Hydrogen peroxide-coupled cis-diol formation catalyzed by naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (NDOS) catalyzes the NAD(P)H and O₂-dependent oxidation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDOS consists of three protein components: a flavo-[2Fe-2S] reductase (NDR), a ferredoxin electron transfer protein (NDF), and an (αβ)₃ oxygenase (NDO) containing a mononuclear iron site and a Rieske-type [2Fe-2S] cluster in each α-subunit. The active site is built across a subunit-subunit boundary, and each subunit contributes one type of metal center. Our previous studies have shown that NDO with both metal centers reduced is capable of an O₂-coupled single turnover to yield the correct cis-diol product in the absence of the NDR and NDF components (Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953). It is shown here that addition of H₂O₂ to NDO allows reaction with naphthalene to rapidly yield the correct product in a "peroxide shunt" reaction that does not require a reduced Rieske cluster. The mononuclear Fe²⁺ center is oxidized during turnover, while the Rieske cluster remains in the oxidized state. Peroxide shunt turnover in the presence of ¹⁸O-labeled H₂O₂, H₂O, or O₂ shows that both oxygen atoms in the product derive primarily from H₂O₂. The peroxide shunt halts after one turnover despite the presence of excess H₂O₂ and naphthalene, but this is not the result of enzyme inactivation. Rather, it appears that the product cannot be released when the mononuclear iron is in the Fe³⁺ state, blocking a second turnover. This work supports the hypotheses that the cis-dihydroxylation activity of NDOS requires only the NDO component, that a peroxo intermediate is formed during normal catalysis, and that product release requires an additional reducing equivalent beyond those necessary for the first turnover.