Figure 2.
Structure and catalytic mechanisms of functional NOS. (A) NOS monomers are capable of transferring electrons from reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), to flavin-adenine-dinucleotide (FAD) and flavin-mononucleotide (FMN) and have a limited capacity to reduce molecular oxygen to superoxide (O2−•).18,163,164 Monomers and isolated reductase domains can bind calmodulin (CaM), which enhances electron transfer within the reductase domain.165 NOS monomers are unable to bind the cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH4) or the substrate l-arginine and cannot catalyze NO production.163,166 (B) In the presence of haem, NOS can form a functional dimer.163,166 Haem is essential for the interdomain electron transfer from the flavins to the haem of the opposite monomer.165,167 Due to differences in the calmodulin-binding domain, elevated Ca2+ is required for calmodulin binding (and thus catalytic activity) in nNOS and eNOS, whereas calmodulin binds to inducible NOS with high affinity even in the absence of Ca2+. When sufficient substrate l-arginine (l-Arg) and cofactor BH4 are present, intact NOS dimers couple their haem and O2 reduction to the synthesis of NO (fully functional NOS). l-Citrulline (l-Cit) is formed as the byproduct. For clarity, the flow of electrons is only shown from the reductase domain of one monomer to the oxygenase domain of the other monomer. NOS enzymes perform two separate oxidation steps, one to form Nω-hydroxy-l-arginine and a second to convert this intermediate to NO.18 All NOS isoforms contain a zinc ion (Zn) coordinated in a tetrahedral conformation with pairs of CXXXXC motifs at the dimer interface. This site is of major importance for the binding of BH4 and l-arginine. Electron transfer from the reductase domain (*) enables NOS ferric (Fe3+) haem to bind O2 and form a ferrous (Fe2+)-dioxy species. This species may receive a second electron (**) preferentially from BH4 or from the reductase domain. The nature of the resulting oxidized BH4 has been identified as the trihydrobiopterin radical (BH3•) or the trihydropterin radical cation protonated at N5 (BH3•H+). The BH3• radical (or radical cation) can be recycled to BH4 by the NOS itself (using an electron supplied by the flavins). Alternatively, there is evidence that reducing agents such as ascorbic acid (AscH, which is present in cells in millimolar concentrations) can reduce the BH3• radical back to BH4103 (Asc·, ascorbate radical).