Solution studies of the aromatic amino acid hydroxylases are consistent with the Few(IV)O intermediate not forming until both the amino acid and tetrahydropterin substrates have bound. Structural studies have shown that the positions of active-site loops differs significantly between the free enzyme and the enzyme-amino acid-tetrahydropterin complex. In tryptophan hydroxylase (TrpH) these mobile loops contain residues 124-134 and 365-371, with a key interaction involving 11e366. The I366N mutation in TrpH results in decreases of 1-2 orders of magnitude in the k(cat) and k(cat)/k(m), values. Single turnover analyses establish that the limiting rate constant for turnover is product release for the wild-type enzyme but is formation of the first detectable intermediate I in catalysis in the mutant enzyme. The mutation does not alter the kinetics of NO binding to the ternary complex nor does it uncouple (FeO)-O-IV formation from amino acid hydroxylation. The effects on the kcat value of wild-type TrpH of changing viscosity are consistent with rate-limiting product release. While the effect of viscosity on the k(cat)/K-O2 value is small, consistent with reversible oxygen binding, the effects on the k(cat)/K-m values for tryptophan and the tetrahydropterin are large, with the latter value exceeding the expected limit and varying with the identity of the viscogen. In contrast, the kinetic parameters of 1366N TrpH show small changes with viscosity. The results are consistent with binding of the amino acid and pterin substrate to form the ternary complex being directly coupled to closure of loops over the active site and formation of the reactive complex. The mutation destabilizes this initial event.