Why do nitric oxide synthases use tetrahydrobiopterin?

We are combining stopped-flow, stop-quench, and rapid-freezing kinetic methods to help clarify the unique redox roles of tetrahydrobiopterin (H4B) in NO synthesis, which occurs via the consecutive oxidation Of L-arginine (Arg) and N-hydroxy-L-arginine (NOHA). In the Arg reaction, H4B radical formation is coupled to reduction of a heme (FeO2)-O-II intermediate. The tempo of this electron transfer is important for coupling (FeO2)-O-II formation to Arg hydroxylation. Because H4B provides this electron faster than can the NOS reductase domain, H4B appears to be a kinetically preferred source of the second electron for oxygen activation during Arg hydroxylation. A conserved Trp (W457 in mouse inducible NOS) has been shown to influence product formation by controlling the kinetics of H,B electron transfer to the (FeO2)-O-II intermediate. This shows that the NOS protein tunes H4B redox function. In the NOHA reaction the role of H4B is more obscure. However, existing evidence suggests that H4B may perform consecutive electron donor and acceptor functions to reduce the (FeO2)-O-II intermediate and then ensure that NO is produced from NOHA. (C) 2002 Elsevier Science Inc. All rights reserved.