IDENTIFICATION OF IRON LIGANDS IN TYROSINE-HYDROXYLASE BY MUTAGENESISOF CONSERVED HISTIDINYL RESIDUES

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substra te. The enzyme is a homotetramer; each monomer contains a single nonhe me iron atom. Five histidine residues are conserved in all tyrosine hy droxylases that have been sequenced to date and in the related eukaryo tic enzymes phenylalanine and tryptophan hydroxylase. Because histidin e has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidine s had been mutated to glutamine or alanine were expressed in Escherich ia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/subunit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with V-max values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/K-m values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of th e H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These resul ts are consistent with H331 and H336 being ligands to the active site iron atom.