(Received for publication, November 21, 1994; and in revised form, June 13, 1995)
From the
Thermus thermophilus HB8 cells grown under reduced
dioxygen tensions contain a substantially increased amount of heme A,
much of which appears to be due to the presence of the terminal
oxidase, cytochrome ba. We describe a purification
procedure for this enzyme that yields
100 mg of pure protein from
2 kg of wet mass of cells grown in
50 µM O
. Examination of the protein by SDS-polyacrylamide
gel electrophoresis followed by staining with Coomassie Blue reveals
one strongly staining band at
35 kDa and one very weakly staining
band at
18 kDa as reported earlier (Zimmermann, B. H., Nitsche, C.
I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5779-5783). By
contrast, treatment of the gels with AgNO
reveals that the
larger polypeptide stains quite weakly while the smaller polypeptide
stains very strongly. These results suggested the presence of two
polypeptides in this protein. Using partial amino acid sequences from
both proteins to obtain DNA sequence information, we isolated and
sequenced a portion of the Thermus chromosome containing the
genes encoding the larger protein, subunit I (cbaA), and the
smaller protein, subunit II (cbaB). The two polypeptides were
isolated using reversed phase liquid chromatography, and their mole
percent amino acid compositions are consistent with the proposed
translation of their respective genes. The two genes appear to be part
of a larger operon, but we have not extended the sequencing to identify
initiation and termination sequences. The deduced amino acid sequence
of subunit I includes the six canonical histidine residues involved in
binding the low spin heme B and the binuclear center
Cu
/heme A. These and other conserved amino acids are placed
along the polypeptide among alternating hydrophobic and hydrophilic
segments in a pattern that shows clear homology to other members of the
heme- and copper-requiring terminal oxidases. The deduced amino acid
sequence of the subunit II contains the Cu
binding motif,
including two cysteines, two histidines, and a methionine, but, in
contrast to most other subunits II, it has only one region of
hydrophobic sequence near its N terminus. Alignment of these two
polypeptides with other cytochrome c and quinol oxidases,
combined with secondary structure analysis and previous spectral
studies, clearly establish cytochrome ba
as a bona fide member of the superfamily of heme- and
copper-requiring oxidases. The alignments further indicate that
cytochrome ba
is phylogenetically distant from
other cytochrome c and quinol oxidases, and they substantially
decrease the number of conserved amino acid residues.
The terminal oxidases of the respiratory electron transfer systems of mitochondria and many bacteria belong to a group of integral membrane proteins that have heme- and copper-containing active centers. These enzymes receive electrons either from quinols or from cytochromes c and direct them in the reduction of dioxygen to water. On average, for each electron transferred, one proton is moved from the interior, negative side of the membrane to the exterior, positive side. In this fashion, much of the free energy of the redox process is conserved in a gradient of membrane potential and pH. The cytochrome c oxidase from bovine heart tissue has served as the prototype of this class of enzymes for many years. However, during the past two decades it has become evident that certain bacterial enzymes possess similar spectroscopic and functional attributes, and indeed, the bovine enzyme is but one member of a superfamily of homologous proteins that have largely identical biological function and are likely to have very similar three-dimensional structures. (See the following references for review: Fee et al.(1986), Anraku(1988), Saraste et al. (1991a), Babcock and Wikström(1992), Ferguson-Miller(1993), Gennis(1993), and Calhoun et al. (1994).) As new family members are characterized, the identification of common structural features as well as divergent structures, especially in phylogenetically distant members, provides useful information in determining the minimal elements required for function, while demonstrating the extent of variation that can be tolerated without loss of function.
Members of this family contain one low spin
cytochrome and a bimetallic structure consisting of a high spin heme in
close proximity to a copper ion. These metals reside in the large
subunit of the complex, subunit I, where they are coordinated to at
least six conserved histidine residues. The cytochrome c oxidases bind two (cf. Kroneck et al.(1990) and
Fee et al.(1995) and references therein) additional copper
ions in the Cu site to conserved histidine and cysteine
residues of subunit II. The quinol oxidases possess a homologous
subunit II, but the conserved residues involved in the Cu
site in the cytochrome c oxidases are not present (cf. Mather et al. (1991) and Lappalainen and
Saraste(1994) and references therein). It is possible that the subunit
II of quinol oxidases serves to bind a quinol and/or a semiquinone
which can transfer electrons to the heme centers. If so, the two types
of oxidases would function similarly with electrons flowing from
cytochrome c (or quinols) into the Cu
site (or
semiquinone binding site), then on to the low spin heme, and finally to
the binuclear site where dioxygen reduction occurs (cf.
Babcock and Wikström(1992)). Tentative
three-dimensional models have been advanced, as have mechanistic
schemes for the reduction of dioxygen (cf. Ferguson-Miller
(1993), Mather et al. (1993a), and Calhoun et
al.(1994)). In addition, the coupling of the electron transfer
processes to proton translocation has been the matter of considerable
speculation (Blair et al., 1986; Wikström et al., 1994).
