Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H+-ATPase- from Bacillus PS3

The properties of the nucleotide binding sites in the isolated beta and alp ha subunits of H+-ATPase from Bacillus PS3 (TF1) have been examined by stud ying the EPR properties of bound VO2+, which is a paramagnetic probe for th e native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with diff erent mixtures of both alpha and beta subunits, and with the catalytic alph a(3)beta(3)gamma subcomplex have been characterized by a combination of EPR , ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by V-51 hyperfine co upling parameters (A(parallel to) = 168 x 10(-4) cm(-1) and A(perpendicular to) = 60 x 10(-4) cm(-1)) that suggest that VO2+ binds to the isolated bet a subunit with at least one nitrogen ligand. Results obtained for the analo gous VO2+ complex with the isolated ex subunit are virtually identical. ESE EM and HYSCORE spectra are also reported and are similar for both complexes , indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A(parallel to)(N-14) = 4.44 MHz, and A(perpendicular to)(N-14) = 4.3 MHz and quadrupole coupling parameters e(2)qQ approximate to 3.18 MHz and eta approximate to 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alp ha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ boun d to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha(3)beta(3) hexamer has a second nitrog en ligand with magnetic properties similar to those of a histidine imidazol e. This situation is analogous to that in the alpha(3)beta(3)gamma subcompl ex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S. , Sigalat, C,, Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 1 4281-14293]. These data are-interpreted in terms of only partially structur ed nucleotide binding sites in the isolated beta and alpha subunits as-comp ared to fully structured nucleotide binding sites in the alpha(3)beta(3) he terohexamer, the alpha(3)beta(3)gamma subcomplex, and the whole TF1 ATPase.