIa
H. sapiens
—
D. melanogaster
—
A. gambiae
—
C. elegans
S. cerevisiae
S. pombe
A. thaliana
—
—
—
COX5c
AT5G61310
(NP_200939)
AT2G47380
(NP_182260)
AT3G62400
(NP_850738)
—
D. discoideum
VIb
VIaH (Q02221),
VIaL (P12074)
CG17280 (NP_611805),
CG30093 (NP_725504)
agCG49440 (XP_316821),
E—NGG00000004851
(XP_319592)
F54D8.2 (Q20779)
COX13 (P32799)
COX13 (O74471)
—
VIc
VIc (P09669)a
VIb (P14854),
VIb.2 (NP_653214)
CG14235 (NP_728294)
cyclope (NP_536787)
agCG51594
(XP_316509)
agCG53860
(XP_317747)
Y71H2AM.5 (NP_497618)
COX12 (Q01519)
COX12 (O94581)
COX6b1 (BAA87883)
COX6b2 (NP_568867)
—
COX9/VIIa (P07255)
COX9 (NP_588061)
—
COX6b3 (NP_194535)
AT1G32710 (NP_174548)
—
COX VIIS (P20610)b,
COX VIIE (P20609)b
DDB0186146
NOTE.—The gene ID according to the organism-specific databases is followed in parentheses by the NCBI accession number of the protein sequences used for
alignments in this study. Multiple sequences indicate the presence of multiple paralogs. Dashes indicate that no significant homologs were found in an organism’s genome
database or trace archive.
a
Other vertebrates have heart-type and liver-type isoforms of these subunits. Subunit VIIIH is a pseudogene in H. sapiens (Goldberg et al. 2003).
b
These subunits are poorly conserved in D. discoideum and were not included in further sequence alignment.
a fold classification. The lack of either sequence or
structural similarity between different subunits suggests
that none of them arose through duplication from another
nuclear-encoded subunit.
Comparison of COX Subunit Composition Across Taxa
Although the three mitochondrial-encoded subunits
are present in all organisms that contain COX, the composition of the holoenzyme varies between taxa. Determination of the crystal structure of bovine COX (Tsukihara
et al. 1996) clarified the composition of the vertebrate
holoenzyme. Recent completion of whole-genome sequencing projects has now made it possible to use the
vertebrate sequences to search for homologs in all eukaryotic kingdoms.
Ludwig and colleagues have postulated that the
number of subunits is correlated with the regulatory
complexity of the enzyme, as suggested by the presence of
four subunits in the bacterium Paracoccus denitrificans,
seven in Dictyostelium discoideum, 11 in S. cerevisiae, and
13 in Bos taurus (Ludwig et al. 2001). Our analysis
suggests that this trend is true within the metazoans as
well. Mammalian subunit VIIb has no homolog in insects
(Szuplewski and Terracol 2001) or in nematodes, and
Caenorhabditis elegans lacks genes homologous to
mammalian subunits VIc, VIIa, and VIII as well (tables
2 and 3). However, failure to find homologs in these other
species may be the result of incomplete sequencing or
annotation of the genome (as is likely with subunit VIIc in
Anopheles gambiae because this subunit is present in both
D. melanogaster and C. elegans but not in Anopheles).
The composition of COX in plants provides further
evidence that nuclear-encoded subunits may have evolved
for taxon-specific regulatory functions. In plants, electrophoresis has shown that COX is composed of at least seven
Table 3
Nomenclature of Nuclear-Encoded COX Subunits VIIa, VIIb, VIIc, and VIII Across Different Taxa
Vertebrates
VIIa
VIIb
H. sapiens
VIIaH (P24310),
VIIaL (P14406)
CG18193 (NP_649793),
CG9603 (Q9VHS2)
agCG56784 (XP_311038),
agCG49762 (XP_310165)
—
COX7 (P10174)
—
—
—
VIIb (P24311),
VIIb.2 (NP_570972)
—
CG2249 (NP_652397)
—
—
agCG47737 (XP_313684)
—
—
—
—
—
F26E4.6 (NP_492596)
COX8 (P04039)
COX8 (NP_594028)
—
—
—
—
—
—
—
D. melanogaster
A. gambiae
C. elegans
S. cerevisiae
S. pombe
A. thaliana
D. discoideum
VIIc
VIIc (P15954)
VIII
VIII (P10176)a,
VIII.3 (NP_892016)
CG7181 (NP_649328)
NOTE.—The gene ID according to the organism-specific databases is followed in parentheses by the NCBI accession number of the protein sequences used for
alignments in this study. Multiple sequences indicate the presence of multiple paralogs. Dashes indicate that no significant homologs were found in an organism’s genome
database or trace archive.
a
Other vertebrates have heart-type and liver-type isoforms of these subunits. Subunit VIIIH is a pseudogene in H. sapiens (Goldberg et al. 2003).
