COMPARATIVE FUNCTIONING OF DIHYDRO-PTERIN AND TETRAHYDROPTERIN IN SUPPORTING ELECTRON-TRANSFER, CATALYSIS, AND SUBUNIT DIMERIZATION IN INDUCIBLE NITRIC-OXIDE SYNTHASE

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 c an support NO synthesis. Here, we characterize pterin-free inducible N OS (iNOS) and iNOS reconstituted with eight different tetrahydro-or di hydropterins to elucidate how changes in pterin side-chain structure a nd ring oxidation state regulate iNOS. Seven different enzyme properti es that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxida tion stale (i.e., they required fury reduced tetrahydropterins) and we re independent of side chain structure: NO synthesis and the ability t o increase heme-dependent NADPH oxidation in response to substrates. I n contrast, five properties were exclusively dependent on pterin side- chain structure or stereochemistry and were independent of pterin oxid ation state: pterin binding affinity, and its ability to shift the hem e iron to its high-spin stale. stabilize the ferrous heme iron coordin ation structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer, These results clarify how structural versus re dox properties of the pterin impact on its multifaceted role in iNOS f unction, In addition, the data reveal that during NO synthesis all pte rin-dependent steps up to and including heme iron reduction can take p lace independent of the pterin ring oxidation state, indicating that t he requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.