Tsutomu Unemoto

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K+-depleted and Na+-loaded Vibrioalginolyticus is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na+-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na+ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na+-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na+ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na+ chemical gradient.

Na+-translocating NADH-quinone reductase of marine and halophilic bacteria

The respiratory chain of marine and moderately halophilic bacteria requires Na+ for maximum activity, and the site of Na+-dependent activation is located in the NADH-quinone reductase segment. The Na+-dependent NADH-quinone reductase purified from marine bacteriumVibrio alginolyticus is composed of three subunits, α, β, and γ, with apparentMr of 52, 46, and 32kDa, respectively. The FAD-containing β-subunit reacts with NADH and reduces ubiquinone-1 (Q-1) by a one-electron transfer pathway to produce ubisemiquinones. In the presence of the FMN-containing α-subunit and the γ-subunit, Q-1 is converted to ubiquinol-1 without the accumulation of free radicals. The reaction catalyzed by the α-subunit is strictly dependent on Na+ and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which is tightly coupled to the electrogenic extrusion of Na+. A similar type of Na+-translocating NADH-quinone reductase is widely distributed among marine and moderately halophilic bacteria. The respiratory chain ofV. alginolyticus contains another NADH-quinone reductase which is Na+ independent and has no energy-transducing capacity. These two types of NADH-quinone reductase are quite different with respect to their mode of quinone reduction and their sensitivity toward NADH preincubation.

Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus.

The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.

FMN is covalently attached to a threonine residue in the NqrB and NqrC subunits of Na+-translocating NADH-quinone reductase from Vibrio alginolyticus

The Na+‐translocating NADH‐quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post‐source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr‐235 in the NqrB subunit and to Thr‐223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide.

Sequencing and the alignment of structural genes in the nqr operon encoding the Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus.

We previously cloned a part of nqr operon encoding the Na+‐translocating NADH‐quinone reductase (NQR) from the marine Vibrio alginolyticus (Hayashi et al., FEBS Lett. 356 (1994) 330–332]. From its nucleotide sequences, four consecutive open reading frames (ORF) encoding the γ‐subunit (27.7 kDa), two unidentified ORFs of 22.6 kDa and 21.5 kDa, and the β‐subunit (45.3 kDa) were recognized. The gene encoding the α‐subunit was located upstream, and together with the recent report by Beattie et al. [FEBS Lett. 356 (1994) 333–338], the nqr operon was found to be constructed from six consecutive structural genes, where nqrl, nqr3 and nqr6 correspond to the α‐, γ‐, and β‐subunits, respectively, of the NQR complex.

K+/H+ antiporter functions as a regulator of cytoplasmic pH in a marine bacterium, Vibrio alginolyticus

The marine bacterium, Vibrio alginolyticus, regulates the cytoplasmic pH at about 7.8 over the pH range 6.0-9.0. By the addition of diethanolamine (a membrane-permeable amine) at pH 9.0, the internal pH was alkalized and simultaneously the cellular K+ was released. Following the K+ exit, the internal pH was acidified until 7.8, where the K+ exit leveled off. The K+ exit was mediated by a K+/H+ antiporter that is driven by the outwardly directed K+ gradient and ceases to function at the internal pH of 7.8 and below. The Na+-loaded cells assayed in the absence of KCl generated inside acidic delta pH at alkaline pH due to the function of an Na+/H+ antiporter, but the internal pH was not maintained at a constant value. At acidic pH range, the addition of KCl to the external medium was necessary for the alkalization of cell interior. These results suggested that in cooperation with the K+ uptake system and H+ pumps, the K+/H+ antiporter functions as a regulator of cytoplasmic pH to maintain a constant value of 7.8 over the pH range 6.0-9.0.

Covalently bound flavin in the NqrB and NqrC subunits of Na+-translocating NADH-quinone reductase from Vibrio alginolyticus

Na+‐translocating NADH‐quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS–PAGE of the purified complex, NqrB and NqrC subunits were found to give yellow–green fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N‐terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed‐phase high performance liquid chromatography. N‐Terminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr‐235 in the NqrB and Thr‐223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin‐linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress.

Identification of six subunits constituting Na+‐translocating NADH‐quinone reductase from the marine Vibrio alginolyticus

We previously reported that the purified Na+‐translocating NADH‐quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of three major subunits, α, β and γ. NQR operon was sequenced and was found to be composed of 6 structural genes. Among these genes, nqr1, nqr3 and nqr6 were identified to code for α‐, γ‐ and β‐subunits, respectively. The protein products from nqr2, nqr4 and nqr5, however, were not reported. The sequence data predicted that these three proteins are very hydrophobic and may be unusual in mobility and staining on SDS‐PAGE. By modifying the detection method of proteins on SDS‐PAGE, we could detect all six subunits encoded by NQR operon in the purified NQR complex. The open reading frame of each subunit was identified from its N‐terminal amino acid sequence.

Mechanosensitive channel functions to alleviate the cell lysis of marine bacterium, Vibrio alginolyticus, by osmotic downshock.

The mechanosensitive channel with large conductance of Escherichia coli is the first to be cloned among stretch‐activated channels. Although its activity was characterized by a patch clamp method, a physiological role of the channel has not been proved. The marine bacterium, Vibrio alginolyticus, is sensitive to osmotic stress and cell lysis occurs under osmotic downshock. We introduced an mscL gene into Vibrio alginolyticus, and the mechanosensitive channel with large conductance functions was found to alleviate cell lysis by osmotic downshock. This is the first report to show a physiological role of the mechanosensitive channel with large conductance.

Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain

The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.