Evolution of Cytochrome C Oxidase 1575
nuclear-encoded subunits (Jansch et al. 1996). However,
only two vertebrate nuclear-encoded subunits (Vb and VIb)
have orthologs in the Arabidopsis thaliana genome, which
suggests that the remaining subunits are unique to plants. Of
these novel subunits, only one, termed 5c, has been
characterized (Hamanaka et al. 1999). Our Blast search of
the A. thaliana genome also found an uncharacterized fourth
paralog of subunit VIb that was not identified in a previous
Southern hybridization screen (Ohtsu et al. 2001). This
omission is likely the result of significant divergence from
the other three paralogs in the region of the probe used for the
screen, which included most of the coding region of
AtCOX6b2.
Origin and Evolution of Nuclear-Encoded
COX Subunits
Like the nuclear-encoded COX subunits, the majority
of genes required for mitochondrial function are encoded
by the nuclear genome. Some of these genes originated in
the ancestral proteobacterium with subsequent transfer to
the host nucleus, whereas others evolved within the
eukaryotic nuclear genome (Berg and Kurland 2000). To
explore the evolutionary origin of the nuclear-encoded
COX subunits, we tested the first possibility by searching
for homologous sequences in the a-proteobacterium
Rickettsia prowazekii, the prokaryote thought to be most
closely related to ancestral mitochondria (Andersson et al.
1998), and the mitochondrial genome of the protozoan
Reclinomonas americana, which contains the largest
number of genes of any mitochondrion studied (Lang et
al. 1997). A Blast search failed to find homologs of any
nuclear-encoded subunits in Rickettsia, which suggests
that these proteins were not present in the ancestral
endosymbiont. The Reclinomonas mitochondrial genome,
although containing COX subunits I, II, and III as well as
a homolog of a nuclear-encoded COX assembly protein,
also does not contain any genes similar to the other
nuclear-encoded subunits (Lang et al. 1997). Together,
these findings suggest that the nuclear subunits did not
originate in the mitochondrial genome.
To determine when the COX subunits may have
arisen in the eukaryotic genome, we searched for
homologs in the genome of the protozoan Giardia lamblia,
an ancient amitochondriate eukaryote that diverged close
to when eukaryotes and prokaryotes split. Blast searches
failed to reveal any putative homologs of nuclear-encoded
COX subunits in Giardia, which suggests that these genes
evolved in eukaryotes after the invasion of the ancestral
endosymbiont. To attempt to find other genes in the
eukaryotic genome from which the nuclear-encoded
subunits may have been derived, we performed PSI-Blast
searches with each of the nuclear-encoded subunits. Two
iterations of each search failed to identify any genes with
significant homology, which suggests extensive divergence from ancestral genes.
Although the evolutionary origin of the nuclearencoded subunits remains a mystery, they have subsequently evolved specialized functions. ClustalW sequence
alignments show that each subunit is orthologous across
all taxa in which it is found (data not shown), but the
presence of multiple paralogs within taxa suggests evolution of regulatory functions. For example, as previously
discussed, alternative paralogs are expressed under normal or hypoxic conditions in both D. discoideum and
S. cerevisiae (Schiavo and Bisson 1989). In vertebrates,
multiple subunits have paralogs that are expressed either
ubiquitously (L, liver-type) or primarily in mature contractile muscles (H, heart-type) (Ewart, Zhang, and Capaldi
1991), whereas paralogs of subunit VIb show differential
tissue expression in plants and mammals (Ohtsu et al.
2001; Huttemann, Jaradat, and Grossman 2003). Similar
functional differences may explain the existence of other
uncharacterized paralogs found in insects and plants
(tables 1–3). There also is evidence for adaptive evolution
of individual paralogs after duplication, as shown by the
inactivation of subunit VIIIH in Old World monkeys
and hominids, possibly linked to optimization of aerobic
energy metabolism (Goldberg et al. 2003).
Conservation of COX Subunits and Implications
for Function
Although limited comparative analyses within taxa
have been undertaken to identify evolutionary patterns in
COX (Schmidt, Goodman, and Grossman 1999; Ludwig et
al. 2001), a broader analysis across diverse taxa increases the
power to detect specific sites that are essential for basic
enzyme function and regulation. To identify regions of
potential functional importance, we aligned sequences from
all taxa in tables 1, 2, and 3, as well as the bovine sequence
for each subunit. Sequences of all known isoforms within
a species were included in the alignment. Subunits present in
only one taxon (Vc in plants, VIII in animals, and VIIb in
humans only) were excluded from the analysis because they
are not essential for function across taxa. For each subunit,
residues were classified as ‘‘identical,’’ ‘‘conserved,’’ or
‘‘nonconserved,’’ on basis of the level of physicochemical
conservation determined by a ClustalW sequence alignment
across all taxa in which the subunit is present.
