Lysine:N6-Hydroxylase: Cofactor Interactions

Recombinant lysine:N6-hydroxylase (rIucD) requires the cofactors FAD and NADPH for its catalytic function of the conversion ofL-lysine to itsN6-hydroxy derivative. In the presence of high concentration of chloride ions (≥ 600 mM), the protein exists in a reversible inactive conformation. Depending on the oxidation state of its thiol functions, rIucD can bind 2,6-dichlorophenol indophenol (DPIP), either covalently or noncovalently, the former type of interaction occurring with protein preparations possessing unmodified thiol groups. Both covalent and noncovalent complexes of rIucD and DPIP appear to be capable of NADPH oxidation in the presence of exogenous DPIP by a phenomenon of exchange of reducing equivalents between the protein-bound dye and that free in the medium. In the presence of FAD, the latter type of complex has been found to function as a diaphorase. The diminution in the catalytic activity of rIucD observed at high concentrations of the flavin cofactor does not appear to be due to an uncoupling of the processes of NADPH oxidation and lysine:N-hydroxylation caused by an exchange of reducing equivalents between the enzyme-bound FAD and that free in the medium.