Nitric oxide synthase stabilizes the tetrahydrobiopterin cofactor radical by controlling its protonation state.

Nitric oxide synthase (NOS), a homodimeric enzyme with a flavin reductase domain and a P450-type heme-containing oxygenase domain, catalyzes the formation of NO from L-arginine, NADPH, and O(2) in a two-step reaction sequence. In the first step, a tetrahydrobiopterin (H(4)B) cofactor bound near one of the heme propionate groups acts as an electron donor to the P450-type heme active site, yielding a one-electron oxidized radical that is subsequently re-reduced. In solution, H(4)B undergoes two-electron oxidation, showing that the enzyme significantly alters the proton- and electron-transfer properties of the cofactor. Multifrequency EPR and ENDOR spectroscopy were used to determine magnetic parameters, and from them the (de)protonation state of the H(4)B radical in the oxygenase domain dimer of inducible NO synthase that was trapped by rapid freeze quench. From 9.5 and 330-416 GHz EPR and from 34 GHz (1)H ENDOR spectroscopy, the g tensor of the radical and the hyperfine tensors of several N and H nuclei in the radical were obtained. Density functional theory calculations at the PBE0/EPR-II level for H(4)B radical models predict different spin density distributions and g and hyperfine tensors for different protonation states. Comparison of the predicted and experimental values leads to the conclusion that the radical is cationic H(4)B(*+), suggesting that NOS stabilizes this protonated form to utilize the cofactor in a unique dual one-electron redox role, where it can deliver an electron to the active site for reductive oxygen activation and also remove an electron from the active site to generate NO and not NO(-). The protein environment also prevents further oxidation and subsequent loss of function of the cofactor, thus enabling the enzyme to perform the unusual catalytic one-electron chemistry.