Characterization of the metal binding environment of catalytic site 1 of chloroplast F1-ATPase from Chlamydomonas

Metal ligands of the VO2+-adenosine diphosphate (ADP) complex bound to high-affinity catalytic site 1 of chloroplast F-1 adenosine triphosphatase (CF1 ATPase) were characterized by electron paramagnetic resonance (EPR) spectroscopy. This EPR spectrum contains two EPR species designated E and F not observed when VO2+-nucleotide is bound to site 3 of CF1. Site-directed mutations beta E197C, beta E197D, and beta E197S in Chlamydomonas CB impair ATP synthase and ATPase activity catalyzed by CF1F0 and soluble CF1, respectively, indicating that this residue is important for enzyme function. These mutations caused large changes in the V-51 hyperfine tensors of VO2+-nucleotide bound to site 1 but not to site 3. Mutations to the Walker homology B aspartate beta D262C, beta D262H, and beta D262T of Chlamydomonas CF1 caused similar effects on the EPR spectrum of VO2+-ADP bound to site 1. These results indicate that the conversion of the low-affinity site 3 conformation to high-affinity site 1 involves the incorporation beta E197 and beta D262 as metal ligands.