Inhibitory activities of lansoprazole against respiration in Helicobacter pylori.

Lansoprazole and its derivative AG-1789 dose-dependently inhibited cellular respiration by an endogenous substrate and decreased the ATP level in Helicobacter pylori cells. The inhibitory action of lansoprazole and AG-1789 against respiration was specific to substrates such as pyruvate and alpha-ketoglutarate and similar to the inhibitory action of rotenone, which is an inhibitor for the mitochondrial respiratory chain. Growth inhibition by lansoprazole and AG-1789 as well as by rotenone was augmented at high oxygen concentrations under atmospheric conditions. Since the 50% inhibitory concentrations of these compounds for the respiration were close to their MICs for H. pylori growth, the growth inhibition might be due to respiratory inhibition by these compounds.

A novel hydrophobic diheme c-type cytochrome. Purification from Corynebacterium glutamicum and analysis of the QcrCBA operon encoding three subunit proteins of a putative cytochrome reductase complex.

Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase activity (heme staining), showed only one band at about 28 kDa. This 28 kDa protein was purified from C. glutamicum membranes by chromatography in the presence of decylglucoside using DEAE-Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an alpha band at 551 nm, and its E(m, 7) was about 210 mV. A QcrCAB operon encoding the subunits of a putative quinol cytochrome c reductase was found 3'-downstream of ctaE encoding subunit III of cytochrome aa(3) in the C. glutamicum genome. The deduced amino acid sequence of qcrC, composed of 283 amino acid residues, contained two heme C-binding motifs and was in agreement with partial peptide sequences obtained from the 28 kDa protein after V8 protease digestion. We propose to name this protein cytochrome cc. The presence of cytochrome cc is a common feature of high G+C content Gram-positive bacteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. QcrA and qcrB of C. glutamicum encode the Rieske Fe-S protein and cytochrome b, respectively, although these proteins were not co-purified with cytochrome cc. The phylogenetic tree of cytochromes b and b(6) show that C. glutamicum cytochrome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinococci and Thermus cytochromes. This indicates that there is a fourth group of bacteria in addition to the three clades: proteobacterial cytochrome b, cyanobacterial b(6) and green sulfur-low G+C Gram-positive bacteria.

Purification and characterization of cytochrome c-553 from Helicobacter pylori.

Helicobacter pylori, a microaerophilic Gram-negative spiral bacterium residing in the human stomach, contains a small size soluble cytochrome c. This cytochrome c was purified from the soluble fraction of H. pylori by conventional chromatographies involving octyl-cellulose and CM-Toyopearl. Its reduced form gave an alpha absorption band at 553 nm, and thus the cytochrome was named H. pylori cytochrome c-553. The cytochrome, giving a band below 10,000 Da upon SDS-PAGE, was determined to have a mass of 8,998 by time of flight mass spectroscopy. Its N-terminal peptide sequence was TDVKALAKS---, indicating that the nascent polypeptide was cleaved to produce a signal peptide of 19 amino acid residues and a mature protein composed of 77 amino acid residues. The cb-type cytochrome c oxidase oxidized ferrocytochrome c-553 of this bacterium actively (V(max) of about 250 s(-1)) with a small K(m) (0.9 microM). Analysis of the effect of the salt concentration on the oxidase activity indicated that oxidation of cytochrome c-553 is highly inhibited under high ionic conditions. The amino acid sequence of H. pylori cytochrome c-553 showed the closest similarity to that of Desulfovibrio vulgaris cytochrome c-553, and these sequences showed a weak relationship to that of the cytochrome c(8)-group among class I cytochromes c.

Menaquinol oxidase activity and primary structure of cytochrome bd from the amino-acid fermenting bacterium Corynebacterium glutamicum.