As expected, the three mitochondrial-encoded subunits
encoding the core enzyme are conserved across diverse
species at comparatively high levels that from 36% to 63%.
Subunit Va, which is 39% conserved and is found in animals
and yeasts, is much more conserved than any of the other
nuclear-encoded subunits. The high level of conservation
suggests that subunit Va has been subject to stronger
purifying selection. Supporting this implication of functional constraint is experimental evidence for ligand
interaction that involves subunit Va (Arnold, Goglia, and
Kadenbach 1998). The evidence is discussed in more detail
in following sections. Subunits VIa and VIb are conserved at
moderate levels (22% and 24%, respectively). Subunit VIb
is one of the two nuclear-encoded subunits shared between
animals, yeasts, and plants and, based on studies of the
crystal structure (Tsukihara et al. 1996), it appears to play
a key role in intermonomer contact, whereas subunit VIa
contains a known interaction site with ATP (Taanman,
Turina, and Capaldi 1994). In contrast, subunit VIIc is only
16% conserved and has no characterized function.
A low proportion of conserved amino acids, however,
does not necessarily indicate a lack of function. Subunit IV is
1576 Das et al.
FIG. 1.—Identical residues highlighted on the bovine heart COX
structure. Residues discussed in the text are numbered according to their
position in the bovine structure. Nuclear subunits are labeled on the right
monomer; hypothetical binding sites for ATP/ADP (in yeast and/or
mammals), T2 (in mammals), human androgen receptor (hAR, in
mammals), the regulatory subunit of protein kinase A (RIa, in mammals),
and Na1/Ca21 (in mammals), as well as the location of the zinc ion, are
indicated on the left. Mitochondrial subunits are in gray; indicated
membrane boundaries are approximate.
proportionately the least conserved of the nuclear-encoded
subunits (9%). Despite the low level of conservation
between the animal and yeast subunit IV sequences, we
concur with previous authors (Huttemann, Kadenbach, and
Grossman 2001) that these subunits are homologs, albeit
highly divergent, on the basis of two results from our
sequence analysis. First, in a search of the NCBI Conserved
Domain Database, the yeast sequences show significant
homology to COX IV domains. Second, in a neighborjoining tree of all COX nuclear-encoded subunits from all
species (unpublished data), the yeast sequences cluster most
closely in a monophyletic group with the subunit IV
sequences of animals, in contrast with the deep divergence
between monophyletic groups of any two other subunits.
Identification of Functional Domains by
Comparative Structural Analysis
To detect specific functional sites in the nuclearencoded subunits, we mapped identical and conserved
amino acids onto the crystal structure of bovine COX. We
then assessed whether these sites correlated with residues
of known function on the basis of biochemical data or
whether they might be novel sites of unknown function
and, thus, targets for future experimental investigation.
The crystal structure of bovine COX in dimeric form
(Tsukihara et al. 1996) is shown in figure 1; all identical
residues in the nuclear-encoded subunits are highlighted.
(All sequence alignments, a complete list of identical and
conserved residues, and stereo images of each subunit with
identical and conserved residues highlighted are included
in Supplementary Material online.) Specific residues are
referred to by their nomenclature in accordance with the
bovine sequence in the PDB structure.
FIG. 2.—Interaction of conserved regions of nuclear-encoded
subunits IV and Va. Wall-eye stereo view of matrix domains of subunits
IV (red) and Va (blue). Identical and conserved residues are labeled and
shown in ball-and-stick models. Dashed line indicates a putative salt
bridge between KIV43 and EIV55, whose side chains are 3.0 Å apart. The
contacts between LIV40, KIV43, LIV51, and EIV55 maintain the helixturn-helix structure in subunit IV that positions WIV48 to extend into
a highly conserved region of subunit Va. This region corresponds to part
of the likely binding site for thyroid hormone T2.
Below, we discuss the structural context and putative
functional significance of the identical residues in each
subunit. In the following analysis, we define amino acids to
be in contact if the minimum atom-atom distance between
their side chains is less than 5 Å (Russell and Barton 1994).
Subunit IV
Subunit IV is the least conserved of the nuclearencoded subunits. The identical and conserved residues fall
into two regions of the subunit—one cluster in the matrix
domain and five residues in the cytosolic domain. In the
matrix domain, WIV48, an identical site, contacts other
identical residues in a highly conserved region of subunit Va
and is likely to be part of the same functional region (fig. 2).
