Transfer intermediate in ferric holoShpM66A/apoHtsAH229A reaction but not in holoShpM153A/apoHtsAH229A reaction. HoloShp mutant was mixed with apoHtsA mutant in 20 mM Tris-HCl in a stopped-flow spectrophotometer. The arrows indicate the direction of the spectral shift with time. A, spectral shift in the reaction of 1.2 μM holoShpM66A with 13 μM apoHtsAH2299A. The black and colored curves are in the initial fast and subsequent slow phases, respectively. B, time course of ΔA420 and ΔA600 for the reaction in panel A. The black traces and gray curves represent the observed data and double exponential fitting curves, respectively. C, spectral shift in the reaction of 0.9 μM holoShpM153A with 13 μM apoHtsAH2299A. D, time course of ΔA410 and ΔA600 for the reaction in panel C. The black traces represent the observed data, and the gray curve of ΔA410 is the single exponential fitting curve.