Oxidation of the α3(βD311C/R333C)3γ Subcomplex of the Thermophilic Bacillus PS3 F1-ATPase Indicates That Only Two β Subunits Can Exist in the Closed Conformation Simultaneously

Abstract

In the crystal structure of the bovine heart mitochondrial The proton-translocating F 0 F 1 -ATP synthases couple proton electrochemical gradients to condensation of ADP with P i in energy-transducing membranes. The F 0 moiety is a membraneembedded protein complex that mediates proton translocation. F 1 is a peripheral membrane protein complex composed of five different subunits with ␣ 3 ␤ 3 ␥␦⑀ stoichiometry. When removed from the membrane, F 1 is an ATPase containing six nucleotide binding sites. Three are catalytic sites that are predominantly on ␤ subunits at ␣/␤ interfaces. The three other sites, called noncatalytic sites, are located predominantly on ␣ subunits at different ␣/␤ interfaces (1-3).The crystal structures of the bovine heart and rat liver mitochondrial F 1 -ATPases as well as that of the ␣ 3 ␤ 3 subcomplex of the TF 1 -ATPase have been determined (4 -6). In the crystals of the bovine heart enzyme (BH-MF 1 ), 1 which form in media containing AMP-PNP, ADP, Mg 2ϩ , and N 3 Ϫ , noncatalytic sites are homogeneously liganded with MgAMP-PNP, whereas catalytic sites are heterogeneously liganded (4). One, designated ␤ T , contains MgAMP-PMP, another, designated ␤ D , contains MgADP and the third catalytic site, designated ␤ E , is empty. Except for small differences in the regions of the terminal phosphates of bound nucleotides, the arrangements of functional amino acid side chains in catalytic sites in ␤ T and ␤ D are essentially identical. In contrast, these residues are arranged differently in ␤ E . In the crystals of the ␣ 3 ␤ 3 subcomplex of TF 1 that form in media free of Mg 2ϩ and nucleotides, the ␣ subunits are in closed conformations corresponding to the liganded ␣ subunits of the mitochondrial enzymes. The ␤ subunits in the ␣ 3 ␤ 3 structure are in open conformations corresponding to ␤ E of BH-MF 1 (5).In the crystals of the rat liver F 1 -ATPase (RL-MF 1 ), which form in media containing ATP, P i , and EDTA, noncatalytic sites are homogeneously occupied with MgATP. In contrast to the crystals of BH-MF 1 , all three catalytic sites of RL-MF 1 contain bound nucleotides in the absence of Mg 2ϩ (6). One catalytic site contains ADP alone, whereas the other two contain ADP and P i . Since Mg 2ϩ is not associated with ADP and P i bound to catalytic sites of RL-MF 1 , side chains in catalytic sites that interact with the Mg 2ϩ ion in the crystal structure of BH-MF 1 are arranged differently in the crystal structure of RL-MF 1 . However, these differences are minor compared with the difference between the closed conformations of ␤ D and ␤ T and the open conformation of ␤ E in BH-MF 1 . In the crystal structure of RL-MF 1 , the conformations of all three ␤ subunits resemble the closed conformation of ␤ D and ␤ T in the crystal structure of BH-MF 1 . Bianchet et al. (6) suggest that the open conformation of ␤ E in the crystal structure of BH-MF 1 is the consequence of low concentrations of nucleotides in the crystallization medium. Extending this argument, they propose that the RL-MF 1 structure with t...

The Fluorescence Spectrum of the Introduced Tryptophans in the α3(βF155W)3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 Cannot Be Used to Distinguish between the Number of Nucleoside Di- and Triphosphates Bound to Catalytic Sites

“…Therefore, it is not unreasonable to assume that occupancy of catalytic sites with different ligands might differently affect the orientation of the side chain of the introduced tryptophan with the side chain of ␤Phe 326 . The finding that [ 3 H]ADP was entrapped in only two catalytic sites when the ␤F155W and ␤Y345W mutants were treated with 2 mM Mg[ 3 H]ADP followed by Al 3ϩ and F Ϫ is consistent with earlier observations indicating that three ␤ subunits cannot simultaneously exist in closed conformations (30,31). For instance, Fig.…”

Large conformational changes of the ɛ subunit in the bacterial F 1 F 0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

“…The close approach of the C-terminal ␣-helix to ␤ subunits explains the crosslinking between Cys-108 and both ␣Cys-411 and ␤Cys-381 reported earlier (20,28,29). Such close interaction between and ␣ or ␤ can be expected to restrict or even prevent conformational changes in the ␤ subunits that are central to the rotation of the central stalk (35,36). Related to this, Hara et al have recently shown that the electrostatic interaction between the C-terminal helix of the subunit and the DELSEED motif in the ␤ subunit is essential for the inhibitory effect of the subunit (37).…”

ATP synthase — a marvellous rotary engine of the cell

“…Conversely, if the γ-subunit is forced to rotate by the F o motor, this can induce changes in the nucleotide-binding states of the β-subunits, resulting in the synthesis of ATP. Indeed, the dynamic occurrence of the CCO (or CCC′) state during catalytic turnover has been shown by the formation of a specific crosslink that can bridge only between two C-form β-subunits in an F 1 molecule 15 , and the activity of F 1 is lost by fixing the β-subunits in the C form 16 .…”