Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase

Tetrahydrobiopterin (BH4), not dihydrobiopterin or biopterin, is a critical element required for NO formation by nitric oxide synthase (NOS). To elucidate how BH4 affects eNOS activity, we have investigated BH4 redox functions in the endothelial NOS (eNOS). Redox-state changes of BH4 in eNOS were examined by chemical quench/HPLC analysis during the autoinactivation of eNOS using oxyhemoglobin oxidation assay for NO formation at room temperature. Loss of NO formation activity linearly correlated with BH4 oxidation, and was recovered by overnight incubation with fresh BH4. Thus, thiol reagents commonly added to NOS enzyme preparations, such as dithiothreitol and beta-mercaptoethanol, probably preserve enzyme activity by preventing BH4 oxidation. It has been shown that conversion of L-arginine to N-hydroxy-L-arginine in the first step of NOS catalysis requires two reducing equivalents. The first electron that reduces ferric to the ferrous heme is derived from flavin oxidation. The issue of whether BH4 supplies the second reducing equivalent in the monooxygenation of eNOS was investigated by rapid-scan stopped-flow and rapid-freeze-quench EPR kinetic measurements. In the presence of L-arginine, oxygen binding kinetics to ferrous eNOS or to the ferrous eNOS oxygenase domain (eNOS,,) followed a sequential mechanism: Fe(II) <----> (FeO2)-O-II --> Fe(III) + O-2(.)- Without L-arginine, little accumulation of the (FeO2)-O-II intermediate occurred and essentially a direct optical transition from the Fe(II) form to the Fe(III) form was observed. Stabilization of the (FeO2)-O-II intermediate by L-arginine has been established convincingly. On the other hand, BH4 did not have significant effects on the oxygen binding and decay of the oxyferrous intermediate of the eNOS or eNOS oxygenase domain. Rapid-freeze-quench EPR kinetic measurements in the presence Of L-arginine showed a direct correlation between BH4 radical formation and decay of the (FeO2)-O-II intermediate, indicating that BH4 indeed supplies the second electron for L-arginine monooxygenation in eNOS.