Different types of heme structures are
utilized by this family of enzymes. Eukaryotic enzymes appear to
contain only heme A, thus the designation cytochrome aa. Rhodobacter sphaeroides (Hosler et al., 1992) and Paracoccus denitrificans (Ludwig
and Schatz, 1980) are examples of bacteria that have a cytochrome aa
. Escherichia coli can accommodate a
heme O in the high spin site and a heme B in the low spin site yielding
the designation cytochrome bo (or oo
or bo
) (cf. Puustinen and
Wikström(1991) Wu et al.(1992), Saiki et al.(1993), and Calhoun et al.(1994)). There are
additional variations on this theme involving hemes A, O, B, and
possibly C (cf. Gennis(1993) and Preisig et
al.(1993)).
In 1988, Zimmermann et al. reported the
existence of cytochrome ba in Thermus
thermophilus HB8 in which heme B occupies the low spin heme site,
heme A the high spin site, while the Cu sites are similar to those of
other members of the cytochrome c oxidase family. This enzyme
is a cytochrome c oxidase and was first described as
containing a single protein subunit having molecular mass
35 kDa
and possessing only a single cysteine in the molecule (Zimmermann et al., 1988). This finding was considered unusual since the
largest subunit of all other enzymes in this family is greater than 50
kDa in size, and at least one additional subunit (subunit II) is always
present. Moreover, the cysteine content was of particular interest
since all previous models for metal coordination in the Cu
site involved two cysteines and two histidines, residing in a
separate polypeptide. It remains to be demonstrated that cytochrome ba
translocates protons.
Here we describe a
purification procedure for the enzyme, show that it consists of two
subunits, identify and sequence the genes that encode the two proteins,
and analyze the deduced amino acid sequences. The major conclusions are
that cytochrome ba is a multisubunit cytochrome c oxidase, and its subunits are homologs of the heme-copper
oxidase superfamily.
Phagemid
vectors pBluescript II SK(+/-) and bacterial strain XL-1
blue were obtained from Stratagene Cloning Systems. Calf intestinal
phosphatase, 5-bromo-4-chloro-3-indoyl
-D-galactopyranoside, and TEMED were obtained from Sigma.
Isopropyl
-thio-D-galactopyranoside and some restriction
enzymes were purchased from Boehringer Mannheim. Additional restriction
enzymes, T4 DNA ligase, and T4 polynucleotide kinase were purchased
from New England Biolabs. Exonuclease III deletion series were
constructed utilizing the Erasabase Kit from Promega. Sequencing
enzymes and reagents were purchased from U. S. Biochemical Corp.
Agarose LE, Tris base, and urea were purchased from Research Organics,
Inc. Nitrocellulose filters (82 mm, round) were purchased from
Schleicher & Schuell. [
-
P]ATP (6000
Ci/mmol) and
-
S-dATP (1000-1500 Ci/mmol) were
purchased from DuPont NEN. Synthetic oligonucleotides were provided by
the DNA synthesis facility at Los Alamos National Laboratories, Life
Sciences Division.
Amino acid analyses and reversed
phase liquid chromatography (RPLC) were carried out at the University
of New Mexico School of Medicine Protein Chemistry Laboratory. The
amino acid analyses were performed using the Pico-Tag system (Waters,
Milford, MA). Samples of cytochrome ba were
shipped to Albuquerque and analyzed without any prior attempt to remove
buffer or detergent. 2-5 µg of protein were pipetted into 6
50-mm glass tubes. After drying, the tubes were placed into a
larger container preloaded with 250 µl of 6 N HCl. The
container was flushed with nitrogen, evacuated, sealed, and placed in
the oven for 20 h at 110 °C. After hydrolysis, acid was removed
under vacuum (Speedvac). Samples for the determination of cysteine were
first oxidized with performic acid (Hirs, 1967) for 18 h at room
temperature. Performic acid was removed in a Speedvac, and the samples
were hydrolyzed as above. For derivatization and detection of amino
acids, samples after hydrolysis were dried, mixed with 10 µl of
redry solution (ethanol, water, triethylamine, 2:2:1), dried again, and
reacted with 20 µl of phenylisothiocyanate (Cohen and Strydom,
1988) reagent (ethanol, water, triethylamine, phenylisothiocyanate) for
20 min at room temperature. Excess reagent was removed under vacuum.
Derivatized samples were dissolved in 100 µl of 0.14 M sodium acetate that had been adjusted to pH 6.4 with acetic acid.
5-20 µl were injected on the column for analysis. Analysis
was performed on a Waters C18 column (3.9
150 mm) using a
gradient elution as described by Bidlingmeyer et al.(1984). A
sample of egg white lysozyme was used as control protein. Quantitation
was obtained using Pierce standard H amino acid calibration mixture.
N-terminal amino acid sequences were obtained from purified protein sent on dry ice to both the Institüt für Biochemie, Aachen, Germany, and to the University of New Mexico Protein Chemistry Facility in Albuquerque, NM, for peptide isolation and analysis. N-terminal peptide sequences and cyanogen bromide cleavage and peptide fragment purification were carried out essentially as described in Buse et al.(1989).