Identical site EIV55 appears to be important for maintaining
a helix-turn-helix structure through its contacts with three
other conserved residues (LIV40, KIV43, and LIV51) on the
adjacent helix and loop. The charged side chains of EIV55
and KIV43 are only 3.0 Å apart and well positioned to form
a stabilizing salt bridge. This fold allows WIV48 to be
positioned to extend into a highly conserved domain of
subunit Va. Other conserved residues in the matrix domain
also maintain contact with subunit Va.
In mammals, two ATP binding sites in subunit IV
have been proposed. One binding site has been modeled in
the matrix domain and involves residues RIV20, RIV73,
TIV75, EIV77, and WIV78, as well as residues from subunits
I and II (Huttemann, Kadenbach, and Grossman 2001).
None of these residues in subunit IV are conserved across
taxa. The second ATP binding site in mammals is located in
the cytosolic domain (Reimann et al. 1988), but the putative
function of the conserved cytosolic residues in subunit
IV is not clear. The sole identical residue in that region,
GIV133, is likely conserved not because of interaction with
other residues or ligands, but rather to provide the structural
flexibility needed for the sharp bend in the backbone chain
in that region. Indeed, analysis of the geometry of this
residue by application of PROCHECK (Laskowski et al.
1993) shows that the C-N-Ca angle of GIV133 deviates
from the ideal angle of 112.58 by 16.78, the largest such
deviation in the COX structure by some 20%.
Evolution of Cytochrome C Oxidase 1577
Subunit Va
The five a-helices of subunit Va form a right-handed
superhelix (Tsukihara et al. 1996), a structure that probably
is maintained across animals and yeasts, as shown by the
distribution of identical and conserved residues throughout
the protein. However, the majority of the identical residues
are clustered in the three a-helices formed by the amino acid
sequence between PVa43 and GVa97. This region, which
includes a number of charged residues, is 48% conserved
(28% identical) and forms an exposed pocket that is also
bordered by WIV48 (fig. 2). Subunit Va has been shown to
bind thyroid hormone 3,5-diiodothyronine (T2) (Arnold,
Goglia, and Kadenbach 1998), and we speculate that this
pocket may be the T2 binding site.
Subunit Vb
The COOH-terminal of subunit Vb contains a zinc site
that, in animals, involves four cysteine residues structurally
arranged in a classical zinc finger motif (Tsukihara et al.
1996). Whereas three of these cysteines are identical across
all taxa, CVb62 is substituted with a glycine residue in plants
(Welchen, Chan, and Gonzalez 2002), as well as in yeasts
and in D. discoideum. However, the sequences for subunit
Vb in Arabidopsis, rice, S. cerevisiae, S. pombe, and D.
discoideum contain a histidine residue at position 67. This
histidine may fulfill the same function as CVb62 in animals,
because histidine residues can also coordinate zinc ions in
classical zinc finger domains (Matthews and Sunde 2002).
We predict that this evolutionary change in animals would
likely require a change in the conformation of the backbone
to properly position the cysteine to coordinate the zinc ion.
Identical residues SVb51 and RVb56 may be critical residues
for attachment to subunit I, supported by several other
conserved residues contacting core subunits I and III. Also,
the b-barrel structure of the COOH-terminal domain
(Tsukihara et al. 1996) may be maintained across taxa, on
the basis of conservation of three residues in contact on the
inner surface (VVb49, VVb58, and YVb89) that appear to
stabilize the structure.
In mammals, subunit Vb interacts with the regulatory
subunit of protein kinase A. This action inhibits COX
activity in a cAMP-dependent process (Yang et al. 1998;
Bender and Kadenbach 2000). In addition, in vitro studies
have shown that subunit Vb, primarily through the COOHterminal, binds the human androgen receptor (hAR),
although the effect of hAR on COX activity is unknown
(Beauchemin et al. 2001). Although these putative regulatory mechanisms have not been tested across taxa, conservation of similar interactions may underlie the maintenance
of functional and structural domains of subunit Vb.
Subunit VIa
The primary conserved region of subunit VIa is a loop
structure on the cytosolic side of the enzyme. This region
correlates with a putative ATP binding site at residues 63
to 68, previously identified by more limited sequence
comparison and based on similarity to other ATP binding–
site motifs (Taanman, Turina, and Capaldi 1994). The
GDGXX(T/S) motif is conserved in all taxa we analyzed,
with slight modifications: G!S at position 1 in C. elegans
and D!E at position 2 in A. gambiae and in D.
melanogaster CG17280. Our analysis identified several
other residues that may also be part of the binding site or
that may help to stabilize the structure. RVIa56, KVIa58,
WVIa62, FVIa70, and NVIa72 are identical amino acids that
are found on either side of the ATP binding motif and that
contact subunits I or III.
