Metal ligands of the VO2+-adenosine diphosphate (ADP) complex bound to high-affinity catalytic site 1 of chloroplast F1 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 βE197C, βE197D, and βE197S in Chlamydomonas CF1 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 51V hyperfine tensors of VO2+-nucleotide bound to site 1 but not to site 3. Mutations to the Walker homology B aspartate βD262C, βD262H, and βD262T 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 βE197 and βD262 as metal ligands.
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