The Intricate Mechanism of Nitric Oxide Synthase

The biological formation of NO is performed by nitric oxide synthase. For the first half of the mechanism, a consensus understanding has been reached, but not for the second half. Several attempts to reach a mechanism in agreement with experimental observations have been reported, but without success. Therefore, another study of that half-reaction has been made here. The main difficulties have been to explain the observed non-stoichiometric action of the cofactor H₄B, and also why only one outside reduction from NADP is enough. A key new finding here is that the first step involves a protonation of the proximal cysteine of the heme. O₂ then binds to iron and forms O₂H with a proton from the NOH ligand of the substrate NHA. A long-range electron transfer from H₄B leads to the formation of the heme bound H₂O₂, which is followed by a dissociation of the OO bond of H₂O₂. The last step studied here was a release of NO and a reverse electron transfer back to H₄B. The results are in very good agreement with experimental observations, which is in line with previous findings for other redox enzymes using the present methodology.