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

•We studied the effect of BH4 and L-arg on O2 induced radicals in iNOSox.•Parallel single turnover stopped-flow/rapid freeze EPR measurements were performed.•We report differences between O2 intermediates formed by 3 NOSs isoforms.

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 (iNOSox) in the presence or absence of BH4 and/or l-arginine. Fully reconstituted iNOSox (+ BH4, + L-Arg) forms a dimer and yields a typical BH4 radical that indicates coupled reaction. iNOSox (− BH4) remains mainly monomeric and produces exclusively superoxide, that is only marginally affected by the presence of l-arginine. iNOSox (+ 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 (eNOSox) [V. Berka, G. Wu, H.C. Yeh, G. Palmer, A.L. Tsai, J. Biol. Chem. 279 (2004) 32243–32251] and ferrous neuronal NOSox (nNOSox) [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.

Graphical abstractRadical formation mechanism of iNOSox in the presence or absence of BH4 and/or l-arginine.Figure optionsDownload full-size imageDownload as PowerPoint slide