The C331A Mutant of Neuronal Nitric-Oxide Synthase Is Defective in Arginine Binding

• DOI: 10.1074/jbc.273.52.34799
• Publication Date: 2002-07-26T15:07:05Z
• Authors: Pavel Martásek; R. Timothy Miller; Qing Liu; Linda J. Roman; John C. Salerno; Catharina Taiko M...; C.S. Raman; Steven S. Gross; Masao Ikeda-Saito; Bettie Sue Siler ...
• Keywords: Omega-N-Methylarginine

It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural functional attributes purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm shifts immediately upon addition a high spin form, C331A-nNOS mutant, exhibited 420 nm. did bind detectable levels either [3H]Nomega-nitro-L-arginine or [3H]BH4, [3H]BH4 reinstated after extended excess L-arginine. On other hand, WT-NOS were identical regard imidazole affinity, CaM rates cytochrome c 2, 6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion low heme concentrations (0.1-10 mM). The estimated Kd two orders magnitude greater than WT-nNOS (>100 microM versus 2-3 microM). Here propose plays important role stabilizing nNOS. Our findings suggest primary dysfunction C331A mutant nNOS, disruption BH4-substrate interactions broadcast from cysteine residue. Prolonged appears cause remodeling protein form similar WT-nNOS, allowing normalized synthetic activity.