The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H202 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H202-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.
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