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, O₂ activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-l-biopterin cofactor (H₄ B), and the H₄ B radical must be reduced back to H₄ B in order for NOS to continue catalysis. Although an NADPH-derived electron is used to reduce the H₄ B radical, how this occurs is unknown. We hypothesized that the NOS flavoprotein domain might reduce the H₄ 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 H₄ 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-H₄ B radical formed in neuronal NOS (nNOS) during Arg hydroxylation. We used several nNOS variants that supported either a slower or faster than normal rate of ferric heme reduction. We found that the rates and extents of nNOS heme reduction correlated well with the rates and extents of 5-methyl-H₄ B radical reduction among the various nNOS enzymes. This supports a model where the heme porphyrin transfers an electron from the NOS flavoprotein to the H₄ B radical formed during catalysis, revealing that the heme plays a dual role in catalyzing O₂ activation or electron transfer at distinct points in the reaction cycle.