Shunsuke Noguchi

Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the α-subunit of Torpedo californicaNa+K+-ATPase

Point mutations of Asp-376 of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase (the site of phosphorylation during the catalytic cycle) to Asn, Glu or Thr led to virtual abolishment of Na+/K(+)-ATPase activity and ouabain-binding capacity. Replacement of Lys-507 of the same subunit (the putative ATP-binding site) by Met resulted in decreases in Na+/K(+)-ATPase activity and ouabain-binding capacity. These results are in agreement with those reported for rabbit sarcoplasmic reticulum Ca2(+)-ATPase (Maruyama, K. and MacLennan, D.H. (1988) Proc. Natl. Acad. Sci. USA 85, 3314-3318).

Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the α-subunit

Currents generated by the Na+/K+ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na+] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had α-subunits truncated before position Lys28 (αΔK28) or Thr29 (αΔT29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K+ show voltage-dependent inhibition by external [Na+]. The apparent K1 values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na+-K+ competition for external binding to the E2P form, apparent K1 values and effective charges for the interaction of the Na+ ions with the E2P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K1 value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K1 value. For αΔK28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS28 is included in the truncation (α·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K1 value is reduced to 107 mM. The K, values for pump inhibition by external Na+, calculated by taking into account Na+-K+ competition, are smaller than the K/12 values determined in the presence of 5 mM [K+]. The difference is more pronounced for those pump variants that have higher Km, values. The variations of the parameters describing inhibition by external [Na+] are qualitatively similar to those described for the stimulation of the pumps by external [K+] in the absence of extracellular [Na+]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na+ and K+ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.

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.

Cytochrome c oxidase contains an extra charged amino acid cluster in a new type of respiratory chain in the amino-acid-producing Gram-positive bacterium Corynebacterium glutamicum

The membranes from Corynebacterium glutamicum cells contain a hydrophobic di-haem C protein as the cytochrome c subunit of the new type of cytochrome bc complex (complex III in the respiratory chain) encoded by the qcrCAB operon [Sone, N., Nagata, K., Kojima, H., Tajima, J., Kodera, Y., Kanamaru, T., Noguchi, S. & Sakamoto, J. (2001). Biochim Biophys Acta 1503, 279-290]. To characterize complex IV, cytochrome c oxidase and its structural genes were isolated. The oxidase is of the cytochrome aa(3) type, but mass spectrometry indicated that the haem is haem As, which contains a geranylgeranyl side-chain instead of a farnesyl group. The enzyme is a SoxM-type haem-copper oxidase composed of three subunits. Edman degradation and mass spectrometry suggested that the N-terminal signal sequence of subunit II is cleaved and that the new N-terminal cysteine residue is diacylglycerated, while neither subunit I nor subunit III is significantly modified. The genes for subunits II (ctaC) and III (ctaE) are located upstream of the qcrCAB operon, while that for subunit I (ctaD) is located separately. The oxidase showed low enzyme activity with extrinsic substrates such as cytochromes c from horse heart or yeast, and has the Cu(A)-binding motif in its subunit II. A prominent structural feature is the insertion of an extra charged amino acid cluster between the beta2 and beta4 strands in the substrate-binding domain of subunit II. The beta2-beta4 loop of this oxidase is about 30 residues longer than that of major cytochrome c oxidases from mitochondria and proteobacteria, and is rich in both acidic and basic residues. These findings suggest that the extra charged cluster may play a role in the interaction of the oxidase with the cytochrome c subunit of the new type of bc complex.

Assembly of the α-subunit of Torpedo californica Na+/K+-ATPase with its pre-existing β-subunit in Xenopus oocytes

Abstract The α- and β-subunits of Torpedo californica Na+/K+-ATPase were expressed in turn in single oocytes by alternately microinjection the specific mRNAs for the α- and β-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing β-subunit, which had been synthesized by injecting mRNA for the β-subunit, could assemble with the α-subunit expressed later in the single oocytes and the resulting αβ complex acquired both ouabain-binding and Na+/K+-ATPase activities. On the other hand, formation of the αβ complex was not detected when the α-subunit was expressed first, followed by the β-subunit. These data suggest that the β-subunit acts as a receptor or a stabilizer for the α-subunit upon the biogenesis of Na+/K+-ATPase.