SDS-PAGE in the presence of 8 M urea was carried out, with minor modifications, according to Downer et al. (1976), using a Hoeffer model SE 500 slab gel system with 1.5-mm spacers or a Bio-Rad Mini-PROTEAN II electrophoresis cell. Total acrylamide concentration was 7%, with a Bis:acrylamide ratio of 1:15. About 15 mm of a 3.5% gel (1:15 Bis:acrylamide) was used as a discontinuous stacking gel on top of the separating gel. The standard denaturing condition used was incubation at 70 °C for 15 min in 4% SDS, 4% 2-mercaptoethanol, 15 mM Tris-Cl, pH 7.8, 5% glycerol, and 4 M urea. The separating gel was run at 7.5% acrylamide, 0.2% Bis, pH 4.3, while the stacking gel was 2.5% acrylamide, 0.62% Bis, and 13.3 µM riboflavin at pH 5.0. Polymerization of the stacking gel was induced by exposure to a fluorescent light, and electrophoresis was run with the cathode at the bottom. The sample was applied in a solution of 20% glycerol, 5 mM MES buffer, pH 6.4, 0.1 mM EDTA, 0.5% lauryl maltoside, with 0.1 mM methyl green used as the tracking dye. Both denaturing and native gels were stained and fixed with 26% isopropanol, 11% glacial acetic acid, and 0.1% Coomassie Blue R, and destained with 10% isopropanol and 10% glacial acetic acid. Silver staining was carried out using the Bio-Rad Silver Stain Plus kit. Nondenaturing polyacrylamide gel electrophoresis in the presence of lauryl maltoside was carried out, with minor modifications, according to Gabriel(1972).
The following conditions for reversed
phase liquid chromatography were found to give adequate recovery of
protein and significant resolution. A Phenomenex C-1 column (4.6
30 mm, 0.5-ml void volume) equilibrated with solvent A (60%
formic acid, aqueous) followed by a linear gradient to 100% solvent B
(60% formic acid, 30% isopropanol, aqueous) and finally by continued
isocratic washing effected a separation of the two subunits of
cytochrome ba
. The presence of protein in the
individual fractions was determined by quantitative amino acid
analyses.
The initial purification steps for cytochromes ba are the same as those we have developed for the
isolation of other respiratory proteins from T. thermophilus membranes (Yoshida et al., 1984). Briefly, 2 kg of cell
paste are blended and extracted with 0.2 M Tris-Cl, pH 7.8, to
obtain cytochrome c
. Membrane spheroplasts are
formed by treating the cell paste with lysozyme at 70 °C. The
spheroplasts are ruptured in a Manton-Gaulin laboratory homogenizer,
and the membranes are purified by a series of centrifugation steps and
extracted with Triton X-100. The detergent extract is loaded onto a 10
60-cm DE52 column preequilibrated at room temperature with 2%
Triton X-100 in Tris-EDTA (TE) buffer. This large column is washed at
room temperature with
6 column volumes 2% Triton X-100 in TE
buffer. Usually, during the washing of the column, a pink-colored band
precedes a brown-colored band. (
)The brown-colored band
contains cytochrome ba
. Occasionally, the bands do
not fully separate on this column, in which case the fractions
containing heme A are combined, and the resulting solution is the
starting material for the purification of cytochrome ba
.
The brown-colored material elutes from the
large column in 5 column volumes, and the cytochrome ba
is recovered from the eluate by batch
adsorption. (
)In this procedure, the solution is adjusted to
pH 6.4, diluted to a conductivity of
200 µmho, and added to
CM-52 resin preequilibrated with 10 mM MES at pH 6.5 (250 ml
of resin/kg of wet cells). The protein-adsorbed resin is poured into a
5 cm
50-cm column and cytochrome was eluted with 1.0 M NaCl, 0.1% Triton X-100, 5 mM MES, pH 6.5, 0.1
mM EDTA. Approximately 500 ml of cytochrome ba
-containing solution is thus obtained. The
solution is then concentrated to
25 ml (1/20 of original volume)
with an Amicon concentrator fitted with a PM-10 membrane. Octyl
glucoside is added to a final concentration of 70 mM, and the
solution is stirred for a minimum of 2 h at 4 °C. The solution is
then dialyzed for at least 12 h against 40 volumes of 0.1% Triton
X-100, 10 mM Tris-Cl, pH 7.8, 0.1 mM EDTA with three
buffer changes. The octyl glucoside treatment and subsequent dialysis
must be repeated at least once. Although not understood, treatment with
octyl glucoside is essential to the success of the purification.
At
this point the solutions of cytochrome ba still
contain significant amounts of cytochrome b
. The
latter is removed as follows. The cytochrome ba
solution is concentrated to
50 ml and loaded at room
temperature onto a 4.6
10-cm DE52 column equilibrated with 0.1%
Triton X-100, 10 mM Tris-Cl, pH 7.8, 0.1 mM EDTA. The
column is washed with equilibrating buffer, and the formation of two
bands is observed: a faster moving pink band containing cytochrome b
and a slower moving brown band containing
cytochrome ba
. Cytochrome ba
is collected as it elutes with care being taken to minimize
contamination by the pink material. The solution thus enriched in
cytochrome ba
is concentrated to
50 ml and
passed through the column as before. After three passages through the
DE52 column, most of the contaminating cytochrome b
is removed, as judged by the ratio of heme B to heme A in
pyridine hemochrome spectra.
