Domain swapping in inducible nitric-oxide synthase. Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer.
Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA. usiddhan@aecom.yu.edu
U
U
Siddhanta
A
A
Presta
B
B
Fan
D
D
Wolan
D L
DL
Rousseau
D J
DJ
Stuehr
D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D004579:Electron Transport; D004926:Escherichia coli; D005415:Flavins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic; D013059:Spectrum Analysis, Raman
D005415:Flavins; D006418:Heme; D009249:NADP; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II