Cytochrome d was spectroscopically detected in membrane fractions of the amino-acid-fermenting, high-G+C gram-positive bacterium Corynebacterium glutamicum. Inhibition of NADH oxidase activity in the membranes by cyanide suggested that the main terminal respiratory oxidase during the stationary phase was a type of cytochrome bd. Cytochrome bd-type quinol oxidase, purified from the membranes, was composed of two subunits. Its reduced form showed absorption peaks at 627, 595, and 560 nm, which were due to haem d, high-spin protohaem, and low-spin protohaem, respectively. The air-oxidised form showed a peak at 645 nm, which might be due to oxygenated ferrous haem d. The spectral features and the size of subunit I are more similar to the properties of cytochromes bd from Proteobacteria, such as Escherichia coli, than to those of cytochrome bd from low-G+C gram-positive bacteria, such as Bacillus stearothermophilus. The menaquinol oxidase activity of the purified cytochrome bd was low, but was enhanced about fivefold by pre-incubating the enzyme with menaquinones. The order of effectiveness of quinols as oxidase substrates was clearly different from that of quinones as the activators of enzyme activity. Furthermore, activation was destroyed by ultraviolet irradiation of the pre-incubated enzyme and then restored by a second incubation with menaquinone. These results indicate that the enzymatic properties of this new oxidase are more similar to the properties of cytochromes bd from low-G+C gram-positive bacterial than to those of proteobacterial counterparts. They also suggest that the enzyme has a second quinone-binding site essential for full activity, in addition to the active centre for substrate oxidation. By using probes based on partial peptide sequences of the subunits, the genes for the two subunits of C. glutamicum cytochrome bd were cloned. The deduced amino acid sequence demonstrated that subunit I lacks the C-terminal half of the Q loop and that the primary structure of C. glutamicum cytochrome bd is more similar to that of other gram-positive bacteria than to proteobacterial cytochromes bd.

Energy-yielding properties of SoxB-type cytochrome bo(3) terminal oxidase: analyses involving Bacillus stearothermophilus K1041 and its mutant strains.

We isolated a K17q8 mutant from K17 mutant cells of Bacillus stearothermophilus which contain SoxB-type cytochrome bo(3) as well as cytochrome bd but not SoxM-type cytochrome caa(3), which is the main terminal oxidase in B. stearothermophilus K1041. The respiration of K17q8 was highly sensitive to as little as 10 microM cyanide, indicating that the main terminal oxidase is cytochrome bo(3). The aerobic growth yield of K17q8 was lower than that of wild-type K1041, but higher than that of parental K17. The H(+)/O ratio of K17q8 was about 5, i.e. a little lower than the 6.1-6.5 of K1041, but higher than the 2.9-3.1 of K17 [Sone et al. (1999) J. Biosci. Bioeng. 87, 495-499]. Analyses of membrane fragments indicated that K17q8 contains about 0.2 nmol cytochrome bo(3) per mg membrane protein, and scarcely any subunits of cytochromes caa(3) and bd. From the membrane fraction of K17q8, cytochrome bo(3) was purified and shown to be composed of two subunits with apparent molecular masses of 56 and 19 kDa. The enzyme contained protoheme IX and heme O, as the main low-spin heme and high-spin heme. Analysis of the substrate specificity indicated that the high-affinity site is very specific to cytochrome c-551, a cytochrome c which is a membrane-bound lipoprotein of thermophilic Bacillus. The I(50) of purified cytochrome bo(3) was determined to be 4 microM, indicating that cytochrome bo(3) among the three terminal oxidases in B. stearothermophilus was most susceptible to cyanide. The respiration of K17q8 was mostly inhibited by the addition of cyanide at this concentration.

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran.

We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of beta-LG. The molar ratios of beta-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The beta-LG-CMD conjugates maintained the retinol-binding activity of native beta-LG. Intrinsic fluorescence study indicated that shielding of the surface of beta-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around (125)Thr-(135)Lys (alpha-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of beta-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-beta-LG antibody response was markedly reduced after immunization with the beta-LG-CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of beta-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the beta-LG-CMD conjugates were similar to those of beta-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of beta-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the beta-LG in these conjugates.

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus.

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity.

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.

Defining the structural domain of subunit II of the heme-copper terminal oxidase using chimeric enzymes constructed from the Escherichia coli bo-type ubiquinol oxidase and the thermophilic Bacillus caa(3)-type cytochrome c oxidase.

