Superoxide generation by endothelial nitric oxide synthase: The influence of cofactors

Abstract

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium͞calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium͞calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, N G -hydroxy-L-arginine, L-thiocitrulline, and L-N G -monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH 4 ) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH 4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of f lavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH 4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

“…Loss of BH4 can uncouple eNOS synthesis [17]. It is hypothesised that in this uncoupled state, electron flow from the reductase domain to the oxygenase domain of eNOS is diverted to molecular oxygen rather than L-arginine [6,30]. This leads to preferential SO production over NO production.…”

“…This redox-active compound can be pro-oxidant, directly leading to superoxide generation that reduces NO bioactivity [42,43]; it can also be anti-oxidant by simply removing the free radical species, i.e. peroxynitrite, rather than having any specific effect on NOS activity or regulation [5,6,31,44].…”

“…Simultaneously, the increased SO production inhibits eNOS NO biosynthesis [3,4] and degrades NO [5], thus further contributing to diabetic endothelial dysfunction. Under these conditions, eNOS 'uncoupling' results in SO formation over NO production [6][7][8].…”

“…BH4 is finally produced through further steps catalysed by 6-pyruvol-tetrahydropterin synthase and sepiapterin reductase [10]. BH4 appears to facilitate electron transfer from the reductase domain to the oxygenase domain of NOS, and maintains the haem prosthetic group in its redox active form [6,7,11]. Furthermore, BH4 also promotes and possibly stabilises eNOS protein in the active homodimer formation [12][13][14][15][16].…”

Protective effect of arginine on oxidative stress in transgenic sickle mouse models

“…However, there is no definitive data on the status of antioxidant enzyme and lipid peroxidation according to the disease severity, as well as on the effect of NO replenishment. Among the likely sources of O 2 •− in SCD are plasma membrane NADPH oxidase [12,44], enzyme NO synthase due to depleted substrate arginine [40] and increased plasma xanthine oxidase (XO) activity [3,41].…”