[ATP synthase: highly organized molecular structure and its marvelous function].
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Adenosine Diphosphate↗
Biomedical subjects
Noriyo Mitome
Publications and source records attributed to Noriyo Mitome.
Thermophilic ATP synthase has a decamer c-ring: indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling.
In a rotary motor F(o)F(1)-ATP synthase that couples H(+) transport with ATP synthesis/hydrolysis, it is thought that an F(o)c subunit oligomer ring (c-ring) in the membrane rotates as protons pass through F(o) and a 120 degrees rotation produces one ATP at F(1). Despite several structural studies, the copy number of F(o)c subunits in the c-ring has not been determined for any functional F(o)F(1). Here, we have generated and isolated thermophilic Bacillus F(o)F(1), each containing genetically fused 2-mer-14-mer c (c(2)-c(14)). Among them, F(o)F(1) containing c(2), c(5), or c(10) showed ATP-synthesis and other activities. When F(1) was removed, F(o) containing c(10) worked as an H(+) channel but F(o)s containing c(9), c(11) or c(12) did not. Thus, the c-ring of functional F(o)F(1) of this organism is a decamer. The inevitable consequence of this finding is noninteger ratios of rotation step sizes of F(1)/F(o) (120 degrees /36 degrees ) and of H(+)/ATP (10:3). This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by H(+) transport at F(o) and elementary events in catalysis at F(1).
Adenosine Triphosphate↗
F(0) of ATP synthase is a rotary proton channel. Obligatory coupling of proton translocation with rotation of c-subunit ring.
Coupling of proton flow and rotation in the F(0) motor of ATP synthase was investigated using the thermophilic Bacillus PS3 enzyme expressed functionally in Escherichia coli cells. Cysteine residues introduced into the N-terminal regions of subunits b and c of ATP synthase (bL2C/cS2C) were readily oxidized by treating the expressing cells with CuCl(2) to form predominantly a b-c cross-link with b-b and c-c cross-links being minor products. The oxidized ATP synthases, either in the inverted membrane vesicles or in the reconstituted proteoliposomes, showed drastically decreased proton pumping and ATPase activities compared with the reduced ones. Also, the oxidized F(0), either in the F(1)-stripped inverted vesicles or in the reconstituted F(0)-proteoliposomes, hardly mediated passive proton translocation through F(0). Careful analysis using single mutants (bL2C or cS2C) as controls indicated that the b-c cross-link was responsible for these defects. Thus, rotation of the c-oligomer ring relative to subunit b is obligatory for proton translocation; if there is no rotation of the c-ring there is no proton flow through F(0).
Bacillus↗
The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F(1)-ATPase.
F1-ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or P(i). Here, using a mutant alpha(3)beta(3)gamma complex of thermophilic F(1)-ATPase (alpha W463F/beta Y341W) and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan, we found that P(i) interfered with the binding of MgATP to F(1)-ATPase, but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F(1)-ATPase during the experiments did not obscure the interpretation because another mutant, which was able to bind nucleotide but not hydrolyse ATP (alpha W463F/beta E190Q/beta Y341W), also gave the same results. The half-maximal concentrations of P(i) that suppressed the MgADP-inhibited form and interfered with MgATP binding were both approximately 20 mm. It is likely that the presence of P(i) at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme-MgADP-P(i) complex, an active intermediate in the catalytic cycle.
Adenosine Diphosphate↗