15-lipoxygenase catalytically consumes nitric oxide and impairs activationof guanylate cyclase

Analysis of purified soybean and rabbit reticulocyte 15-lipoxygenase (15-LO X) and PA317 cells transfected with human 15-LOX revealed a rapid rate of l inoleate-dependent nitric oxide ((NO)-N-.) uptake that coincided with rever sible inhibition of product ((13S)-hydroperoxyoctadecadienoic acid, or (13S )-HPODE) formation. No reaction of (NO)-N-. (up to 2 mu M) with either nati ve (E-red) or ferric LOXs (0.2 mu M) metal centers to form nitrosyl complex es occurred at these (NO)-N-. concentrations. During HPODE-dependent activa tion of 15-LOX, there was consumption of 2 mol of (NO)-N-./mol of 15-LOX. S topped flow fluorescence spectroscopy showed that (NO)-N-. (2.2 mu M) did n ot alter the rate or extent of (13S)-HPODE-induced tryptophan fluorescence quenching associated with 15-LOX activation. Additionally, (NO)-N-. does no t inhibit the anaerobic peroxidase activity of 15-LOX, inferring that the i nhibitory actions of (NO)-N-. are due to reaction with the enzyme-bound lip id peroxyl radical, rather than impairment of (13S)-HPODE-dependent enzyme activation. From this, a mechanism of 15-LOX inhibition by (NO)-N-. is prop osed whereby reaction of (NO)-N-. with EredLOO. generates E-red and LOONO, which hydrolyzes to (13S)-HPODE and nitrite (NO2-). Reactivation of E-red, considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of (NO)-N-. with 15-LOX inhibited (NO)-N-.-depende nt activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated (NO)-N-. production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of (NO)-N-. with lipoxygenase peroxyl radical intermediates will result in modulation of both (NO)-N-. bioavailab ility and rates of production of lipid signaling mediators.