How azide inhibits ATP hydrolysis by the F-ATPases

Abstract

In the structure of bovine F1-ATPase determined at 1.95-Å resolution with crystals grown in the presence of ADP, 5 -adenylylimidodiphosphate, and azide, the azide anion interacts with the ␤-phosphate of ADP and with residues in the ADP-binding catalytic subunit, ␤DP. It occupies a position between the catalytically essential amino acids, ␤-Lys-162 in the P loop and the ''arginine finger'' residue, ␣-Arg-373, similar to the site occupied by the ␥-phosphate in the ATP-binding subunit, ␤TP. Its presence in the ␤DP-subunit tightens the binding of the side chains to the nucleotide, enhancing its affinity and thereby stabilizing the state with bound ADP. This mechanism of inhibition appears to be common to many other ATPases, including ABC transporters, SecA, and DNA topoisomerase II␣. It also explains the stimulatory effect of azide on ATP-sensitive potassium channels by enhancing the binding of ADP.mitochondria ͉ oxidative phosphorylation ͉ inhibition ͉ mechanism

How the regulatory protein, IF 1 , inhibits F 1 -ATPase from bovine mitochondria

“…The diffraction properties of crystals of the F 1 -I1-60His complex were improved by shrinking the unit cell, especially in the a dimension, by changing the relative humidity from 98.5% to 98.0-97.5% by controlled dehydration. Similar benefits derived from controlled dehydration of crystals of bovine F 1 -ATPase grown in the presence and absence of azide have been described (6,14). Quaternary structural changes relative to the reference (14,15) and (F 1 -IF 1 ) 2 structures can be attributed to increased lattice contacts (see SI Text).…”

“…The coiled-coil region is not required for inhibitory activity, and residues 14-47 have been defined by deletion analysis as the minimal inhibitory sequence (13). Therefore, as presented in this article, bovine F 1 -ATPase has been inhibited by an active monomeric N-terminal fragment of bovine IF 1 consisting of residues 1-60 plus a C-terminal histidine 6 sequence, known as I1-60His. The complex between I1-60His and F 1 -ATPase has been crystallized and solved by x-ray crystallography at 2.1-Å resolution, thereby revealing the precise mode of binding of the inhibitor to F 1 -ATPase in the inhibited complex.…”

“…Several pieces of evidence indicate that in the active enzyme, ATP hydrolysis takes place at the ␤ DP catalytic site and not the ␤ TP catalytic site (6,17). For example, in the ground-state analog structure, BeF 3 Ϫ .F 1 (17), the guanidinium of the catalytically essential ''arginine finger'' residue ␣Arg-373 (15), and consequently the attacking nucleophilic water molecule, are significantly further away from the analog of the ␥-phosphate of ATP in the ␤ TP -subunit than in the ␤ DPsubunit, hence disfavoring ATP hydrolysis in the ␤ TP -subunit.…”

“…In the various structures of F 1 -ATPase, the side chain of ␣Arg-373 adopts either an ''inward pointing'' position or an ''outward pointing'' position. In both the ␤ DP and ␤ TP catalytic sites of the reference structure, of the ADP-azide inhibited enzyme (6,14,15), and of ground-state and transition-state analog structures formed with metallo-fluorides (17)(18)(19), ␣Arg-373 points in toward the catalytic site with its guanidinium group interacting with the ␤-and ␥-phosphates (or their ␥-phosphate analogs, BeF 3 Ϫ or AlF 4 Ϫ , or with azide). This position is consistent with its proposed role in stabilizing the pentavalent intermediate in ATP hydrolysis (15).…”

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F 1 –ATPase from bovine heart mitochondria

“…Crystals of bovine F 1 -ATPase were grown in the presence of ADP and magnesium ions, as described previously (10), except that the concentration of free magnesium ions in the mother liquor was diminished by the inclusion of phosphonate, a chelator of divalent metal cations. The structure of the resulting bovine F 1 -ATPase complex, known as F 1 -PH, was solved by molecular replacement with data to 2.6 Å.…”