To probe the location of the quinol oxidation site and physical interactions for inter-subunit electron transfer, we constructed and characterized two chimeric oxidases in which subunit II (CyoA) of cytochrome bo-type ubiquinol oxidase from Escherichia coli was replaced with the counterpart (CaaA) of caa(3)-type cytochrome c oxidase from thermophilic Bacillus PS3. In pHNchi5, the C-terminal hydrophilic domain except a connecting region as to transmembrane helix II of CyoA was replaced with the counterpart of CaaA, which carries the Cu(A) site and cytochrome c domain. The resultant chimeric oxidase was detected immunochemically and spectroscopically, and the turnover numbers for Q(1)H(2) (ubiquinol-1) and TMPD (N,N, N',N'-tetramethyl-p-phenylenediamine) oxidation were 28 and 8.5 s(-1), respectively. In pHNchi6, the chimeric oxidase was designed to carry a minimal region of the cupredoxin fold containing all the Cu(A) ligands, and showed enzymatic activities of 65 and 5.1 s(-1), and an expression level better than that of pHNchi5. Kinetic analyses proved that the apparent lower turnover of the chimeric enzyme by pHNchi6 was due to the higher K(m) of the enzyme for Q(1)H(2) (220 microM) than that of cytochrome bo (48 microM), while in the enzyme by pHNchi5, both substrate-binding and internal electron transfer were perturbed. These results suggest that the connecting region and the C-terminal alpha(1)-alpha(2)-beta(11)-alpha(3) domain of CyoA are involved in the quinol oxidation and/or physical interactions for inter-subunit electron transfer, supporting our previous proposal [Sato-Watanabe, M., Mogi, T., Miyoshi, H., and Anraku, Y. (1998) Biochemistry 37, 12744-12752]. The close relationship of E. coli quinol oxidases to cytochrome c oxidase of Gram-positive bacteria like Bacillus was also indicated.

Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus.

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151-158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a more widely distributed type including the B. stearothermophilus enzyme, suggesting that the latter type is evolutionarily older.

Cloning of Bacillus stearothermophilus ctaA and heme A synthesis with the CtaA protein produced in Escherichia coli.

The Bacillus stearothermophilus ctaA gene, which is required for heme A synthesis, was found upstream of the ctaBCDEF/caaEABCD gene cluster as in B. subtilis and B. firmus. The deduced protein sequence indicate that CtaA is a 35-kDa intrinsic membrane protein with seven hydrophobic segments. Alignment of CtaA sequences showed conserved residues including histidines that may be involved in heme B binding and substrate binding. Expression of ctaA in E. coli resulted in increased formation of a membrane-bound b-type cytochrome, heme A production, and severe growth inhibition. Furthermore, B. stearothermophilus CtaA produced in E. coli was found to catalyze the conversion of heme O to heme A in vitro.

Characterization of a cb-type cytochrome c oxidase from Helicobacter pylori.

Helicobacter pylori is a microaerophilic Gram-negative spiral bacterium residing in human stomach. A cb-type cytochrome c oxidase that terminates the respiratory chain was purified to near homogeneity by solubilizing the membranes with Triton X-100 and applying anion exchange, Cu-chelating, and gel filtration chromatographies. Redox- and CO-difference spectra and pyridine ferrohaemochrome analysis showed the enzyme to contain three haems C, one low-spin protohaem, and one high-spin protohaem that probably forms a dioxygen-reducing bimetalic center with a copper atom. The enzyme actively oxidizes soluble cytochrome c from this bacterium (TNmax of about 250 s-1) with a Km of 0.9 microM. Yeast cytochrome c and N,N,N',N'-tetramethyl p-phenylenediamine (TMPD) are also oxidized at similar maximal velocities with larger Km's. Oxygen pulse experiments on resting cells in the presence of ascorbate plus TMPD or L-lactate indicated that this sole terminal oxidase pumps H+, although the H+ pumping activity by proteoliposomes reconstituted from the enzyme and P-lipids was low. Two main bands with haem C at 58 and 26 kDa were observed upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and succeeding protein and haem staining. Sequencing of the operon encoding the subunits of the enzyme revealed the presence of ccoNOQP. N-terminal analysis of the 58 kDa band showed 15 or 13 amino acids coinciding with the amino acid sequences deduced from the DNA of ccoN and ccoO. CcoN, the largest subunit bearing two protohaems and copper, and ccoO, a mono-haem cytochrome subunit form a protein complex with an apparent molecular mass of 58 kDa, even in the presence of sodium dodecyl sulfate. The 26 kDa band is tentatively assumed to be ccoP with two haems C.

Direct correlationship between proton translocation and growth yield: an analysis of the respiratory chain of Bacillus stearothermophilus.

