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

Background: F-1-ATPase is the globular domain of F1F0-ATP synthase that catalyses the hydrolysis of ATP to ADP and phosphate. The crystal structure of bovine F-1-ATPase has been determined previously to 2.8 Angstrom 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 mechanism, F-1-ATPase was inhibited by reaction with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and the structure of the inhibited complex (F-1-NBD) determined by X-ray crystallography. Results: In the structure the three beta subunits adopt a different conformation with different nucleotide occupancy. NBD-CI 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. 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.