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.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) on activities of enzyme complexes in the electron transport system were studied using isolated mitochondrial preparations from C57BL/6J mouse brains. Both MPTP and MPP+ dose-dependently inhibited activity of NADH-ubiquinone oxidoreductase (EC 1.6.5.3). The inhibition was reversible. Preincubation of freeze-thawed mitochondria with MPTP or MPP+ had no effect on the inhibition; however, when nonfrozen mitochondria were used, NADH-ubiquinone oxidoreductase activity was reduced to 46% of that in the nonincubated sample after a 5-min preincubation with MPTP and to 77% of that in the nonincubated sample after a 5-min preincubation with MPP+. Kinetic analyses revealed that inhibition of MPTP was noncompetitive and that of MPP+ uncompetitive with respect to NADH. On the other hand, inhibition of MPTP was uncompetitive and that of MPP+ noncompetitive with respect to ubiquinone. Succinate-ubiquinone oxidoreductase (complex II), dihydroubiquinone-cytochrome c oxidoreductase (complex III), and ferrocytochrome c-oxygen oxidoreductase (EC 1.9.3.1) activities were either slightly inhibited or not inhibited by MPTP or MPP+. The significance of these findings is discussed in relation to the mechanism of MPTP-induced neuronal degeneration.

In F(o)F(1) (F(o)F(1)-ATP synthase), proton translocation through F(o) drives rotation of the oligomer ring of F(o)-c subunits (c-ring) relative to F(o)-a. Previous reports have indicated that a conserved arginine residue in F(o)-a plays a critical role in the proton transfer at the F(o)-a/c-ring interface. Indeed, we show in the present study that thermophilic F(o)F(1s) with substitution of this arginine (aR169) to other residues cannot catalyse proton-coupled reactions. However, mutants with substitution of this arginine residue by a small (glycine, alanine, valine) or acidic (glutamate) residue mediate the passive proton translocation. This translocation requires an essential carboxy group of F(o)-c (cE56) since the second mutation (cE56Q) blocks the translocation. Rotation of the c-ring is not necessary because the same arginine mutants of the 'rotation-impossible' (c(10)-a)F(o)F(1), in which the c-ring and F(o)-a are fused to a single polypeptide, also exhibits the passive proton translocation. The mutant (aR169G/Q217R), in which the arginine residue is transferred to putatively the same topological position in the F(o)-a structure, can block the passive proton translocation. Thus the conserved arginine residue in F(o)-a ensures proton-coupled c-ring rotation by preventing a futile proton shortcut.

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (⌬pH) and the electrical potential differences (⌬⌿) across membranes. The optimum rate of synthesis reached 85 s ؊1 , and the H ؉ /ATP ratio of 4.0 ؎ 0.1 was obtained. ATP was synthesized at a considerable rate solely by ⌬pH, indicating ⌬⌿ was not absolutely required for synthesis. Consistent with the H ؉ /ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-Å resolution showed the presence of 12 Vo-c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases. ATP synthase ͉ rotary motor ͉ membrane protein ͉ bioenergetics ͉ two-dimensional crystal M embers of the F o F 1 and V-ATPase superfamily (rotary ATPase/synthase) use a rotary catalytic mechanism to perform their specific function (1, 2). The F o F 1 mainly catalyzes ATP synthesis in mitochondria, chloroplasts, and aerobic bacteria (3, 4). In contrast, V-ATPases exist in the endomembranes of all eukaryotic cells and in the plasma membrane of some specific eukaryotic cells functioning as a proton pump with a variety of cellular functions (2). The homologues of eukaryotic V-ATPases are also found in the plasma membrane of some bacteria (5, 6). Like the F o F 1 , V-ATPase from the thermophilic eubacterium Thermus thermophilus catalyzes ATP synthesis (7,8). In addition, it has the simplest known subunit structure (Fig. 1a) and is thus an excellent model for studying the mechanism of action of these important molecules. Subunits A and B of V-ATPase are the counterparts of subunits ␤ and ␣ of F o F 1 ATPase. Three copies of each subunit are arranged around the central rotor, which is made of single copies of subunits D and F. The A 3 B 3 DF moiety, termed V 1 , is responsible for the ATP hydrolysis or ATP synthesis reaction. The remaining subunits, V o -a (sometimes referred to as subunit I), V o -c (sometimes referred to as subunit L), and V o -d, E, and G form the V o domain of T. thermophilus V-ATPase (9). The V o -c subunits, which are folded into two transmembrane helices, constitute a membraneembedded oligomeric ring structure (10). The V o -c rotor ring and subunit V o -a form a proton channel, as seen in the c rotor ring of the F o -a subunit, despite low sequence similarity between the proteins.The basic mechanism of ATP synthesis for F o F 1 is well understood, as described below. Briefly, the ring of the F o -c subunit oligomer and ␥-subunits of F 1 comprise the central rotor, and together these rotate as a single body (11). The transmembrane electrochemical potential gradient of proton [⌬ H ϩ ϭ PMF ϫ F (PMF, proton motive force; F, Faraday constant)] drives rotation of the roto...

Bacterial operons for F1Fo-ATP synthase typically include an uncI gene that encodes a function-unknown small hydrophobic protein.When we expressed a hybrid F 1Fo (F1 from thermophilic Bacillus PS3 and Na ؉ -translocating Fo from Propionigenium modestum) in Escherchia coli cells, we found that uncI derived from P. modestum was indispensable to produce active enzyme; without uncI, csubunits in F 1Fo existed as monomers but not as functional c11-ring. When uncI was expressed from another plasmid at the same time, active F 1Fo with c11-ring was produced. A plasmid containing only uncI and c-subunit gene produced c 11-ring, but a plasmid containing only c-subunit gene did not. Direct interaction of UncI protein with c-subunits was suggested from copurification of His-tagged UncI protein and c-subunits, both in the state of c 11-ring and c-monomers. Na ؉ induced dissociation of His-tagged UncI protein from c 11-ring but not from c-monomers. These results show that UncI is a chaperone-like protein that assists c 11-ring assembly from c-monomers in the membrane.protein folding ͉ membrane protein ͉ Na ϩ transport