Intramolecular electron transfer from biopterin to FeII-O2 complex in nitric oxide synthases occurs at very different rates between bacterial and mammalian enzymes: Direct observation of a catalytically active intermediate.

Nitric oxide synthase (NOS) is a cytochrome P450-type mono‑oxygenase that catalyzes the oxidation of L-arginine to nitric oxide. We previously observed that intramolecular electron transfer from biopterin to Fe2+-O2 in Deinococcus radiodurans NOS (DrNOS) using pulse radiolysis. However, the rate of electron transfer in DrNOS (2.2 × 103 s-1) contrasts with a reported corresponding rate (11 s-1) in a mammalian NOS determined using rapid freeze-quench (RFQ) EPR. We applied pulse radiolysis to Bacillus subtilis NOS (bsNOS) and to rat neural NOS oxygenase domain NOS (mNOS). Concurrently, RFQ EPR was used to trap a pterin radical during single-turnover enzyme reactions of the enzymes. By using the pulse radiolysis method, hydrated electrons (eaq-) reduced the heme iron of NOS enzymes. Subsequently, ferrous heme reacted with O2 to form a Fe2+-O2 intermediate. In the presence of pterin, the intermediate of bsNOS was found to convert to other intermediate in the time range of milliseconds. A similar process was determined to have occurred after pulse radiolysis of the pterin-bound mNOS, though the rate was much slower. The intermediates of all of the NOS enzymes further converted to the original ferric form in the time range of seconds. When using the RFQ method, pterin radicals were formed very rapidly in both DrNOS and bsNOS in the time range of milliseconds. In contrast, the pterin radical in mNOS was observed to form slowly, at a rate of ∼20 s-1.