Structure and function of human hemoglobin covalently labeled with periodate-oxidized adenosine triphosphate

Periodate-oxidized adenosine triphosphate (o-ATP), a ribose ring-opened dialdehyde derivative of ATP, reacts specifically with human deoxyhemoglobin to give a single major covalently modified product after reduction with sodium borohydride. This product, designated di-ATP Hb, was isolated using ion-exchange chromatography and shown to have incorporated two molecules of o-ATP/tetramer. Peptide mapping and x-ray crystallography at 2.8-Å resolution indicate that a covalent adduct is formed between the ligand and residues Lys-82 EF6 of each β chain in the organic phosphate-binding site of the molecule. di-ATP Hb exhibits a significantly decreased oxygen affinity (P₅₀ = 20.8 mm Hg versus 5.8 mm Hg control; 50 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol, pH 7.4, 0.1 M C, 20°C). The subunit cooperativity of di-ATP Hb is also reduced (n(max) = 1.9 versus 2.7 control).