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

AbstractBackground: 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 å resolution. The enzyme comprises five different subunits in the stoichiometry α3β3γδϵ; the three catalytic β subunits alternate with the three α subunits around the centrally located single γ subunit. To understand more about the catalytic mechanism, 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.Results: In the structure the three β subunits adopt a different conformation with different nucleotide occupancy. NBD-Cl reacts with the phenolic oxygen of Tyr311 of the βE subunit, which contains no bound nucleotide. The two other catalytic subunits βTP and βDP contain bound adenylyl-imidodiphosphate (AMP-PNP) and ADP, respectively. The binding site of the NBD moiety does not overlap with the regions of βE that form the nucleotide-binding pocket in subunits βTP and βDP nor does it occlude the nucleotide-binding site. Catalysis appears to be inhibited because neither βTP nor βDP can accommodate a Tyr311 residue bearing an NBD group.Conclusions: 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