Subunit VIa is also adjacent to subunit VIb near the
dimer interface. YVIa50 is an identical residue that contacts
residues GVIb79 (identical) and FVIb81 (conserved) in
subunit VIb, which suggests that it is critical for the
interface of the two subunits. Of the 10 residues at the NH2terminal of subunit VIa that contact subunit I of the opposite
monomer, only AVIa4 is conserved. This arrangement
indicates some variation in how the intermonomer contact
in that region is maintained. Finally, six identical and
conserved residues are distributed throughout the transmembrane helix of subunit VIa. Although functional studies
in yeast and mammals suggest a second ATP binding site on
the matrix side of VIa (Anthony, Reimann, and Kadenbach
1993; Beauvoit et al. 1999), the orientation of these
conserved residues along the helix does not make them
obvious candidates for a putative binding site. Instead, they
may be more important for maintaining the structure of the
helix or contact with subunit III.
Subunit VIb
Subunit VIb is composed of three a-helices connected
by relatively tight turns. VIb is the only subunit that is
situated completely on the side of the intermembrane space
and is the primary nuclear subunit responsible for intermonomer contact. The importance of this role is reflected in the
high level of sequence conservation across taxa. The region
of contact is from residues 39 to 53, which forms the turn
between two helices that extend into the intermembrane
space (Tsukihara et al. 1996). GlyVIb47 is identical across all
taxa, perhaps because of the constraints of the tight structure
of the turn. The two extending helices are maintained in
a near antiparallel geometry by two disulfide bridges
between CVIb29 to CVIb64 and CVIb39 to CVIb53, all of
which are identical. Several other identical and conserved
residues likely preserve the orientation of the third helix as
well, whereas conserved COOH-terminal amino acids
contact subunit VIa.
Although the amino acids that are essential for the
conformation of this domain are all identical, the intervening residues vary between species. For example,
mammals have three extra amino acids in the loop at
positions 42 to 44, and both the testis-specific isoform in
mammals (Huttemann, Jaradat, and Grossman 2003) and
the novel isoform in Arabidopsis are highly divergent from
the common isoforms of their respective species in this
region. Such lineage-specific and tissue-specific diversity
suggests that this region may be evolving for some
physiological function.
An interaction site for cytochrome c has been
proposed on the cytosolic side of the enzyme. The
proposed interaction involves acidic residues from subunits I, II, III, and VIb (Tsukihara et al. 1996). Of the
1578 Das et al.
amino acids from subunit VIb that are hypothesized to
interact with cytochrome c, DVIb74 is conserved across
taxa, whereas EVIb78 is not. Because DVIb74 is the only
conserved residue on the external surface of the third helix,
our analysis supports an important role for this amino acid
in interacting with cytochrome c. There are also three
identical sites in the NH2-terminal region. Although these
residues (19 to 21) may merely stabilize the conformation
of the subunit, they are on the margin of an open pocket on
the cytosolic surface. This region may have functional
significance because it is bordered by identical residues
from subunits I and III as well.
Subunit VIc
The identical and conserved sites in subunit VIc do
not correlate with any known function. Two identical sites,
FVIc50 and YVIc51, are located at the cytosolic end of the
transmembrane helix and contact subunits II and IV. The
neighboring amino acids on subunits II and IV are not
conserved, however, so there is no selection on interacting
pairs. The third identical residue in subunit VIc is located
in the cytosolic a-helix, directly above the other two
residues. As KVIc58 is near no other subunit, these three
identical residues together may be important for some
ligand interaction.
Subunit VIIa
Three identical sites in subunit VIIa occur at the sharp
turn between the transmembrane helix and the NH2-terminal
a-helix. These residues are likely to be required to maintain
the structure of the subunit and its orientation with respect to
other subunits; for example, PVIIa19 is located at the sharp
bend in the turn, whereas QVIIa13 forms a polar interaction
with the conserved residue TVb14. VVIIa14, which is also
conserved, contacts an identical residue on subunit III,
EIII60. Less explicable is the identity of LVIIa31, which
contacts YIII55 and QIII56. These two residues in subunit III
are not conserved in plants and yeasts but are identical
within animals, as well as adjacent to WIII57, which is
identical across taxa. Therefore, some function may be
associated with this region.
