tailieunhanh - Báo cáo khoa học: Fast ferrous heme–NO oxidation in nitric oxide synthases

During catalysis, the heme in nitric oxide synthase (NOS) binds NO before releasing it to the environment. Oxidation of the NOS ferrous heme–NO complex by O2 is key for catalytic cycling, but the mechanism is unclear. We utilized stopped-flow methods to study the reaction of O2 with ferrous heme–NO complexes of inducible and neuronal NOS enzymes. | ỊFEBS Journal Fast ferrous heme-NO oxidation in nitric oxide synthases Jesus Tejero Jerome Santolini and Dennis J. Stuehr Department of Pathobiology Lerner Research Institute Cleveland Clinic Foundation Cleveland OH USA Keywords enzyme mechanism heme protein heme-thiolate nitric oxide redox Correspondence D. J. Stuehr Department of Pathobiology NC-22 The Cleveland Clinic Foundation Lerner Research Institute 9500 Euclid Ave. Cleveland OH 44195 USA Fax 1 216 636 0104 Tel 1 216 445 6950 E-mail stuehrd@ Present address Laboratoire de Stress Oxydant et Detoxication iBiTec-S Commissariat a l Energie Atomique Saclay Gif-sur-Yvette Cedex France During catalysis the heme in nitric oxide synthase NOS binds NO before releasing it to the environment. Oxidation of the NOS ferrous heme-NO complex by O2 is key for catalytic cycling but the mechanism is unclear. We utilized stopped-flow methods to study the reaction of O2 with ferrous heme-NO complexes of inducible and neuronal NOS enzymes. We found that the reaction does not involve heme-NO dissociation but instead proceeds by a rapid direct reaction of O2 with the ferrous heme-NO complex. This behavior is novel and may distinguish heme-thiolate enzymes such as NOS from related heme proteins. Received 27 April2009 revised 1 June 2009 accepted 16 June 2009 doi Introduction Nitric oxide NO is a signaling and effector molecule in the neural vascular and immune systems 1 . Three related NO synthases NOS EC generate NO from L-arginine L-Arg in mammals inducible NOS iNOS endothelial NOS eNOS and neuronal NOS nNOS 2-5 . NOS-like enzymes also exist in some Gram-positive bacteria 6 7 . All mammalian NOS are homodimers and each subunit consists of an N-terminal oxygenase domain that binds iron protoporphyrin IX heme 6R -5 6 7 8-tetrahydro-L-biop-terin H4B and L-Arg and a C-terminal reductase domain that binds FmN fad and NADPH. The two domains are connected to one another by an intervening