Hiroshi Omote

Molecular imaging of Escherichia coli F0F1-ATPase in reconstituted membranes using atomic force microscopy

The structure of Escherichia coli F0F1‐ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping‐mode operation. The majority of F0F1‐ATPases were visualized as spheres with a calculated diameter of , and a height of from the membrane surface. F0 sectors were visualized as two different ring‐like structures (one with a central mass and the other with a central hollow of depth) with a calculated outer diameter of . The two different images possibly represent the opposite orientations of the complex in the membranes. The ring‐like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.

Regulation and Reversibility of Vacuolar H+-ATPase

Arabidopsis thaliana vacuolar H+-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H+-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (ΔμH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (ΔpH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (ΔΨ). Chimeric vacuolar membranes showed ATP synthesis coupled with ΔμH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A1 and exhibited two apparent K m values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates ΔpH but not ΔΨ, suggesting that ΔpH is essential for ATP synthesis.

Synthase (H(+) ATPase): coupling between catalysis, mechanical work, and proton translocation.

Coupling with electrochemical proton gradient, ATP synthase (F(0)F(1)) synthesizes ATP from ADP and phosphate. Mutational studies on high-resolution structure have been useful in understanding this complicated membrane enzyme. We discuss mainly the mechanism of catalysis in the beta subunit of F(1) sector and roles of the gamma subunit in energy coupling. The gamma-subunit rotation during catalysis is also discussed.

β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP Synthase) Is an Essential Residue for Cooperative Catalysis

Glu-β185 of the Escherichia coli H+-ATPase (ATP synthase) β subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-β185 were less than 0.2% of the wild type one; the Asp-β185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-β185 F1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F1. The divalent cation dependences of the S-carboxymethyl-β185 and Asp-β185 ATPase activities were altered from that of the wild type. The Asp-β185, Cys-β185, S-carboxymethyl-β185, and Gln-β185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S-carboxymethyl-β185 and Asp-β185 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-β185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg•ADP complex. These results suggest that Glu-β185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-β185 is located close to the catalytic site in the higher order structure (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628).

CONFORMATIONAL TRANSMISSION IN ATP SYNTHASE DURING CATALYSIS : SEARCH FOR LARGE STRUCTURAL CHANGES

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at βGly-149 was suppressed by the second mutations at the outer surface of the β subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that β/α and β/γ subunits interactions are important for the energy coupling between catalysis and H+ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the γ subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.

A glycine-rich sequence in the catalytic site of F-type ATPase

Affinity labeling and genetic studies on the glycine-rich sequence of the β subunit ofE. coli F-type ATPase are discussed. A model of the structure of the enzyme near the γ phosphate moiety is proposed.

Catalysis and energy coupling of H+-ATPase (ATP synthase): Molecular biological approaches

The molecular biological approach has provided important information for understanding the F0F1 H(+)-ATPase. This article focuses on our recent results on the catalytic site in the beta subunit, and the roles of alpha/beta subunit interaction and amino/carboxyl terminal interaction of the gamma subunit in energy coupling. Extensive mutagenesis of the beta subunit revealed that beta Lys-155, beta Thr-156, beta Glu-181 and beta Arg-182 are essential catalytic residues. beta Glu-185 is not absolutely essential, but a carboxyl residue may be necessary at this position. A pseudo-revertant analysis positioned beta Gly-172, beta Ser-174, beta Glu-192 and beta Val-198 in the proximity of beta Gly-149. The finding of the roles of beta Gly-149, beta Lys-155, and beta Thr-156 emphasized the importance of the glycine-rich sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser, E. coli beta residues between beta Gly-149 and beta Thr-156) conserved in many nucleotide binding proteins. The A subunits of vacuolar type ATPases may have a similar catalytic mechanism because they have conserved glycine-rich and Gly-Glu-Arg (corresponding to beta Gly-180-beta Arg-182) sequences. The results of these mutational studies are consistent with the labeling of beta Lys-155 and beta Lys-201 with AP3-PL, and of beta Glu-192 with DCCD [15]. The DCCD-binding residue of a thermophilic Bacillus corresponds to beta Glu-181, an essential catalytic residue discussed above. The defective coupling of the beta Ser-174-->Phe mutant was suppressed by the second mutation alpha Arg-296-->Cys, indicating the importance of alpha/beta interaction in energy coupling. The gamma subunit, especially its amino/carboxyl interaction, seems to be essential for energy coupling between catalysis and transport judging from studies on gamma Met-23-->Lys or Arg mutation and second-site mutations which suppressed the gamma Lys-23 mutation. Thus the conserved gamma Met-23 is not absolutely essential but is located in the important region for amino/carboxyl interaction for energy coupling.

Mutational Analysis of ATP Synthase An Approach to Catalysis and Energy Coupling

The ATP synthases (FoF1) of Escherichia coli, mitochondria, and chloroplasts (for reviews, see Futai and Omote, 1996; Fillingame, 1990; Senior, 1990; Futai et al., 1989) are closely similar and have conserved basic subunit structures: a catalytic sector F1—α3β3γδe—and a transmembrane proton pathway Fo—a b 2 c 10–12. The ATP synthesis by FoF1 is driven by a transmembrane electrochemical proton gradient established by the electron transfer chain. The gradient is required for catalytic turnover: ATP release from and ADP plus Pi (phosphate) binding to the catalytic site in the β subunit. The cooperativity between the three catalytic sites has been established. However, elucidation of the mechanism of energy coupling between catalysis and the electrochemical proton gradient is still at an early stage.

F-type H+-ATPase: Catalysis and Proton Transport

F0F1 H+ATPase (or F-type ATPase) catalyzes ATP synthesis or hydrolysis coupling with proton translocation (for reviews, see Futai et al., 1989; Senior, 1990; Fillingame, 1990). The F-type ATPase of Escherichia coli is similar to those found in inner mitochondrial or chloroplast thylakoid membranes, and has contributed greatly to the understanding of this complicated enzyme. The catalytic site of the enzyme is in the P subunit or at the interface between the α and β subunits of the membrane extrinsic F1 sector. The proton pathway is formed from the a, b, and c subunits of the membrane intrinsic Fo sector. The γ, δ, and e subunits of F1 are required functionally and structurally to connect the catalytic subunits to the Fo sector. The mechanism of ATP hydrolysis can be studied using purified F1 (F1 — ATPase).