Subunit VIIc
Four identical residues in subunit VIIc are paired in
Pro-Phe combinations in two locations. One pair occurs in
the irregular NH2-terminal domain on the matrix side; the
position of PVIIc12 at a bend in this region suggests that
this pair is required for the structure. The transmembrane
helix of subunit VIIc changes its angle of inclination
midway through the helix. The second pair, with PVIIc36,
is located at this bend in the helix and is supported by
another identical residue, FVIIc33, which again suggests
that it is conserved for structural integrity. Other conserved
residues at the COOH-terminal end of the transmembrane
helix interact with conserved residues in subunit I.
Evidence for Selection on Functional Domains
Because our analysis of sequence conservation across
taxa identified many residues that did not correlate with
regions of known function, we performed two global
analyses to assess whether these conserved residues are
likely to be playing a functional or structural role and
whether any selective pressures could be influencing the
evolution of the enzyme as a whole. We first examined
whether similar biochemical classes of amino acids were
more likely to be conserved within different taxa. We then
explored the spatial location of amino acids conserved
across all taxa to determine whether they were positioned
in a nonrandom distribution that would support selective
constraints based on structure or function.
Groups of amino acids can be classified by the different
roles they tend to play within the context of a protein’s
structure and function (Betts and Russell 2003). Variation in
substitution rates of different amino acids can shed light on
the selective forces acting on a protein. On the basis of the
relative importance of different functional or structural
constraints, amino acids that are more likely to be involved
in polar interactions (for example in salt bridges or in ligand
interaction) or nonpolar interactions (such as hydrophobic
packing or aromatic ring stacking) may be conserved at
different frequency. To determine whether similar functional constraints are acting on different evolutionary
lineages, we analyzed within-taxon sequence alignments
(human-Drosophila, S. cerevisiae–S. pombe, and Arabidopsis-rice) for similar patterns of amino acid conservation.
To predict the expected distribution of conserved
residues given an equal probability of conservation for all
amino acids, we calculated the average frequency of amino
acid occurrence across all subunits within each lineage. The
distribution of amino acids identical within each taxon was
significantly different from the average distribution in
yeasts and in animals but not in plants (v2 test; yeasts, P ¼
0.017; animals, P ¼ 0.023; plants, P ¼ 0.639), as might
be expected on the basis of the comparatively recent
divergence of monocots and eudicots. However, in all
taxa, the aromatic amino acids Phe, Tyr, and Trp were
overrepresented among identical residues by at least 15%
compared with the number expected on the basis of overall
amino acid frequency, as were Gly and Cys (table 4). All
of these amino acids play key roles in structural stability:
the bulky side chains of aromatic residues are often key
components of hydrophobic cores and can participate in
energetically favorable stacking interactions; cysteines are
important for disulfide bonds and for binding metals; and
glycine is conformationally flexible and occurs at tight
turns in structures (Betts and Russell 2003). This pattern
across independently evolving lineages suggests that, in
general, hydrophobic amino acids in COX are conserved
to maintain the structure of functional sites and possibly to
maintain the structure of interfaces between subunits. In
contrast, no strong trends are evident across taxa in the
conservation of hydrophilic amino acids. However, those
hydrophilic residues that are conserved are still likely to
play important roles; for example, salt bridges tend to be
conserved for specific functional or structural purposes
rather than by any general rule based on solvent exposure,
proximity to active sites, or location in the protein
(Schueler and Margalit 1995).
Visual inspection of the COX structure showed that
identical and physicochemically conserved residues occur
Evolution of Cytochrome C Oxidase 1579
Table 4
Distribution of Identical Amino Acids Within Taxa
Animals
Yeasts
Plants
Observed Expected Observed Expected Observed Expected
Aliphatic
Gly
Ala
Val
Leu
Ile
Pro
29
28
18
33
9
28
24.2
32.3
22.2
31.9
14.5
18.2
25
20
10
32
7
23
19.9
24.9
19.7
26. 2
15.4
17.1
22
26
18
15
6
19
16.3
30.1
19.8
13.2
7.0
18.1
Aromatic
Phe
18
Tyr
19
Trp
11
15.3
13.2
6.4
21
14
12
17.3
12.1
7.1
10
8
7
7.9
5.5
4.3
10
2
7.4
8.5
8
2
4.9
5.9
7
0
4.6
1.9
Hydroxyl
Ser
12
Thr
9
21.9
15.8
14
13
20.7
17.7
7
13
14.0
15.0
Hydrophilic
Lys
26
His
6
Arg
32
Asp
19
Glu
24
Asn
12
Gln
10
26.5
8.5
26.7
15.3
20.3
13.0
12.8
22
12
26
16
27
11
11
25.4
7.5
20.5
15.1
21.8
14.6
12.0
11
10
12
13
39
5
5
14.4
7.3
12.6
14.4
34.6
5.8
6.1
Sulfur
Cys
Met
NOTE.—Amino acids are grouped by chemical classification. Sequences used
for within-taxon alignment were H. sapiens and D. melanogaster in animals, S.
cerevisiae and S. pombe in yeasts, and A. thaliana and O. sativa in plants. Bold text
indicates amino acids that are overrepresented by at least 15%; italicized text
indicates underrepresentation by at least 15%.
in spatial proximity to each other, which suggests that
many of these residues may indeed be positioned to form
functional sites (fig. 3A). We explored this observation by
calculating whether identical and conserved residues have
a tendency to cluster together. Clustering of conserved
amino acids would suggest that selection acts to maintain
functional or structural interactions between residues.