Buffer exchange and concentration of
the cytochrome ba solution is accomplished by
repeated concentration and dilution cycles using Amicon 350-ml
concentrators fitted with PM-10 membranes at 4 °C using 10 mM Hepes, pH 7.1, 0.1 mM EDTA, 0.1% Triton X-100 as the
dilution buffer. The solution is loaded onto a 3.2
90-cm CM-52
column (350 ml of resin/kg of starting wet cell weight) preequilibrated
with 0.1% Triton X-100, 10 mM Hepes, pH 7.1, 0.1 mM
EDTA. This column is washed with 3 column volumes of equilibrating
buffer and developed with 7 column volumes of a linear gradient of 0 to
0.10 M NaCl in the equilibrating buffer (shown in Fig. 1).
Figure 1:
Elution profile of Thermus cytochrome ba-containing fractions from the
final cation exchange column. Absorbance at 416 nm and conductivity of
the individual fractions (
8 ml in volume) eluted from a
TE-buffered CM-52 column with a 0 to 0.1 M NaCl gradient (see
``Experimental Procedures'').
Figure 4:
Design of the oligodeoxynucleotide probes
used in the isolation of Thermus cytochrome ba genes. A, partial amino acid sequences from holo protein (Major and Minor N termini) and the N terminus from a
purified CNBr fragment, determined as described under
``Experimental Procedures.'' B, the underlined portion of the latter sequence was used to direct the synthesis of
two overlapping probes as indicated. The codon frequency is given for
the corresponding codon(s) in which X = G + C, i.e. both nucleotides were incorporated at that position
during the synthesis. C, a portion of cbaB sequence
that was used to identify and clone the BamHI fragment
containing the complete cbaB sequence.
PEPPLOT was used for hydropathy analysis by the method of (Kyte and Doolittle, 1982), and PEPTIDESORT was used to calculate the amino acid composition encoded by the ORFs and some predicted properties of the individual proteins. The programs GAP and LINEUP were used to generate alignments of multiple amino acid sequences with some manual editing.
Figure 2:
Demonstration of differential staining of Thermus cytochrome ba subunits by
Coomassie Blue and silver ions. Left, lanesa and b were loaded with a total of 2 µg of molecular
weight standards (see below) and 10 µg of cytochrome ba
, respectively, and the gel stained with
Coomassie Blue. Note the weakly staining band at
18 kDa. Right, lanes a` and b` were loaded with 2
µg of the molecular weight standards and 10 µg of cytochrome ba
, respectively, and stained with silver (Bio-Rad
Silver Stain Plus). Note the strong staining of the
18-kDa band
relative to the
35-kDa band. Experimental details are given under
``Experimental Procedures.'' Molecular weight standards as
indicated by the arrows are, from the top:
phosphorylase b, 97,400; bovine serum albumin, 66,200; hen egg
white ovalbumin, 45,000; bovine carbonic anhydrase, 31,000; soybean
trypsin inhibitor, 21,500; hen egg white lysozyme, 14,400. On the right side the top arrow indicates
35,000 and
the bottom arrow
18,000 Da.
Figure 3:
Reversed phase chromatography profile of Thermus cytochrome ba (A continuous
recording of absorbance at 214 nm versus fraction number). The
column used was a Phenomenex C-1 (0.5-ml void volume) and was
equilibrated with 40% H
O and 60% formic acid. The
experiment was carried out as follows: 150 µg cytochrome ba
were dissolved in 100 µl of 60% formic acid
and applied to the column. A linear gradient was immediately begun to
bring the eluant to 10% H
O, 60% HCOOH, and 30% isopropanol
over a period of 20 min. The flow rate was 1 ml/min, and the portion of
eluting solvent at the detector is indicated by the sloping
line. The column was then washed isocratically for an additional
20 min with the final solution. A portion of each fraction was tested
for the presence of protein. No protein was found in tubes 1-15
but protein was found in tubes 15 and above. Several fractions were
combined for quantitative protein amino acid analysis: fractions
1, 2, and 3 as indicated by the horizontal
bars. See ``Experimental Procedures'' for details. The
results are presented in Table 1as mole percent and are compared
with values deduced from the translated gene
sequences.
The partial amino acid sequences initially obtained
from the purified cytochrome ba are shown in Fig. 4A. N-terminal sequencing of the holoprotein
resulted in a major and a minor sequence, with the latter occurring at
25% of the majority sequence. The consistent difference in yield
from the two polypeptides made it possible to ``read'' the
sequence of the major component for as many as 29 cycles. In addition,
a purified peptide was isolated following cyanogen bromide cleavage
which provided an amino acid sequence useful in probe construction. The
11-amino acid sequence YWLLPNLTGKP (underlined) from this peptide was
used to design two partially overlapping, 8-fold degenerate oligomeric
probes as shown in Fig. 4B. The strategy for gene
isolation was to conduct genomic hybridization with probes 1 and 2 in
separate but identical gels in order to identify genomic restriction
fragments that hybridized to both probes, which would then be cloned.
