Tryptophan hydroxylase (TrpH) uses a non-heme mononuclear iron center to catalyze the tetrahydropterin-dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH•Fe(II), TrpH•Fe(II) •tryptophan, TrpH•Fe(II)•6MePH4•tryptophan, and TrpH•Fe(II)•6MePH4•phenylalanine complexes with O 2 were monitored by stopped-flow absorbance spectroscopy and rapid quench methods. The second-order rate constant for the oxidation of TrpH•Fe(II) has a value of 104 M-1 s-1 irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH•Fe(II)•6MePH4•tryptophan complex with oxygen are consistent with the initial step being reversible binding of oxygen, followed by the formation with a rate constant of 65 s-1 of an intermediate I that has maximal absorbance at 420 nm. The rate constant for decay of I, 4.4 s-1, matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quench analyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 s-1 and that overall turnover is limited by a subsequent slow step, presumably product release, with a rate constant of 0.2 s-1. All of the data with tryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate, the reaction can be described by three steps: a second-order reaction with oxygen to form I, decay of I as tyrosine forms, and slow product release.
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