We plotted the frequency of pairwise minimum atomatom distances of less than 30 Å for all identical and
conserved amino acids as well as for an equal number of
randomly selected residues (fig. 3B). There is an excess of
conserved amino acids at distances of less than 15 Å from
each other; amino acids within this distance are likely to be
part of the same structural feature or functional region. This
result, along with the overrepresentation of structurally
important residues among conserved amino acids, supports
the premise that these conserved residues act to maintain
the structural integrity of functional sites. Even when
the highly conserved subunits Va and VIb are excluded
from the analysis, direct contacts (defined as less than 5 Å)
still occur at twofold higher frequency among conserved
residues than in the randomized data set (data not shown).
Discussion
What are the roles of the nuclear-encoded subunits in
COX? Experimental approaches have revealed many
FIG. 3.—Conserved residues are nonrandomly clustered in putative
functional domains. (A) Amino acids in subunits Vb and VIIc are shown
in space-filling display to illustrate how conserved residues (colored green
[Vb] and blue [VIIc]) tend to be located in close proximity to identical
residues (colored red [Vb] and orange [VIIc]). (B) A histogram of
pairwise distances between the 284 identical and conserved residues in
the COX dimer (solid bars) and between an equal number of randomly
chosen residues (open bars). Compared with the randomly chosen
residues, conserved sites occur more frequently at distances of less than
20 Å from each other.
intriguing functions for the nuclear-encoded subunits in
model organisms, such as environmental regulation,
tissue-specific function, and ATP-dependent allosteric
regulation (Schiavo and Bisson 1989; Ewart, Zhang, and
Capaldi 1991; Taanman, Turina, and Capaldi 1994). Other
studies have employed phylogenetic techniques to measure
rates of substitution in the nuclear-encoded subunits to
identify residues likely to be functional or coevolving with
known interacting proteins or cofactors (Schmidt et al.
2001; Goldberg et al. 2003). However, these previous
studies have been confined largely to one organism or to
a group of relatively closely related species, which limits
our understanding of how widespread these regulatory
mechanisms are and of which ones are essential for basic
regulation across diverse taxa. Our broader analysis allows
us to detect highly conserved sites to identify essential
functional domains of the enzyme.
1580 Das et al.
Early in the past decade, the focus on finding functions
of the nuclear-encoded subunits shifted to lower eukaryotes,
in part because experimental studies of higher eukaryotes
were proving difficult (Capaldi et al. 1990). Since then,
researchers have begun to recognize the diversity of
regulatory mechanisms provided by the nuclear-encoded
subunits, which appear to fine-tune the basic redox reaction
of COX to different physiologies. With the availability of
whole-genome sequences, we have been able to perform
a broad search for subunit homologs across taxa that
revealed different subunit compositions of the holoenzyme,
even within the metazoans. The roles of evolutionarily novel
subunits merit closer investigation, as preliminary evidence
indicates interesting functional roles. For example, expression of subunit VIIb is depressed in the intestines of mice
deficient in gastrointestinal pacemaker cells (Takayama et
al. 2001), which suggests a regulatory role consistent with
origin along the deuterostome lineage. Subunit VIII evolved
before the protostome-deuterostome split, yet subsequent
duplication in the vertebrates followed by inactivation of
one paralog in humans implies relatively rapid evolution of
function (Goldberg et al. 2003); this subunit remains
uncharacterized in invertebrates. Plants, in particular,
appear to have an almost entirely novel set of nuclearencoded subunits. Initial characterization of plant subunits
Vc and VIb show differential tissue expression, and the
novel divergent paralog of Arabidopsis subunit VIb that was
identified in our analysis may reveal new regulatory
properties.
The question of how this diversity of nuclear-encoded
subunits evolved remains a conundrum. There is evidence
for recent transfer of COX genes from the mitochondrial to
the nuclear genome. During the evolution of plants, subunit
II has been transferred to the nucleus in the legume lineage,
with some species maintaining two active copies and other
species silencing either the nuclear or the mitochondrial
copy (Palmer et al. 2000). This plasticity may be facilitated
by the large size of the plant mitochondrial genome. We
were unable to find any support for a similar mitochondrial
origin of the nuclear-encoded subunits or of any other
nuclear genes from which the subunits may have been
derived. Therefore, we conclude that they arose in the
nuclear genome after the acquisition of mitochondria and
have diverged extensively from any ancestral genes.
