Nucleotide binding to active and 4-chloro-7-nitrobenzofurazan-inhibited forms of chloroplast F1-ATPase ― an NMR study

Abstract (1) Chloroplast F1-ATPase (CF1) was labelled on both a single tyrosine and at least two cysteine residues by 4-chloro-7-nitrobenzofurazan (Nbf-Cl). The ATPase activity was inhibited by 80–90%. Prior treatment of latent CF1 with N-ethylmaleimide (NEM), to block accessible thiol groups, reduced the Nbf-cysteine modification to the equivalent of one residue per enzyme molecule. Only one tyrosine residue, identified here as tyrosine 328 on one beta subunit, is modified by Nbf-Cl. This is analogous to the tyrosine residue which reacts with Nbf-Cl on the related enzyme from mitochondria (MF1). (2) 31P and 1H nuclear magnetic resonance (NMR) studies of CF1 identified a single tight binding site for the Ca2+ complex of the ATP analogue 5′-adenylylimidodiphosphate (AMPPNP) on each of latent, trypsin activated, and NEM modified CF1. No NMR signals could be detected from from this site. Tight binding of Ca-AMPPNP to latent and trypsin activated CF1 was also inferred from its low inhibition constant (Ki ≈ 12 μM). NMR also showed that addition of excess Ca-AMPPNP to both latent and NEM-labelled CF1 displaced one tightly bound adenine nucleotide. The 31P signals from nucleotides ADP or ATP bound at this single tight site were too broad to be observed but could readily be detected after denaturation of the enzyme with detergent. (3) In contrast to the active forms of CF1, the Nbf-Cl modified enzyme was found to have one ADP and one ATP bound. 31P-NMR signals from the ADP, but not the ATP, could be detected. These signals had the linewidths expected for a bound ligand. In the presence of excess Ca-AMPPNP the bound nucleotide slowly dissociated from the Nbf labelled enzyme at a rate which correlated with loss of the Nbf group from tyrosine 328. This was accompanied by a loss of signal intensity equivalent to one mol of Ca-AMPPNP per mol enzyme.