Figure 3.

Detection of an early intermediate in the reaction of the TrpH reaction. A: Stopped-flow traces at 400 nm during the reaction of 125 μM TrpH/Fe(II)·500 μM tryptophan·1000 μM 6MePH₄ with 230 μM (squares), 400 μM (circles), or 650 μM O₂ (triangles). The lines are from a global fit of the data in Figures 2A, 3A, and 4A to the mechanism in Scheme 7 using the rate constants in Table 1. B: Calculated spectra of the initial TrpH/Fe(II)·tryptophan·6MePH₄ complex (1), the intermediate formed in the initial reaction with O₂ (2), and the species formed upon decay of this intermediate (3). TrpH·Fe(II)(350 μM)·500 μM tryptophan·1000 μM 6MePH₄ and 625 μM O₂ (final concentrations) were mixed in the stopped-flow instrument, and the reaction was monitored at 5 nm intervals from 360 to 470 nm. The resulting data were fit globally (Specfit32) to a two step mechanism to obtain the spectra. C: Absorbance changes during the reaction of 350 μM TrpH/Fe(II)·550 μM phenylalanine·550 μM 6MePH₄ with 625 μM (triangles), 450 μM (circles), or 300 μM O₂ (squares). The lines are from a global fit of the data in Figures 2B, 3C, and 4B to the mechanism in Scheme 8 using the rate constants in Table 2. In A and C, only a portion of the points are shown, and the individual traces are offset for clarity. Conditions as described for Figure 1.