Tetrahydrobiopterin redox cycling in nitric oxide synthase: evidence supports a through‐heme electron delivery

The nitric oxide synthases ( NOS ) catalyze a two‐step oxidation of l ‐arginine (Arg) to generate NO . In the first step, O 2 activation involves one electron being provided to the heme by an enzyme‐bound 6 R ‐tetrahydro‐ l ‐biopterin cofactor (H 4 B), and the H 4 B radical must be reduced back to H 4 B in order for NOS to continue catalysis. Although an NADPH ‐derived electron is used to reduce the H 4 B radical, how this occurs is unknown. We hypothesized that the NOS flavoprotein domain might reduce the H 4 B radical by utilizing the NOS heme porphyrin as a conduit to deliver the electron. This model predicts that factors influencing NOS heme reduction should also influence the extent and rate of H 4 B radical reduction in kind. To test this, we utilized single catalytic turnover and stop‐freeze methods, along with electron paramagnetic resonance spectroscopy, to measure the rate and extent of reduction of the 5‐methyl‐H 4 B radical formed in neuronal NOS (n NOS ) during Arg hydroxylation. We used several n NOS variants that supported either a slower or faster than normal rate of ferric heme reduction. We found that the rates and extents of n NOS heme reduction correlated well with the rates and extents of 5‐methyl‐H 4 B radical reduction among the various n NOS enzymes. This supports a model where the heme porphyrin transfers an electron from the NOS flavoprotein to the H 4 B radical formed during catalysis, revealing that the heme plays a dual role in catalyzing O 2 activation or electron transfer at distinct points in the reaction cycle.