Hybridization of these probes to Thermus genomic DNA that
had been digested with several restriction enzymes identified BamHI and HindIII fragments, both of about 4 kb in
length (coincidentally), that appeared to be good candidates for
cloning. Cloning of these two fragments was carried out as described
under ``Experimental Procedures.'' However, this particular BamHI fragment was not stable under our conditions. About 90%
of the 4 kb (all but 500 bp) was lost in the plasmids recovered
from two transformants, and restriction analysis indicated that the
deletion event was similar in both clones. In contrast, the HindIII fragment remained stable in our constructs, and
various subclones were prepared for use in the generation of templates
for DNA sequencing (see ``Experimental Procedures'').
DNA
sequence analysis of the HindIII fragment indicated two ORFs
relevant to cytochrome ba, with one reading frame
(ORF2) interrupted by the 5`-end of the HindIII fragment. In
order to obtain the complete DNA sequence of the interrupted reading
frame, an oligomeric probe complementary to the 5`-region of ORF2 (SUII-VRQEGP, in Fig. 4C) was synthesized and
used to isolate a
4.3-kb BamHI fragment (the upstream BamHI fragment) which contains the complete ORF2 sequence, and
overlaps substantially with the HindIII fragment. Fig. 5shows the relationship of the cloned restriction fragments
to the Thermus chromosomal region containing the cytochrome ba
genes and summarizes the sequencing strategy.
With the exception of 169 nucleotides at the 5`-end of ORF2 (upstream
of the HindIII site), all sequences have been read at least
once on both strands. However, the sequencing gel from which the 5`-end
of ORF2 was determined was particularly clear, and the
``major'' N-terminal sequence is encoded in this region.
Therefore, we have good confidence in the reported sequence. The
primary DNA sequence and the deduced amino acid sequences are shown in Fig. 6.
Figure 5:
Physical location of and cloning and
sequencing strategies for the genes of T. thermophilus cytochrome ba. The upper line shows
the relationship of the cloned BamHI and HindIII
genomic fragments and the cytochrome ba
subunit
genes and provides a partial restriction map. The lower line and arrows show the sequencing strategy for the cbaBA genes. The location of the DNA site that hybridizes with
oligonucleotide probes 1 and 2 is marked with an asterisk (*).
Restriction enzyme sites: B, BamHI; H, HindIII; X, XhoI.
Figure 6: Nucleotide sequence of the T. thermophilus genes encoding subunit II (cbaB) and subunit I (cbaA) and the deduced amino acid sequences. The amino acid sequences are given below the nucleotide sequence in the single letter code, numbering is from the initial methionine of each polypeptide. Peptide sequences obtained from cyanogen bromide fragments and N-terminal sequencing are underlined. The first five amino acids in subunit I (MAVRA) are not detectable in the purified protein by N-terminal sequencing; the subsequent sequence (SEISRVY . . .) is detectable. Putative ribosome binding sites are underlined for subunit II at position 15 and for subunit I at position 525.
The two reading frames discovered in the DNA sequence
were named cbaA (encoding ba subunit I)
and cbaB (encoding ba
subunit II), and
the following evidence is presented to support this designation. The
two N-terminal peptides whose sequences were originally determined from
the purified protein (the major and ``minor'' N-terminal
sequences; Fig. 4), the peptide sequence obtained from the
cyanogen bromide fragment (from which probes 1 and 2 were derived; Fig. 4), as well as four additional peptide sequences determined
more recently and provided to us by Dr. G. Buse (
)are found
in the predicted amino acid sequences of these two ORFs (underlined in Fig. 6). Identification of six sequenced peptides within
the larger gene provides substantial evidence that ORF1, which is
designated cbaA, encodes one of the components found in
holo-cytochrome ba
, and also serves to establish
the reading frame within the gene. Similarly, the major N-terminal
sequence occurs in ORF2, which is designated cbaB.
Additional evidence that cbaA and cbaB represent
expressed T. thermophilus genes was provided by codon
preference analysis, which is based on the nonrandom codon usage
frequencies found in expressed genes and was implemented as described
under ``Experimental Procedures'' (resulting graph not
shown). The codon preference statistic for each of the three possible
forward reading frames was computed using an averaging window of 25
bases. ORF1 (putative cbaA) and ORF2 (putative cbaB)
coincidentally were both in reading frame 1. The average codon
preferences (p values) found were: frame 1, 1.2765; frame 2,
0.5222; and frame 3, 0.4347, while the average codon preference for a
random sequence using our Thermus codon preference table was
0.624. There are only 29 unusual codons present out of 731 codons
within the ORFs in reading frame 1, while 251 and 346 unusual codons
are found in the corresponding regions of frames 2 and 3, respectively.
This analysis provides clear statistical evidence that cbaA and cbaB are genes. Finally, the amino acid compositions
predicted by the two ORFs compare favorably with those of the two
principal protein fractions identified by reversed phase liquid
chromatography of purified cytochrome ba.
Table 1compares the mole percent amino acid compositions
predicted from translation of the gene sequences with those obtained
experimentally. ()The combined fractions 21-31 from
the RPLC experiment (Fig. 3) have an experimental amino acid
composition predicted by translating the cbaB gene. The
translated gene sequence indicates that subunit I has 562 amino acids
and an exact molecular weight of 62,527 (not including the hemes or
copper ions). The protein is extremely hydrophobic, consisting of
52 mol % of hydrophobic amino acids, and has a calculated pI of
10.4 with a net charge of +9. Protein in fraction 15 of Fig. 3has an amino acid composition that corresponds to
translation of the cbaA gene. The cbaA product
consists of 168 amino acids and has an exact molecular weight of
18,563; we have termed this subunit II. In contrast to subunit I,
subunit II is a relatively hydrophilic protein with a calculated pI of
6.5 and a net charge of -3 at pH 7. Thus, the overall charge of
the I + II complex will be +6, a property that likely
accounts for the fact that this protein binds to a cation exchange
resin during purification.
