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.

EXPERIMENTAL PROCEDURES

Materials

()

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.

Protein Methods

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.

Growth of Cells and Purification of Cytochrome ba

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'').

Molecular Biology Methods

DNA Preparation

Probe Design and Genomic Hybridization

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.

Cloning Genomic Restriction Fragments

Subcloning, Exonuclease III Deletion Series, and DNA Sequencing

Computer Analyses

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.

RESULTS

Purification of Cytochrome ba

Subunit Composition of Cytochrome ba

SDS-PAGE

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.

RPLC

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% HO 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% HO, 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.

CbaB Is Homologous to Cytochrome c/Quinol Oxidase Subunits II

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).

CbaA Is Homologous to Cytochrome c/Quinol Oxidase Subunits I

()

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.

DISCUSSION

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.

Subunit II

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 NO reductase (cf. Viebrock and Zumft(1988)).

Subunit I

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.

Amino Acid Conservation

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 (HO) and/or water movement, particularly Thr-318 (Thermus caa numbering) (Thomas et al., 1993b). However, this may involve the ``scalar'' protons, those involved in HO 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).

Phylogenetic Considerations

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.

FOOTNOTES

*

Supported by National Institutes of Health Grant GM35342 (to J. A. F.) and abstracted, in part, from the Ph.D. theses of J. A. K. and B. H. Z. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]

§

Recipient of an Associated Western Universities graduate scholarship. Present address: Oregon Health Sciences University, Pediatric Metabolic Laboratory, L473, 3181 SW Sam Jackson Park Rd., Portland, OR 97202.

¶

Present address: Dept. of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00936-5067.

**

Present address: Dept. of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078.

§§

To whom correspondence should be addressed: Dept. of Biology, Mail Code 0322, University of California at San Diego, La Jolla, CA 92093. Tel.: 619-534-4424; Fax: 619-534-0936; jfee{at}jeeves.ucsd.edu.

()

The abbreviations used are: MES, 2-(N-morpholino)ethanesulfonic acid; bis, bisacrylamide; RPLC, reversed phase liquid chromatography; PAGE, polyacrylamide gel electrophoresis; TEMED, N,N,N`,N`-tetramethylenediamine; ORF, open reading frame; bp, base pair(s); kb, kilobase pair(s).

()

The pink-colored material is cytochrome b, whose purification and partial characterization has been described in B. Zimmermann's Ph.D. thesis (Zimmermann, 1988).

()

All procedures are scaled for the amount of protein derived from 2 kg of wet cells and are performed at 4 °C, unless otherwise noted.

()

G. Buse, personal communication.

()

Our preliminary report (Zimmermann et al., 1988) indicated that approximately half the cytocyhrome ba subjected to acid hydrolysis could be accounted for in the resulting amino acids that eluted from the ion exchange column, compared to predictions from the gene sequences. We therefore repeated these analyses both under the standard conditions used earlier and by including pretreatment of the protein with 30% isopropanol, 60% formic acid as done in the RPLC experiment. Both sets of analyses (data not shown) are consistent with the composition predicted from translation of the two genes (cbaA and cbaB). Moreover, when all extant data are compared as mole percent of individual amino acids, they are, within experimental error, identical. If the previously reported data are multiplied by a small constant (2), a composition is obtained that is consistent with the predictions of the gene sequences. What was different between the two analyses? Our notebooks and correspondence revealed that the protein was dialyzed against 50% ethanol prior to analysis, ostensibly to remove excess detergent that accrues during concentration of the protein. The solution provided for analysis was centrifuged, with only the supernatant being subjected to acid hydrolysis. The only reasonable explanation for the discrepancy is that some protein precipitation went undetected and was removed by centrifugation prior to acid hydrolysis. In the current analyses, holoprotein was analyzed directly without the dialysis step.

()

Dr. Robert Gennis provided us with a recent alignment of 65 different cytochrome c oxidase subunit I sequences. This includes the sequences FixN and SoxA.

()

G. Schäfer, personal communication.

()

Not all heme-copper oxidases have been experimentally shown to pump protons. This discussion assumes that proton pumping is an inherent feature of members of the family.

ACKNOWLEDGEMENTS

Drs. Winslow Caughey, Ólöf Einarsdóttir and Gerd LaMar generously supplied purified proteins for use as standards. We thank Terry Mulcahey for assistance with the amino acid analyses. Dr. Robert Blankenship provided invaluable discussions concerning reverse phase chromatography of membrane proteins. We are grateful to Dr. G. Buse for providing some of the amino acid sequence information used in this study and thank members of the Life Sciences Division at the Los Alamos National Laboratory for providing the many oligonucleotide primers used in sequencing the genes and for the use of their VAX computer. We thank Dr. Robert Gennis for providing us with his most recent compilation of subunit I sequences. We gratefully acknowledge the participation of Elizabeth Haberer in the isolation of the ``upstream'' BamHI fragment during the summer of 1992; Carmen Nitsche provided excellent technical assistance during the early phases of this work. We thank Professors Paul Saltman and Masaki Hayashi for the use of their laboratory space. Special thanks are due Paul Saltman and the University of California, San Diego, Department of Biology for their continuing support and encouragement of this work.

---

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