Thermophilic bacilli contain cytochrome caa3-type cytochrome c oxidase as the main terminal oxidase in the respiratory chain. A mutant strain, named K-17, lacking cytochrome caa3 and exhibiting very low N,N,N',N'-tetramethyl-p-phenylene diamine oxidase activity, was isolated by random mutation from Bacillus stearothermophilus K1041 (Sakamoto, J. et al., FEMS Microbiol. Lett., 143, 151-158, 1996). Comparing this mutant with the parent strain K1041, we observed the following differences in energy-yielding properties. (i) K-17 gave an cell yield less than one half of that of the wild type, although the doubling time of K-17 was only a little slower than that of the parent strain. (ii) In cellular respiration, the H+/O ratio of K-17 was 2.9-3.1, while that of the wild type was 6.1-6.5. (iii) A low concentration of cyanide inhibited endogenous respiration of the wild-type cells partly with a concomitant reduction of the H+/O ratio to around 3, while it did not significantly affect the respiration rate and the H+/O ratio of the K-17 cells. (iv) Cytochrome bd-type quinol oxidase seemed to operate in the wild-type cells when a low concentration (below 0.5 mM) of cyanide was added, while this enzyme is the main terminal oxidase in K-17. The K-17 cells also contained cytochrome b(o/a)3-type cytochrome c-551 oxidase. These results demonstrated that the combination of the enzymes involved in the respiratory chain determines the H+/O ratios of the cell and consequently the growth yield of the bacteria.

The cbaAB genes for bo3-type cytochrome c oxidase in Bacillus stearothermophilus.

Structural genes were cloned for cytochrome bo3-type cytochrome c oxidase recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. Sequencing and Northern blotting analyses indicated that the two genes cbaA and cbaB composed an operon encoding for subunits I and II, respectively, and that the oxidase was SoxB-type. They are the first genes for a SoxB-type cytochrome c oxidase whose natural substrate is known.

Did archaeal and bacterial cells arise independently from noncellular precursors? A hypothesis stating that the advent of membrane phospholipid with enantiomeric glycerophosphate backbones caused the separation of the two lines of descent.

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively.

A novel cytochrome b(o/a)3-type oxidase from Bacillus stearothermophilus catalyzes cytochrome c-551 oxidation.

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated several mutants from B. stearothermophilus defective in the caa3-type oxidase activity [Sakamoto, J. et al (1996) FEMS Microbiol. Lett. 143, 151-158]. A novel oxidase was isolated from membrane preparations of one of the mutants, K17. The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme O, heme A, and Cu in a ratio of 1:0.7:0.2:3. CO difference spectra indicate that the high-spin heme is mainly heme O. These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A. The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus. The turnover rate of the activity (Vmax = 190 s[-1]) and its affinity for cytochrome c-551 (Km = 0.15 microM) were much higher than those for yeast and equine heart cytochromes c. The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a Ki value of 19 microM. The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase. Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the heme-copper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.

Characterization of two terminal oxidases in Bacillus brevis and efficiency of energy conservation of the respiratory chain.

The respiratory chain of Bacillus brevis was analyzed. Resting cells showed an H+/O ratio of 4.8-5.2 (5.01+/-0.26), when measured using an oxygen pulse method with endogenous substrates. This value is intermediate between those of Bacillus subtilis (about 4), which predominantly expresses cytochrome aa3-type quinol oxidase, and Bacillus stearothermophilus (about 6), which has quinol cytochrome c reductase plus caa3-type cytochrome c oxidase. Measurement of respiration with various substrates, and its inhibition by cyanide suggested that aa3-type quinol oxidase and caa3-type cytochrome c oxidase operate simultaneously in the respiratory chain of B. brevis. Both terminal oxidases were isolated by solubilizing B. brevis membranes with Triton X-100, and fractionating the extract using DEAE-Fractgel and gel-filtration columns. The quinol oxidase (aa3) was composed of four subunits (57, 34, 23, and 15 kDa), like its counterpart of B. subtilis, while three subunits (52, 34, and 22 kDa) were identified in the cytochrome c oxidase (caa3) preparation in B. stearothermophilus.

Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the CuA-cytochrome c domain from the thermophilic Bacillus caa3-type cytochrome c oxidase.

The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.