H-1 AND P-31 NUCLEAR-MAGNETIC-RESONANCE AND KINETIC-STUDIES OF THE ACTIVE-SITE STRUCTURE OF CHLOROPLAST CF1 ATP SYNTHASE

The interaction of nucleotides and nucleotide analogues and their metal complexes with Mn2+ bound to both the latent and dithiothreitol-activated CF1, ATP synthase has been examined by means of steady-state kinetics, water proton relaxation rate (PRR) measurements, and 1H and 31P nuclear relaxation measurements. Titration of both the latent and activated Mn2+−CF1 complexes with ATP, ADP, Pi, Co(NH3)4ATP, Co(NH3)4ADP, and Co(NH3)4AMPPCP leads to increases in the water relaxation enhancement, consistent with enhanced metal binding and a high ternary complex enhancement. Steady-state kinetic studies are consistent with competitive inhibition of CF1 by Co(NH3)4AMPPCP with respect to CaATP. The data are consistent with a Ki for Co(NH3)4AMPPCP of 650 µM, in good agreement with a previous Ki of 724 µM for Cr(H2O)4ATP [Frasch, W., & Selman, B. (1982) Biochemistry 21, 3636–3643], and a best fit KD of 209 µM from the water PRR measurements. 1H and 31P nuclear relaxation measurements in solutions of CF1 and Co(NH3)4AMPPCP were used to determine the conformation of the bound substrate analogue and the arrangement with respect to this structure of high- and low-affinity sites for Mn2+. The bound nucleotide analogue adopts a bent conformation, with the low-affinity Mn2+ site situated between the adenine and triphosphate moieties and the high-affinity metal site located on the far side of the triphosphate chain. The low-affinity metal forms a distorted inner-sphere complex with the ß-P and γ-P of the substrate. The distances from Mn2+ to the triphosphate chain are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen bonded water molecules or residues from the protein. © 1990, American Chemical Society. All rights reserved.