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The vital signalling molecule nitric oxide is produced by mammalian NOS enzymes in two steps. L-arginine is converted to N-hydroxy-L-arginine NOHA, which is converted to NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor tetrahydrobiopterin (H4B) activates dioxygen at the heme site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochromes P450, a heme-iron oxo species may be formed, or direct reaction between a heme-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. Here we analyse the two reaction steps using the G586S mutant of nNOS, which introduces an additional H-bond in the active site and provides an additional proton source. In the mutant, H4B activates dioxygen as in the wild-type enzyme, but an interesting intermediate heme species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-Arg) indicating that the extra H-bond enables it to discriminate between the two monooxygenation steps. This implies that the two steps follow different chemical mechanisms.