Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O 2 , 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 ␣ 3 ␤ 3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe 2؉ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O 2 . However, upon reduction of the Rieske cluster and exposure to naphthalene and O 2 , ϳ0.85 cisdiol 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.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 (1-4). These enzymes initiate the biodegradation of some of the most recalcitrant aromatic compounds that enter the environment from both natural and industrial sources, making their study of great importance for progress in bioremediation practices (5, 6). In addition, the inherent enantio-and regiospecificity of these enzymes make them useful for synthetic applications (7,8). Several of these multicomponent dioxygenases are known that differ in the number of components and the number and type of subunits in the oxygenase component that contains the active site. However, 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. NCIB 9816-4, which catalyzes the reaction shown in Scheme 1 to yield (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (9).The complete NDOS consists of three protein components: the 36-kDa reductase protein (NDR) that contains a FAD molecule and a plant-type [2Fe-2S] cluster, the 14-kDa ferredoxin electron transfer protein (NDF) that contains a [2Fe-2S] Rieske cluster, an...

Previous studies have shown that narL+ is required for nitrate induction of nitrate reductase synthesis and for nitrate inhibition of fumarate reductase synthesis in Escherichia coli. We cloned narL on a 5.1-kilobase HindlIl fragment. Our clone also contained a previously unidentified gene, which we propose to designate as narX, as well as a portion of narK. Maxicell experiments indicated that narL and narX encode proteins with approximate Mrs of 28,000 and 66,000, respectively. narX insertion mutations reduced nitrate reductase structural gene expression by less than twofold. Expression of 40(narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent offnr. Expression of 'F(narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations. A 4(narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ andfnr'. Analysis of these operon fusions indicated that narL and narX are transcribed counterclockwise with respect to the E. coli genetic map and that narK is transcribed clockwise.Escherichia coli will use several terminal electron acceptors for respiration, including oxygen, nitrate (NO3-), and fumarate. Use of these electron acceptors is hierarchical: aerobiosis prevents nitrate and fumarate reductase synthesis, and nitrate prevents the anaerobic formation of fumarate reductase (18,21,46,53; V. Stewart, Microbiol. Rev., in press).The nitrate reductase enzyme complex consists of three membrane-bound subunits and also contains molybdenum cofactor and heme (Stewart, in press). The structural genes for the polypeptide subunits are termed narC (formerly narG [2]), narH, and narI and are organized in an operon at the nar (formerly chlC) locus at 27 min on the E. coli genetic map (2,9,16,32,38,51; Stewart, in press). Genetic studies indicate that the organization of this operon is hemApromoter-narC-narH-narl -trp (9, 51).Nitrate reductase synthesis is induced by anaerobiosis, and during anaerobic growth, it is further induced by nitrate (46). Studies with F(nar-lac) operon fusions (14, 50) and measurements of nar mRNA synthesis (28) indicate that both anaerobiosis and nitrate act to control narCHI operon transcription. The effect of anaerobiosis is thought to be mediated through FNR (the fnr gene product), a positiveacting transcription factor required for the synthesis of several anaerobic respiratory enzymes (26, 43; Stewart, in press) including nitrate reductase (14, 50) and fumarate reductase (18, 21). The physiological signal for FNR-mediated transcription activation is unknown (56).A mutation termed narL215::TnlO prevents nitrate induction of nitrate reductase synthesis and of F(narC-lacZ) expression (50) but has no effect on control by anaerobiosis. This mutation is complemented in trans by a specialized transducing bacteriophage, k pchlC3 (50). Genetic mapping indicates that narL is located upstream of the nar operon, between hemA and narCHI; a gene of unknown function, narK, maps between narL and...

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The Rieske iron-sulfur proteins have reduction potentials ranging from -150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein structure. However, the increasing number of available crystal structures for Rieske iron-sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F9816-4), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F9816-4, determined using a highly oriented pyrolytic graphite electrode, was -150+/-2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F9816-4 is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.

(40,43). The organization of the nar locus is shown in Fig. 1.Nitrate reductase synthesis is subject to dual positive regulation (38,40). Anaerobic induction requires the fnr gene product, Fnr (reviewed in reference 41), while nitrate induction requires the narL gene product, NarL (40,43). Li and DeMoss (26,27) in the same direction (Fig. 1) Fig. 1).Structures of narX and narL and their predicted products. The narX open reading frame begins with ATG at position 188 and terminates with TAA at position 1822 (Fig. 2). The narL open reading frame begins with ATG at position 1815 and terminates with TGA at position'