Nitric oxide synthase (NOS) catalyzes the oxygenation of arginine to nitric oxide (NO) and citrulline via the intermediate N- hydroxyarginine (NHA). The enzyme is unique in requiring 5 factors for activity, including tetrahydrobiopterin (BH4) which is tightly bound in close proximity to the heme prosthetic group. In addition to NO synthesis, neuronal NOS catalyzes the uncoupled activation of oxygen (especially when BH4 or arginine are not saturating) to form the toxic oxidants, superoxide, peroxide and peroxynitrite. The enzyme is active only in the dimeric form. One of the many roles proposed for NO is that it acts as a neurotransmitter in the brain, and appears to be involved in the physiology of learning and memory. Clarification of the action of BH4 in nitric oxide synthesis is therefore important in understanding neurotransmission and some of its abnormal aspects. It is well established that BH4 has marked physical effects on NOS, including the promotion of arginine binding and stabilization of the active dimeric state. However, little is known of the chemical reactivity of NOS-bound BH4 and the possibility, for example, that it may be a stoichiometric redox reactant in the synthesis of NO. We have directly examined the oxidation of BH4 bound to neuronal NOS and the reduction of its oxidation product(s) under conditions of normal multiple turnovers in which NOS plays a catalytic role, and under single turnover conditions in which NOS acts as a substrate. For these studies we used a sensitive method developed by this group for the specific determination of NOS-bound BH4 in the presence of its oxidation product, quinonoid BH2. It was demonstrated for the first time that during normal multiple turnovers of NOS, NOS- bound BH4 does not remain in a static state, but cycles between the reduced and oxidized forms; the redox state of NOS bound BH4 appears to be determined by the balance between oxidation of BH4 by superoxide, peroxide and peroxynitrite on the one hand, and reductive regeneration of BH4 on the other. These studies demonstrate that this pterin at least has the potential to act in a redox manner as a stoichiometric electron donor in the NOS reaction mechanism. This possibility was examined further under single turnover conditions. Under these conditions, the electron carriers of NOS are not regenerated as they are during multiple turnovers of NOS, so that it is possible to make a quantitative comparison of electrons donated by NOS-bound components such as BH4, and electrons utilized in oxygenation. Previous studies showed that the NOS-bound flavin semiquinone free radical can provide all the electrons required for arginine oxygenation. Although this conclusion argues against BH4 acting as an electron donor for arginine oxygenation, it raises a problem in explaining the mechanism by which the second electron required for this oxygenation can be provided by our preparations of NOS which typically contained only 1 flavin semiquinone free radical per NOS dimer. We and other workers suggested that NOS-bound BH4 might provide this electron. Under single turnover conditions, it was found that the oxidation of BH4 was greater in the presence of arginine than NHA, consistent with the proposal that NOS-bound BH4 is donating an electron for arginine oxygenation to NHA (requiring 2 electrons), but not for NHA oxygenation to citrulline and NO (requiring 1 electron). However, the ratio of the amount of BH4 oxidized to the amount of arginine converted to NHA was not constant with time, as would be expected for this proposal. These combined studies show for the first time that NOS-bound BH4 can undergo redox reactions and at least establish the feasibility of this pterin to act as a stoichiometric electron carrier in the NOS reaction mechanism. In the absence of definitive evidence for or against a stoichiometric redox role of NOS-bound BH4 our best assessment at this time is that NOS- bound BH4 provides a means of removing the toxic oxidants formed during uncoupled oxygen activation by NOS. We plan to follow up on these preliminary studies, using a much simpler single turnover system in which arginine is oxygenated to NHA by the oxyferrous complex of NOS. We collaborated with the Positron Emission Tomography (PET) Department at NIH in examining the inhibitory effects on NOS of six dithiocarbamic acids and their esters. These compounds were selected on the basis of their predicted interaction of one or both sulfur atoms of the dithiocarbamate with the heme iron of NOS and are potentially useful in examining the role of NOS in healthy and pathological states by PET. The compounds may be categorized into two groups: linear compounds, which are free to rotate; and those derived from 2-aminomethylpyrrolidines, which are conformationally restricted. All six compounds showed an ability to inhibit NOS activity by their interaction at the arginine binding site. Further, both conformationally restricted S-methyl esters were unique in showing a substantial loss of inhibitory activity in the presence of added BH4. This indicates that inhibition by these two compounds involves interaction at a second site, namely that involved with BH4 binding, or interaction with the cofactor itself. None of the compounds exhibited remarkably potent inhibition of NOS, and no further structure function studies are planned.
