BINDING OF ATP TO UNCOUPLING PROTEIN OF BROWN FAT MITOCHONDRIA AS STUDIED BY MEANS OF SPIN-LABELED ATP DERIVATIVES

ATP derivatives spin-labeled (SL) at C8, N6, C2' or C3' were employed in binding studies with the uncoupling protein of brown fat mitochondria. Substitution of the ribose strongly impaired binding, whereas labeling of the adenine moiety allowed for tight and functional complex formation. Detailed binding studies with C8-SL-ATP confirmed the known pH and Mg2+ dependence with a stoichiometry of one C8-SL-ATP bound per 66 kDa dimer. Corresponding studies of the uncoupling protein after modification with N-ethylmaleimide or diazobenzene-4-sulfonic acid revealed distinct differences in their effects on nucleotide binding and gating.