We studied catalysis by tetrahydrobiopterin (H4B)-free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation toN ω-hydroxy-l-Arg (NOHA) and citrulline in both NADPH- and H2O2-driven reactions. Citrull…