The role of tetrahydrobiopterin in the activation of oxygen by nitric-oxide synthase

We have studied the reaction of reduced nitric-oxide synthase (NOS) with mo lecular oxygen at -30 degrees C. In the first reaction cycle (from L-Arg to hydroxy-L-Arg), an oxygen adduct complex formed rapidly. Experiments in th e absence of the reductase domain demonstrated that this complex was then f urther reduced by one electron stemming from the cofactor tetrahydrobiopter in (BH4). Spectral evidence suggested an iron(IV) porphyrin pi-cation radic al as an intermediate. The nature of the oxidized BH4 was identified by EPR as a BH3(.) radical. Within the second cycle (from hydroxy-L-Arg to citrul line and NO), an iron(III)-NO complex could be identified clearly by its sp ectral characteristics. The strict requirement of BH4 for its formation sug gests that BH4 plays a redox role, although transient, also in the second r eaction cycle. (C) 2000 Elsevier Science S.A. All rights reserved.