Functional activity of oligosaccharide-deficient (Na,K)ATPase expressed in Xenopus oocytes

(Na,K)ATPase from Torpedo californica was expressed in Xenopus laevis oocytes in the presence of tunicamycin by injecting mRNAs for the α‐ and β‐subunits derived from the cloned cDNAs into the oocytes. The oligosaccharide‐deficient ATPase thus synthesized was transported to the oocytes plasma membrane, where it exhibited virtually the same ATPase activity, ouabain‐binding capacity and 86Rb+ transport activity as the fully glycosylated enzyme. We conclude that the oligosaccharide chains on the β‐subunit has no effect on the catalytic activities of (Na,K)ATPase.

The functional roles of disulfide bonds in the β-subunit of (Na,K)ATPase as studied by site-directed mutagenesis

The β‐subunit of Torpedo californica (Na,K)ATPase contains seven cysteine residues; one (Cys46) is in the single transmembrane segment and the other six (Cys127, Cys150, Cys160, Cys176, Cys215 and Cys278) are in the extracellular domain and form three highly conserved disulfide bonds. Aβ‐subunit mutant with replacement of Cys46 by Ser could assemble with the α‐subunit, and the resulting αβ‐complex was catalytically active. Mutants in which either the N‐terminal side or both Cys residues of the Cys127‐Cys150 bond were replaced by Ser could also tightly assemble with the α‐subunit, but the resulting αβ‐complex was catalytically inactive. On the other hand, disruption of either the Cys160‐Cys176 or Cys215‐Cys278 bond by substituting the N‐terminal side only or both Cys residues with Ser led to a β‐subunit that could not assemble with the α‐subunit. We conclude that the structure of the β‐subunit around the Cys160‐Cys176 and Cys215‐Cys278 loops is indispensable for assembly with the α‐subunit, whereas the Cys127‐Cys150 loop is not essential for assembly but is required for enzyme activity.

Bacillus stearothermophilus qcr Operon Encoding Rieske FeS Protein, Cytochrome b6, and a Novel-type Cytochrome c1 of Quinol-cytochrome c Reductase

The qcr of Bacillus stearothermophilus K1041 encoding three subunits of the quinol-cytochrome c oxidoreductase (cytochrome reductase, bc1 complex) was cloned and sequenced. The gene (qcrA) for a Rieske FeS protein of 19,144 Da with 169 amino acid residues, and the gene (qcrC) for cytochrome c1 of 27,342 Da with 250 amino acid residues were found at adjacent upstream and downstream sides of the previously reported qcrB (petB) for cytochrome b of subunit 25,425 Da with 224 residues (Sone, N., Sawa, G., Sone, T., and Noguchi, S.(1995) J. Biol. Chem. 270, 10612-10617). The three structural genes for thermophilic Bacillus cytochrome reductase form a transcriptional unit. In the deduced amino acid sequence for the FeS protein, the domain including four cysteines and two histidines binding the 2Fe-2S cluster was conserved. Its N-terminal part more closely resembled the cyanobacteria-plastid type than the proteobacteria-mitochondria type when their sequences were compared. The amino acid sequence of cytochrome c1 was not similar to either type; the thermophilic Bacillus cytochrome c1 is composed of an N-terminal part corresponding to subunit IV with three membrane-spanning segments, and a C-terminal part of cytochrome c reminiscent of cytochrome c-551 of thermophilic Bacillus. The subunit IV in the enzyme of cyanobacteria and plastids is the counterpart of C-terminal part of cytochrome b of proteobacteria and mitochondria. These characteristics indicate that Bacillus cytochrome bc1 complex is unique.

Assembly of a hybrid from the α subunit of Na+K+-ATPase and the β subunit of H+K+-ATPase

Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.

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.