The nitric oxide synthases are large, modular, dimeric enzymes composed of a reductase domain, which is related to cytochrome P450 reductase, and a structurally unique oxygenase domain containing a Cys-ligated haem. Both the neuronal and endothelial isoforms are activated by the reversible binding of calmodulin (CaM) at elevated intracellular Ca2+ levels to produce NO as part of a number of cell signalling pathways. CaM binds to the linker region between the two domains and activates the enzyme by inducing intramolecular electron transfer. Protein-engineering experiments have shown that a series of unusual autoinhibitory inserts found only in the CaM-dependent NOS isoforms control both CaM binding and the structural rearrangement it induces. These lie in the reductase domain of the enzyme and include a 40-amino-acid autoinhibitory loop in the FMN-binding module, a 30-amino-acid extension to the C-terminus and the CaM-binding site itself. The substrate (NADPH) also plays an important role in defining the CaM-dependence of the reductase domain by inducing a tight conformational lock in the absence of CaM. Both the substrate and the conformational lock appear to be released on CaM binding; the resultant domain mobility leads to activation.
- cytochrome c
- electron transfer
- nitric oxide
- reductase domain
Enzyme Mechanism – A Structural Perspective, a Biochemical Society Focused Meeting held at St. Andrews University, 12–14 January 2003
Abbreviations used: NOS, nitric oxide synthase; nNOS, iNOS, eNOS, neuronal, induced and endothelial NOS respectively; nNOSrd, nNOS reductase domain; nNOStr, nNOS truncation mutant; H4B, (6R)-5,6,7,8-tetrahydro-l-biopterin; CaM, calmodulin; k2nd, second-order rate constant.
- © 2003 Biochemical Society