Rotary Catalytic Mechanism Research Articles

Coupling H(+) transport to rotary catalysis in F-type ATP synthases: structure and organization of the transmembrane rotary motor.

H(+)-transporting F(1)F(o)-type ATP synthases utilize a transmembrane H(+) potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating beta subunits of the extramembranous F(1) sector of the enzyme, synthesis being driven by rotation of the gamma subunit in the center of the F(1) molecule between the alternating catalytic sites. The H(+) electrochemical potential is thought to drive gamma subunit rotation by first coupling H(+) transport to rotation of an oligomeric rotor of c subunits within the transmembrane F(o) sector. The gamma subunit is forced to turn with the c(12) oligomeric rotor as a result of connections between subunit c and the gamma and epsilon subunits of F(1). In this essay, we will review recent studies on the Escherichia coli F(o) sector. The monomeric structure of subunit c, determined by nuclear magnetic resonance (NMR), is discussed first and used as a basis for the rest of the review. A model for the structural organization of the c(12) oligomer in F(o), deduced from extensive cross-linking studies and by molecular modeling, is then described. The interactions between the the a(1)b(2) 'stator' subcomplex of F(o) and the c(12) oligomer are then considered. A functional interaction between transmembrane helix 4 of subunit a (aTMH-4) and transmembrane helix 2 of subunit c (cTMH-2) during the proton-release step from Asp61 on cTMH-2 is suggested. Current a-c cross-linking data can only be explained by helix-helix swiveling or rotation during the proton transfer steps. A model that mechanically links helix rotation within a single subunit c to the incremental 30 degrees rotation of the c(12) oligomer is proposed. In the final section, the structural interactions between the surface residues of the c(12) oligomer and subunits epsilon and gamma are considered. A molecular model for the binding of subunit epsilon between the exposed, polar surfaces of two subunits c in the oligomer is proposed on the basis of cross-linking data and the NMR structures of the individual subunits.

Subunit organization of the stator part of the F0 complex from Escherichia coli ATP synthase.

Membrane-bound ATP synthases (F1F0) catalyze the synthesis of ATP via a rotary catalytic mechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potential is supposed to propel rotation of a subunit c ring of F0 together with subunits gamma and epsilon of F1, thereby forming the rotor part of the enzyme, whereas the remainder of the F1F0 complex functions as a stator for compensation of the torque generated during rotation. This review focuses on our recent work on the stator part of the F0 complex, e.g., subunits a and b. Using epitope insertion and antibody binding, subunit a was shown to comprise six transmembrane helixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circular dichroism (CD) spectroscopy, the secondary structure of subunit b incorporated into proteoliposomes was determined to be 80% alpha-helical together with 14% beta turn conformation, providing flexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplex was shown to be active in proton translocation and functional F1 binding revealing the native conformation of the polypeptide chain. Chemical crosslinking in everted membrane vesicles led to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32C could be crosslinked to subunit a, indicating a close proximity of subunits a and b near the membrane. Further evidence for the proposed direct interaction between subunits a and b was obtained by purification of a stable ab2 subcomplex via affinity chromatography using His tags fused to subunit a or b. This ab2 subcomplex was shown to be active in proton translocation and F1 binding, when coreconstituted with subunit c. Consequences of crosslink formation and subunit interaction within the F1F0 complex are discussed.

The structure of bovine mitochondrial F1-ATPase: an example of rotary catalysis

There is now compelling evidence in support of a rotary catalytic mechanism in F1-ATPase, and, by extension, in the intact ATP synthase. Although models have been proposed to explain how protein translocation in F0 results in rotation of the gamma-subunit relative to the alpha 3/beta 3 assembly in F1 [22], these are still speculative. It seems likely that a satisfactory explanation of this mechanism will ultimately depend on structural information on the intact ATP synthase.

Bovine F1-ATPase covalently inhibited with 4-chloro-7-nitrobenzofurazan: the structure provides further support for a rotary catalytic mechanism.

F1-ATPase is the globular domain of F1F0-ATP synthase that catalyses the hydrolysis of ATP to ADP and phosphate. The crystal structure of bovine F1-ATPase has been determined previously to 2.8 A resolution. The enzyme comprises five different subunits in the stoichiometry alpha 3 beta 3 gamma delta epsilon; the three catalytic beta subunits alternate with the three alpha subunits around the centrally located single gamma subunit. To understand more about the catalytic mechanisms, F1-ATPase was inhibited by reaction with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and the structure of the inhibited complex (F1-NBD) determined by X-ray crystallography. In the structure the three beta subunits adopt a different conformation with different nucleotide occupancy. NBD-Cl reacts with the phenolic oxygen of Tyr311 of the beta E subunit, which contains no bound nucleotide. The two other catalytic subunits beta TP and beta DP contain bound adenylyl-imidodiphosphate (AMP-PNP) and ADP, respectively. The binding site of the NBD moiety does not overlap with the regions of beta E that form the nucleotide-binding pocket in subunits beta TP and beta DP nor does it occlude the nucleotide-binding site. Catalysis appears to be inhibited because neither beta TP nor beta DP can accommodate a Tyr311 residue bearing an NBD group. The results presented here are consistent with a rotary catalytic mechanism of ATP synthesis and hydrolysis, which requires the sequential and concerted participation of all three catalytic sites. NBD-Cl inhibits the enzyme by preventing the modified subunit from adopting a conformation that is essential for catalysis to proceed.