The predicted amino acid sequences also
provide a possible explanation of the mixed N-terminal sequences
obtained chemically from samples of the holoprotein (Fig. 4).
The major sequence clearly arises from the N terminus of cbaB,
and the substantial yield suggests that the N terminus of subunit II is
not blocked. The minor sequence was deduced from
phenylthiohydantoin-derivatives that were approximately 25% abundant as
those of the major product in each sequencing cycle. This sequence
begins just five amino acids downstream of the DNA-deduced N terminus
of subunit I, suggesting that 75% of the subunit I molecules have
a blocked N terminus while
25% are modified in a
post-translational event that removes the five N-terminally encoded
amino acids (MAVRA). Thus, full-length cbaA polypeptides are
apparently blocked at the N terminus. This phenomenon was observed in
two independent preparations of the enzyme.
Figure 7:
Alignment of selected cytochrome c oxidase subunit II amino acid sequences with that of the T.
thermophilus subunit II. Only portions of the sequences are shown.
The numbers at the beginning of each sequence indicate the amino acid
position in the respective full sequence. A, hydrophobic
segments determined by the method of Kyte and Doolittle(1982) are
represented as continuous equal signs above the amino acid sequences.
These are putative transmembrane helices, and most subunit II sequences
have two such regions. The subunit II sequences from the S.
acidocaldarius quinol oxidase (Lübben et
al., 1992) and T. thermophilus cytochrome ba have only one such region. B, the two
conserved cysteines and histidines and the single methionine, thought
to be metal ligands in the Cu
center, are conserved in
cytochrome c oxidases, including T. thermophilus cytochrome ba
, but not in the quinol
oxidases. These are indicated by the vertical arrows at the bottom of the figure. The C termini of the T. thermophilus (Mather et al., 1991) and the B. subtilis (Saraste et al., 1991b) sequences extend to include a
cytochrome c sequence; this is indicated by CYT.C placed at the end of these sequences.
A one-/three-dimensional topological
model of the cytochrome ba subunit II, which
embodies the hydrophobicity analysis, is shown in Fig. 8. This
model illustrates the proposed cytoplasmic N terminus, the single
transmembrane region which presumably anchors subunit II within the
membrane, and the hydrophilic portion of the molecule containing the
``Cu
motif.'' Malmström and
co-workers have recently provided evidence that the portion of subunit
II outside the membrane has an overall secondary-structure composition
similar to the ``blue'' copper protein, azurin (Wittung et al., 1994).
Figure 8:
Topological presentation of the T.
thermophilus cytochrome ba subunit II amino
acid sequence. The sequence is oriented with the C-terminal domain
containing the putative copper-binding site on the periplasmic side of
the bacterial plasma membrane (labeled Out) by analogy with
other cytochrome c oxidase subunits II (e.g. see
Bisson et al.(1982) and Chepuri and Gennis(1990)). The N
terminus of the subunit then resides on the cytoplasmic side of the
membrane (labeled In). The single hydrophobic stretch is
presented as a helical net bounded by the membrane. The amino acids are
represented as the single-letter code enclosed in a small
circle, with the exception of conserved residues, which are
enclosed in small squares. The putative ligands to the
Cu
center are denoted with carats. The sequence
number is indicated every 25th position next to the corresponding amino
acid. Charged residues are indicated by an enclosed + (Lys or Arg)
or - (Asp or Glu).
Figure 9:
Alignment of selected cytochrome c oxidase subunit I amino acid sequences with that of the T.
thermophilus subunit I. Only portions of the sequences are shown,
summarizing an alignment of the T. thermophilus cytochrome ba subunit I sequence with 19 other subunit I
sequences (the complete alignment is presented elsewhere (Keightley,
1993) (see also text). The Roman numerals above the alignments
refer to hydrophobic segments of sequence determined by the method of
(Kyte and Doolittle, 1982) and delineated by continuous equal
signs; these are not meant to imply specific boundaries of the
putative transmembrane helices. Gaps in the alignment are indicated by periods. Completely conserved amino acids are underlined; the six histidines proposed to be ligands to the
low spin heme and to the high spin heme/Cu
pair are
emboldened and underscored with vertical arrows. Note that there is a
nearly conserved histidine at position 376.
