Investigating the structure of nucleotide binding sites on the chloroplast F-1-ATPase using electron spin resonance spectroscopy

The relative structure and binding properties of nucleotide binding sites of the latent, nonactivated chloroplast F-1(CF1)ATPase have been investigated by employing ESR spectroscopy using 2-N-3-2',3'-SL-ATP (2-N-3-SL-ATP), a spin-labeled photoaffinity analog of ATP. These results are compared to data obtained in analogous experiments using CF1 that was either depleted of its epsilon-subunit or activated by different methods. Nonactivated (na) CF1 in complex with 2-N-3-SL-ATP exhibits ESR spectra typical for enzyme-bound spin labels. At increased 2-N-3-SL-ATP concentration, a second spectral component for enzyme-bound spin label is observed. The line-shape of the second signal indicates an environment of the enzyme-bound radical that differs from the spin-labeled nucleotide bound first. It can be explained by an enzyme-bound radical bound in a way that allows for higher mobility, e.g. a nucleotide binding site in an ''open'' or ''loose'' conformation. Maximal binding of about 5 mol 2-N-3-SL-ANP per mol of enzyme has been reached. Similar results are obtained when using enzyme that has been either previously depleted of the epsilon-subunit or treated with the reducing agent dithiothreitol (DTT) in the cold. Upon heat-activation of CF1 in the presence of ATP and the presence or absence of the reducing agent DTT, the line-shape of the ESR spectra is observed to be quite different from the non-heat-treated enzyme forms. The ''loose'' or ''open'' nucleotide binding site described above (or at least an environment of the enzyme similar to this site) is observed to be accessible to 2-N-3-SL-ATP even at substoichiometric concentrations of the nucleotide analog. The results presented indicate that the enzyme form of CF1 generated after heat treatment in the presence of ATP with or without DTT exhibits altered binding specificities mainly with respect to the sequence of occupation of two different types of nucleotide binding sites.