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Stopped‐flow spectrophotometric and resonance Raman analyses of aldoxime dehydratase involved in carbon–nitrogen triple bond synthesis

2005; Wiley; Volume: 579; Issue: 6 Linguagem: Inglês

10.1016/j.febslet.2005.01.037

1873-3468

Ken-Ichi Oinuma, Hideyuki Kumita, Takehiro Ohta, Kazunobu Konishi, Yoshiteru Hashimoto, Hiroki Higashibata, Teizo Kitagawa, Yoshitsugu Shiro, Michihiko Kobayashi,

Protein Interaction Studies and Fluorescence Analysis

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.