The 562-amino acid sequence of CbaA was subjected to
hydrophobicity analysis and compared to that of the 513 amino acid
human cytochrome aa subunit I (Anderson et
al., 1981) (data not shown). Visual comparison of these two
profiles revealed a similar periodicity of hydrophobic segments, and Fig. 9shows that there are conserved amino acid residues placed
in similar locations in and about these hydrophobic segments. There are
12 hydrophobic segments in known eukaryotic cytochrome oxidase subunits
I and in many of the prokaryotic enzymes for which sequences are
available. The cbaA gene appears to encode one additional
hydrophobic segment that extends the C terminus of the protein by about
50 amino acid residues. The C-terminal extension in cbaA fails
to show significant homology to the corresponding extensions in other
subunit I sequences, or in the N-terminal region of more typical
subunits III, but similarity in this region is generally low among
those sequences as well (Mather et al., 1993a). Variation in
the number of hydrophobic segments in the subunits of prokaryotic
copper/heme oxidases has been noted previously (see
``Discussion'').
Overall, the sequence alignments (Fig. 9) and hydrophobicity analysis (not shown) indicate that
the T. thermophilus cbaA gene encodes a protein that exhibits
significant primary sequence homology and has potential secondary
structural similarity to other members of the cytochrome c/quinol oxidase superfamily. A one-/three-dimensional
topological model of the cytochrome ba subunit I
is shown in Fig. 10. Such a model is also predicated on the
findings of Chepuri and Gennis(1990) which show that the subunit I of
cytochrome bo alternates from the in side to the out side of the membrane as drawn here for subunit I of ba
. This arrangement brings the six conserved
histidines into the same register as suggested in all other models for
the structure of subunit I.
Figure 10:
Topological presentation of the T.
thermophilus cytochrome ba subunit I amino
acid sequence. The sequence is oriented with the N terminus on the
cytoplasmic side (down) and the C terminus on the periplasmic
side (up) of the plasma membrane by analogy to other oxidase
subunits I. The presentation of the individual amino acids and other
details are as described in the legend to Fig. 8. Hydrophobic
segments are presented as helical nets spanning the membrane; they were
arbitrarily set to include 20 residues and labeled sequentially with a Roman numeral. Putative helices II, VI, VII, and X contain the
six conserved histidine residues. The seventh, nearly conserved
histidine residue between IX and X, is inscribed in an oval (position 376). Note that eukaryotic and many bacterial subunits I
lack the XIIIth hydrophobic segment.
The results presented here have changed our perception of Thermus cytochrome ba. We were originally
misled by the behavior of the protein in SDS gels. Specifically, the
small subunit is very weakly stained by Coomassie Blue and the larger
subunit runs at an unusually low molecular weight during SDS-PAGE.
Since the initial report (Zimmermann et al., 1988), other
examples of anomalous staining and SDS-gel mobility of cytochrome
oxidase proteins have been observed. Subunit II of Sulfolobus
acidocaldarius cytochrome aa
shows a similar
differential staining with Coomassie Blue and silver ions. (
)The polypeptide encoded by cbaA is 62 kDa while
the actual protein runs at
35 kDa during SDS-PAGE. Similar
anomalies have been observed: Thermus cytochrome caa
(Fee et al., 1980; Mather et
al., 1993a), Bacillus PS3 cytochrome caa
(Sone and Yanagita, 1982), E. coli cytochrome bo (Chepuri et al., 1990) and S. acidocaldarius cytochrome aa
(Lübben et al., 1992) are a few examples of oxidases that contain
subunits which exhibit anomolous gel mobility in SDS-PAGE. In each
case, the bands run faster than expected, resulting in underestimates
of molecular weights. Among other examples, the Sulfolobus enzyme was originally described as a 38-kDa single subunit quinol
oxidase (Anemüller and Schäfer,
1990). The same enzyme was later described as a complex of three
subunits, with the ``38-kDa protein'' being a subunit I with
an actual size of 58 kDa (Lübben et al.,
1992). In general, mobility in SDS-PAGE is not a reliable method to
estimate the size of these strongly hydrophobic proteins. The
availability of the DNA sequence information, isolation of the
individual proteins and improved protein analyses clearly show that the Thermus cytochrome ba
consists of two
subunits.
Cytochrome ba has spectroscopic features indicating the presence of a normal
Cu
(Zimmermann, 1988), and more recent work has
demonstrated that this is a binuclear Cu center (Fee et al.,
1995). Among cytochrome c oxidase subunits II there are five
conserved residues likely to be ligands to the Cu
center.
In the Thermus ba
sequence these are His-114,
Cys-149, Cys-153, His-157, and Met-160, each of which aligns nicely
with corresponding residues in other subunit II sequences. As noted
above, however, the negatively charged residue between the two Cys
residues has been replaced with a Gln residue; in this regard, ba
may resemble the N
O reductase (cf. Viebrock and Zumft(1988)).
Having the typical 12 hydrophobic
sequences plus one additional C-terminal segment, cytochrome ba represents yet another variation on this theme,
and one might predict the existence of an additional gene in Thermus which encodes a truncated subunit III. We have
examined the possibility of homology between the sequence represented
in the C-terminal extension and subunits III and found none. This is
expected, however, because subunits III are quite dissimilar in their N
termini (cf. Mather et al. (1993a)). The reading
frame that may begin after subunit I (see Fig. 4) could
represent ``cbaC,'' encoding a subunit III lost
during purification of the enzyme. This organization of reading frames
(subunit II, followed on the chromosome by subunit I, and then subunit
III) is common at the gene loci of other members of this protein family (cf. Castresana et al.(1994)), and studies are
underway to test this hypothesis. Alternatively, as is the case in Paracoccus denitrificans (Saraste et al., 1986), the
putative subunit III gene could be elsewhere on the chromosome.
