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

Matt D. Wolfe, John D. Lipscomb

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92 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)829-835
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number2
DOIs
StatePublished - Jan 10 2003

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