Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to Fe^II–O₂ Complex

Abstract

Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of l-arginine (Arg) to nitric oxide (NO) through a reaction intermediate N-hydroxy-l-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from Deinococcus radiodurans was investigated using pulse radiolysis. Radiolytically generated hydrated electrons reduced the heme iron of NOS within 2 μs. Subsequently, ferrous heme reacted with O to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 10 M s. In the tetrahydrofolate (HF)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 10 s. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of HF, this step did not proceed, suggesting that HF was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted to the original ferric form with a first-order rate constant of 4 s. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s). These data suggested that a common catalytically active intermediate involved in the substrate oxidation of both Arg and NHA may be formed during catalysis. In addition, we investigated the solvent isotope effects on the kinetics of the intermediate after pulse radiolysis. Our experiments revealed dramatic kinetic solvent isotope effects on the conversion of the intermediate to the ferric form, of 10.5 and 2.5 for Arg and NHA, respectively, whereas the faster phases were not affected. These data suggest that the proton transfer in DrNOS is the rate-limiting reaction of the intermediate with the substrates.

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

“…This process was similar to that observed during single-turnover reactions in stopped-flow measurements. 208 These results indicated that the intermediate observed after pulse radiolysis is the same intermediate during hydroxylation of Arg.…”

“…Schematic diagram showing intramolecular electron transfer from biopterin to Fe 2+ –O 2 (PDB code 1M7B). Reproduced from ref . Copyright 2018 American Chemical Society.…”

“…This was also supported by solvent isotope effects (10.5) measured on the intermediate kinetics after pulse radiolysis. 208 These results strongly suggested that the ratelimiting step of substrate oxidation involved proton-assisted O− O bond cleavage.…”

“…207 To follow the reaction intermediates for substrate oxidation, we applied pulse radiolysis techniques to NOS from Deinococcus radiodurans (DrNOS). 208 Scheme 1 shows the reaction scheme after pulse radiolysis of DrNOS. The ferric heme iron of NOS was reduced to form the ferrous form very rapidly.…”

“…Pulse radiolysis experiments allow the extremely rapid reduction the heme iron of hemoproteins, enabling observation of O 2 or CO binding process to the ferrous heme. This method reveals the fast rate constants of O 2 with DrNOS (2.8 × 10 8 M –1 s –1 ) and the Met95Ala mutant of E. coli direct oxygen sensor ( Ec DOS) (1.1 × 10 7 M –1 s –1 ), the reactions of which reached completion within the instrumental dead time of the stopped-flow method. In early studies of the pulse radiolysis method, the quaternary structure of methemoglobin was characterized by monitoring the kinetics of O 2 binding to the valence hybrid. − …”