The six conserved
histidine residues in subunit I are now generally agreed to be ligands
to the low spin heme site (cytochrome b in ba) and to the high spin heme/Cu
special pair (cf. Gennis(1992) and Calhoun et
al.(1994)). Questions remain regarding the role of the nearly
conserved His-376 in the IX/X loop (ba
numbering),
which is absent in the Bradyrhizobium japonicum CoxA (Bott et al., 1990; Gabel and Maier, 1990) and CoxN (Bott et
al., 1992) and S. acidocaldarius (SoxM)
(Lübben et al., 1994b) sequences.
Ferguson-Miller, Gennis, and colleagues (Calhoun et al., 1993;
Thomas et al., 1993a; 1993b) have carried out site-directed
mutation work in this area of the sequence, and the results indicate
that this histidine is not essential for oxidase activity. However,
other results indicate this residue is close to the a
/Cu
pair (Hosler et al.,
1994), and we have previously speculated that His-376 may serve as a
direct ligand to Cu
in the cyanide complexed form of
cytochrome ba
(Surerus et al., 1992).
Based on these studies and the sequence alignments presented here, it
is reasonable to assign Thermus cytochrome ba
His-72 (in helix II) and His-386 (helix X) as ligands to
cytochrome b, His-384 (helix X) as the axial ligand to
cytochrome a
, and His-282 and His-283 (helix VII)
and His-233 (helix VI) are probably ligands to Cu
. The
relation of His-376 to Cu
remains undefined, but the
evidence that four histidines coordinate Cu
in the cyano
complex of cytochrome ba
is quite strong (Surerus et al., 1992).
Assuming that the polytopic structure
suggested for the subunit I sequences is generally correct (see Fig. 10), the information in Table 2provides few
candidates for the formation of unique and conserved arrangements of
hydrophilic amino acids within the membrane domain for the purpose of
effecting the proton translocation activity of these enzymes. ()Other workers have suggested that specific residues within
helix VIII may interact with the active centers and are responsible for
proton (H
O
) and/or water movement,
particularly Thr-318 (Thermus caa
numbering)
(Thomas et al., 1993b). However, this may involve the
``scalar'' protons, those involved in H
O
formation. The absence of any other conserved residues in this region
of the sequence would seem to argue against conserved, specialized
structures within the membrane. Nevertheless, the general character of
the potential helical regions is relatively constant throughout subunit
I sequences with I, II, IV, V, VII, IX, X, and XII being quite
hydrophobic (after excluding the coordinated histidines), while helices
III, VI, VIII, and XI tend to be more hydrophilic. The results of
site-directed mutagenesis studies (Gennis(1992) and Calhoun et
al.(1994) and references therein) and statistical analysis of
sequence variation within each helix (Mather et al., 1993a)
have led to a proposed two-dimensional arrangement of the 12 helices.
This places the low spin heme between helices II and X, the high spin
heme is also bound to helix X, and the open coordination position on
the high spin heme is exposed to helices VI and VII (binding
Cu
) and helix VIII, which may provide a generally
hydrophilic region that traverses much of the membrane but is not
necessarily constructed from specific hydrophilic amino acids.
The
information in Table 2suggests possible conservation of
functionality in some of the loops between the helices. The loops IV/V,
V/VI, VII/VIII, and X/XI have no conserved residues. However, the
remaining loops and the N and C termini appear to contain largely
conserved residues or possible functional equivalents. For example, in
loop VI/VII there is a conserved Lys/Arg; in loop IX/X there is a
conserved Thr/Ser and a nearly conserved Asp/Glu; while in loop XI/XII
two adjacent Arg residues are conserved. These residues are proper
targets for site-directed mutagenesis. Interestingly, Asp-135 in the E. coli bo oxidase and Asp-132 in Rhodobacter cytochrome aa (an almost fully conserved
residue in the II/III loop) has been mutated to Asn to form enzymes
that have full electron transferring activity but lack proton pumping
activity (Thomas et al., 1993b; Fetter et al., 1995).
It is interesting that S. acidocaldarius SoxB reportedly has
no acidic functionality in this region, suggesting that if Asp-135 (E. coli) is directly involved in proton pumping, alternative
mechanisms can exist.
In general, these observations
suggest that the ``loops'' between the helices may form
specialized structures that serve to ``gate'' the movement of
protons in to and out of the membrane plane under the influence of the
redox status of the metals, and that the actual movement of a proton
across the membrane may be rather indirectly coupled to electron
transfer events at the metals, for example through conformational
changes in the protein. Such an arrangement seems to exist in
bacteriorhodopsin (Singer, 1990; Krebs and Khorana, 1993).
Of more immediate importance to this work is the
fact that, while cytochrome ba is a member of the
heme-Cu oxidase superfamily, it is phylogenetically quite distant from
any other member of the group and might be expected to display some
unique properties. Indeed, Surerus et al.(1992) showed that
the reaction of cyanide with cytochrome ba
reduces
and ligates the cytochrome a
fully exposing the
electronic spin of Cu
. This behavior has not been observed
with other heme-Cu oxidases.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]