By aligning sequences of the nuclear-encoded subunits from highly divergent species and mapping identical
and physicochemically conserved sites onto the protein
structure, we have been able to classify regions of interest
of the protein into three broad categories: (1) known
functional sites that have diverged among species, (2)
known functional sites that are conserved across species,
and (3) conserved sites of unknown function. Although we
do not speculate further on the third class, we discuss
below the most intriguing examples of the first two.
One case of divergence of known functional regions
involves the sites at which nucleotides can bind to regulate
COX activity. Equilibrium analysis of COX with ATP and
ADP have revealed 10 nucleotide binding sites in the bovine
monomer (Napiwotzki et al. 1997). Two of these sites, the
matrix and cytosolic ATP binding sites on subunit IV, have
been studied experimentally in mammals (Napiwotzki et al.
1997; Huttemann, Kadenbach, and Grossman 2001). In
yeasts, ATP has also been shown to inhibit COX activity
through binding on both the matrix and the cytosolic sides of
subunit VIa (Anthony, Reimann, and Kadenbach 1993;
Taanman, Turina, and Capaldi 1994; Beauvoit et al. 1999).
Our analysis shows that among the locations of these four
nucleotide binding sites, only the cytosolic domain of
subunit VIa is conserved across animals and yeasts. The
others have either arisen independently in each lineage or
have diverged through compensatory mutations that preserve the function of the region.
An example of an unpredicted conserved region is the
putative binding site for thyroid hormone T2 on subunit Va
in mammals (Arnold, Goglia, and Kadenbach 1998). T2
increases COX activity by interfering with allosteric
inhibition by ATP. Our analysis reveals that the binding
site for T2 is highly conserved across animals and yeasts,
a surprising result because insects and yeasts do not produce
thyroid hormones. What might be the analogous ligand in
these other species? In insects, a likely functional analog is
juvenile hormone (JH) (Wheeler and Nijhout 2003);
L-3,5,39-triiodothyronine (T3), an adduct of T2, can act
via a putative JH receptor in insect follicle cell membranes
(Kim et al. 1999). Presumably a similar analog also acts
in yeasts.
On the basis of the proximity of subunits IV and Va, T2
has been proposed to affect COX activity by interacting with
the ATP binding site in the matrix domain of subunit IV
(Ludwig et al. 2001). Because this ATP binding site does not
appear to be conserved across taxa, we argue that this
mechanism is unlikely to be a widespread phenomenon,
unless a matrix ATP binding site in subunit IV has been
independently maintained in different taxa after duplication. Alternatively, an allosteric interaction may occur
with the conserved domain in the cytosolic domain of subunit VIa.
In addition to residues in these known binding sites or
those with obvious interactions with other subunits or
cofactors, our analysis has identified essential structural
elements as well as many identical and conserved amino
acids whose functions are unclear and are worthy of future
study. Some of these structural elements and amino acids
may be catalytic residues in uncharacterized active sites.
However, our results show that hydrophobic and nonpolar
amino acids, which are more likely to be involved in
structural scaffolding rather than in substrate interactions,
tend to be conserved at frequencies higher than expected
within all taxa. Because these identical and conserved
residues are nonrandomly distributed in clusters throughout the protein, we suggest that these groups of interacting
amino acids may be conserved to maintain the structural
foundation for evolving catalytic sites.
Supplementary Material
The following supplementary material can be found
online at the journal’s web site:
1. Multiple sequence alignments in interleaved format for
all nuclear-encoded subunits (HTML).
2. Multiple sequence alignments in sequential format for
all nuclear-encoded subunits (text).
Evolution of Cytochrome C Oxidase 1581
3. Complete list of all identical and conserved residues
(tab-delimited text).
4. PyMOL scripts for the complete structure and all
individual subunits with identical and conserved
residues highlighted (HTML).
5. Stereo images of the complete structure and all
individual subunits with identical and conserved
residues highlighted (PDF).
Acknowledgments
We thank Greg Davis, Alistair McGregor, members
of the Stern lab, and three anonymous reviewers for
helpful comments. This work was supported by a Howard
Hughes Medical Institute Predoctoral Fellowship to J.D.
and a David and Lucile Packard Foundation Fellowship
and a National Institutes of Health grant to D.L.S.
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Michele Vendruscolo, Associate Editor
Accepted May 5, 2004