On stopped-flow analysis of aliphatic aldoxime dehydratase (OxdA), a novel hemoprotein, a spectrum derived from a reaction intermediate was detected on mixing ferrous OxdA with butyraldoxime; it gradually changed into that of ferrous OxdA with an isosbestic point at 421 nm. The spectral change on the addition of butyraldoxime to the ferrous H320A mutant showed the formation of a substrate-coordinated mutant, the absorption spectrum of which closely resembled that of the above intermediate. These observations and the resonance Raman investigation revealed that the substrate actually binds to the heme in OxdA, forming a hexa-coordinate low-spin heme.