Comparison of oxygen-induced radical intermediates in iNOS oxygenase domain with those from nNOS and eNOS

作者：Berka Vladimir^*; Liu Wen; Wu Gang; Tsai Ah Lim

来源：Journal of Inorganic Biochemistry, 2014, 139: 93-105.

DOI：10.1016/j.jinorgbio.2014.06.011

• Nitric oxide synthase
• NOS oxygenase domain
• Rapid-freeze quench EPR
• Oxygen induced radicals
• Reactive oxygen species
• Superoxide

Inducible nitric-oxide synthase (iNOS) produces the reactive oxygen and nitrogen species (ROS/RNS) involved in bacteria killing and is crucial in the host defense mechanism. However, high level ROS/RNS can also be detrimental to normal cells and thus their production has to be tightly controlled. Availability or deficiency of tetrahydrobiopterin (BH4) cofactor and L-arginine substrate controls coupling or uncoupling of NOS catalysis. Fully coupled reaction, with abundant BH4 and L-arginine, produces NO whereas the uncoupled NOS (in the absence of BH4 and/or L-arginine) generates ROS/RNS. In the current work we focus on direct rapid freeze EPR to characterize the structure and kinetics of oxygen-induced radical intermediates produced by ferrous inducible NOS oxygenase domain (iNOSo) in the presence or absence of BH4 and/or L-arginine. Fully reconstituted iNOS(ox) (+BH4, +L-Arg) forms a dimer and yields a typical BH4 radical that indicates coupled reaction. iNOS(ox) (BH4) remains mainly monomeric and produces exclusively superoxide, that is only marginally affected by the presence of L-arginine. iNOS (+BH4, L-Arg) exists as a monomer/dimer mixture and yields both BH4 radical and superoxide. Present study is a natural extension of our previous work on the ferrous endothelial NOSox (eNOS(ox)) [V. Berka, G. Wu, H.C. Yeh, G. Palmer, A.L. Tsai, J. Biol. Chem. 279 (2004) 32243-32251] and ferrous neuronal NOSox (nNOS(ox)) [V. Berka, L.H. Wang, A.L Tsai, Biochemistry 47 (2008) 405-420]. Overall, our data suggests different regulatory roles of L-arginine and BH4 in the production of oxygen-induced radical intermediates in NOS isoforms which nicely serve individual functional role. Published by Elsevier Inc.

• 出版日期2014-10