Abstract
Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O2, and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the alpha3beta3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe2+ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O2. However, upon reduction of the Rieske cluster and exposure to naphthalene and O2, approximately 0.85 cis-diol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.
Highlights
Rieske nonheme iron dioxygenases catalyze a remarkable reaction in which dioxygen is cleaved and both atoms are inserted across a double bond of an unactivated aromatic nucleus to yield a cis-dihydrodiol [1,2,3,4]
Ever, all of the enzyme systems share the common features of a reductase component that can accept and pass on two electrons from reduced pyridine nucleotide, an electron transport system that may be encompassed into the reductase, and an oxygenase that has both Rieske [2Fe-2S] clusters and mononuclear iron centers
One of the most thoroughly studied of the Rieske nonheme iron dioxygenases is naphthalene 1,2-dioxygenase (NDOS)1 isolated from Pseudomonas sp
Summary
Chemicals—All chemicals were purchased from either Sigma, Aldrich, or Matheson and used without purification. (ϩ)-cis-(1R,2S)-Dihydroxy-1,2-dihydronaphthalene (naphthalene cis-dihydrodiol) was prepared as described previously [26]. The reaction was quenched by transferring the mixture to an Eppendorf tube, which was heated by immersion in a 90 °C water bath for 2 min This method completely denatured the protein, as judged by a complete loss of the characteristic red-brown color of the enzyme(s) and total loss of activity. The syringes were loaded with solutions as described above (with or without naphthalene), but the reaction was monitored with a diode array detector or a single wavelength detector at 464 nm. Spin quantitation of the g ϭ 4.3 species formed during single turnover was performed by using the Fe3ϩ-EDTA standard and collecting both the experimental and standard spectra at 20 K At this temperature, for S ϭ 5/2 species with E/D ϭ 1/3 and Ϫ1Ͻ D Ͻ 1, the middle Kramer’s doublet (MS ϭ Ϯ3/2) is approximately maximally occupied. Using the temperature dependence of the g ϭ 4.3 species of the NDO single turnover, we have determined D to be 0.76 Ϯ 0.02 cmϪ1
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
