EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature.
Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.
Kominami
S
S
Faculty of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima, 739-8521, Japan. kominam@ipc.hiroshima-u.ac.jp
Yamazaki
T
T
Koga
T
T
Hori
H
H
D000818:Animals; D003080:Cold Temperature; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005290:Ferric Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics; D052248:Nitric Oxide Synthase Type I; D010777:Photochemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D005290:Ferric Compounds; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat