The "bridging" aspartate 178 in phthalate dioxygenase facilitates interactions between the Rieske center and the iron(II)--mononuclear center

Phthalate dioxygenase (PDO) and its reductase are parts of a two-component Rieske dioxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate (DHD). Aspartate D178 in PDO, located near its ferrous mononuclear center, is highly conserved among Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer between the mononuclear iron and Rieske center in naphthalene dioxygenase [Parales et al. (1999) J. Bacteriol. 181, 1831-1837] and in substrate binding and oxygen reactivity in anthranilate dioxygenase [Beharry et al. (2003) Biochemistry 42, 13625-13636]. The effects of substituting D178 in PDO with alanine or asparagine on the reactivity of the Rieske centers, phthalate hydroxylation, and coupling of Rieske center oxidation to DHD formation were studied previously [Pinto et al. (2006) Biochemistry 45, 9032-9041]. This work describes effects that D178N and D178A substitutions have on the interactions between the Rieske and mononuclear centers in PDO. The mutations affected protonation of the Rieske center histidine and conformation of subunits within the PDO multimer to create a more open structure with more solvent-accessible Rieske centers. When the Rieske centers in PDO were oxidized, D178N and D178A substitutions disrupted communication between the Rieske and Fe-mononuclear centers. This was shown by the lack of perturbations of the UV-vis spectra on phthalate binding to the D178N and D178A variants, as opposed to that observed in WT PDO. However, when the Rieske center was in the reduced state, communication between the centers was not disrupted. Phthalate binding similarly affected the rates of oxidation of the reduced Rieske center in both WT and mutant PDO. Nitric oxide binding at the Fe(II)-mononuclear center, as detected by EPR spectrometry of the Fe(II) nitrosyl complex, was regulated by the redox state of the Rieske center. When the Rieske center was oxidized in either WT or D178N PDO, NO bound to the mononuclear iron in the presence or absence of phthalate. However, when the Rieske center was reduced, NO bound only when phthalate was present. These findings are discussed in terms of the "communication functions" performed by the bridging Asp-178.     

Distal end of 105-125 loop - A putative reductase binding domain of phthalate dioxygenase

The phthalate dioxygenase system consists of the dioxygenase, PDO, which contains a Rieske [2Fe-2S] center and a Fe(II)-mononuclear center, and the reductase, PDR. Involvement of the distal end of the 105-125 loop of PDO in its interaction with PDR was tested by substituting charged residues in the loop with alanines and by replacing the conserved tryptophan-94. Compared to wild-type PDO, all variants had lower catalytic activity and the Rieske centers were reduced more slowly by reduced PDR. The rates of oxidation of the Rieske centers by oxygen, which represent electron transfer between the Rieske and mononuclear centers, were essentially unaffected. These results suggest that positively charged residues of the distal end of the 105-125 loop are collectively involved in PDR binding with the PDO. Contrary to expectations, Trp94 variants were not directly involved in electron transfer between PDR and PDO. The tryptophan appears to have mainly a structural role, apparently preserving the hydrophilic environment of the Rieske center.     

Chemistry of the catalytic conversion of phthalate into its cis-dihydrodiol during the reaction of oxygen with the reduced form of phthalate dioxygenase

The phthalate dioxygenase system, a Rieske non-heme iron dioxygenase, catalyzes the dihydroxylation of phthalate to form the 4,5-dihydro-cis-dihydrodiol of phthalate (DHD). It has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one mononuclear Fe(II) center per monomer, and a reductase (PDR) that contains flavin mononucleotide (FMN) and a plant-type ferredoxin [2Fe-2S] center. This work shows that product formation in steady-state reactions is tightly coupled to electron delivery, with 1 dihydrodiol (DHD) of phthalate formed for every 2 electrons delivered from NADH. However, in reactions of reduced PDO with O(2), only about 0.5 DHD is formed per Rieske center that becomes oxidized. Although the product forms rapidly, its release from PDO is slow in these reactions with oxygen that do not include reductase and NADH. EPR data show that, at the completion of the oxidation, iron in the mononuclear center remains in the ferrous state. In contrast, naphthalene dioxygenase (NDO) [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953] and benzoate dioxygenase (BZDO) [Wolfe, M. D., Altier, D. J., Stubna, A., Popescu, C. V., Munck, E., and Lipscomb, J. D. (2002) Biochemistry, 41, 9611-9626], related Rieske non-heme iron dioxygenases, form 1 DHD per Rieske center oxidized, and the mononuclear center iron ends up ferric. Thus, both electrons from reduced NDO and BZDO monomers are used to form the product, whereas only the reduced Rieske centers in PDO become oxidized during production of DHD. This emphasizes the importance of PDO subunit interaction in catalysis. Electron redistribution was practically unaffected by the presence of oxidized PDR. A scheme is presented that emphasizes some of the differences in the mechanisms involved in substrate hydroxylation employed by PDO and either NDO or BZDO.     

Rates of the phthalate dioxygenase reaction with oxygen are dramatically increased by interactions with phthalate and phthalate oxygenase reductase

The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form its cis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDO(red)) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O(2) (1.4 x 10(-3) s(-1) at 125 microM O(2) and 22 degrees C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDR(ox) to reduced PDO-APO increases the reactivity of the Rieske center with O(2). When no PDR or phthalate is present, the oxidation of the Rieske center in native PDO(red) [which contains Fe(II) at the mononuclear site] occurs in two phases (approximately 1 and 0.1 s(-1) at 125 mM O(2), 23 degrees C), both much faster than in the absence of Fe(II), presumably because in this case O(2) reacts at the mononuclear Fe(II). Addition of PDR(ox) to native PDO(red) resulted in a large fraction of the Rieske center being oxidized at 5 s(-1), and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s(-1). With both PDR(ox) and phthalate present, most of the PDO(red) (approximately 80-85%) oxidizes at 42 s(-1), with the remaining oxidizing at approximately 5 s(-1). Thus, the binding of phthalate or PDR(ox) to PDO(red) each results in greater reactivity of PDO with O(2). The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.     

Histidine ligand protonation and redox potential in the rieske dioxygenases: role of a conserved aspartate in anthranilate 1,2-dioxygenase

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be approximately 100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.     

Electron paramagnetic resonance measurements of the ferrous mononuclear site of phthalate dioxygenase substituted with alternate divalent metal ions: direct evidence for ligation of two histidines in the copper(II)-reconstituted protein.

The metalloenzyme phthalate dioxygenase (PDO) contains two iron-based sites. A Rieske-type [2Fe-2S] cluster serves as an electron-transferring cofactor, and a mononuclear iron site is the putative site of substrate oxygenation. A reductase, which contains FMN and a plant-type [2Fe-2S] ferredoxin domain, transfers electrons from NADH to the Rieske center. Any of the metal ions, Fe(II), Cu(II), Co(II), Mn(II), and Zn(II), can be used to populate the mononuclear site, but only Fe(II) is competent for effecting hydroxylation. Nevertheless, studies of how these metal ions affect both the EPR spectra of the reduced Rieske site and the kinetics of electron transfer in the PDO system indicated that each of these metal ions binds tightly and affects the protein similarly. In this study, EPR spectra were obtained from samples in which iron of the mononuclear site was replaced with Cu(II). The use of (63)Cu(II), in combination with PDO obtained from cultures grown on media enriched in (15)N [using ((15)NH(4))(2)SO(4) as a sole nitrogen source], [delta,epsilon-(15)N]histidine, as well as natural abundance sources of nitrogen, enabled detailed spectral analysis of the superhyperfine structure of the Cu(II) EPR lines. These studies clearly show that two histidines are coordinated to the mononuclear site. Coupled with previous studies [Bertini, I., Luchinat, C., Mincione, G., Parigi, G., Gassner G. T., and Ballou, D. P. (1996) J. Bioinorg. Chem. 1, 468-475] that show the presence of one or two water molecules coordinated to the iron, it is suggested that the mononuclear site is similar to several other mononuclear nonheme iron proteins, including naphthalene dioxygenase, for which crystal structures are available. The lack of observable EPR interaction signals between Cu(II) in the mononuclear site and the reduced Rieske center of PDO suggest that the two sites are at least 12 A apart, which is similar to that found in the naphthalene dioxygenase crystal structure.     

X-ray absorption spectroscopy of the [2Fe-2S] Rieske cluster in Pseudomonas cepacia phthalate dioxygenase. Determination of core dimensions and iron ligation

We have employed X-ray absorption spectroscopy to obtain structural information about the Rieske Fe/S center in the phthalate dioxygenase (PDO) from Pseudomonas cepacia. Native PDO contains a dinuclear Rieske Fe/S center and an additional mononuclear Fe site. In order to study selectively the Fe/S cluster, we measured data for samples in which the mononuclear site was either depleted of metal or reconstituted with Co or Zn. Our results demonstrate that the iron environment in the Rieske cluster is structurally indistinguishable from that found in other Fe/S clusters, thus strongly supporting the suggestion that the unusually high reduction potentials for Rieske clusters are due to electrostatic rather than structural effects. The average Fe-Fe distance is 2.68 (3) A for both oxidized and reduced Rieske clusters. The average Fe-S distance is 2.24 (2) A in the oxidized cluster and 2.28 (2) A in the reduced cluster. Careful analysis of the EXAFS Debye-Waller factors suggests that the bridging and terminal Fe-S distances for the oxidized cluster are 2.20 and 2.31 A, respectively. Taken together with recent ENDOR results, these studies provide a detailed structural model for the Rieske [2Fe-2S] centers.     

Similar enzymes, different structures: phthalate dioxygenase is an alpha3alpha3 stacked hexamer, not an alpha3beta3 trimer like "normal" Rieske oxygenases

Phthalate dioxygenase (PDO) is a member of a class of bacterial oxygenases that contain both Rieske [2Fe-2S] and Fe(II) mononuclear centers. Recent crystal structures of several Rieske dioxygenases showed that they exist as alpha(3)beta(3) multimers with subunits arranged head-to-tail in alpha and beta stacked planar rings. The structure of PDO, which consists of only alpha-subunits, remains to be solved. Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ in the mechanism of catalysis. Gel filtration and analytical centrifugation experiments, supplemented with mass spectrometric analysis (both ESI-MS and ESI-GEMMA), in this work showed a hexameric arrangement of subunits in the PDO multimer. Our proposed model for the subunit arrangement in PDO postulates two alpha(3) planar rings one on top the other, similar to the alpha(3)beta(3) arrangement in other Rieske dioxygenases. Unlike other Rieske dioxygenases, this arrangement brings two Rieske and two mononuclear centers, all on separate subunits, into proximity, allowing their cooperation for catalysis. Potential reasons necessitating this unusual structural arrangement are discussed.     

Aspartate 205 in the catalytic domain of naphthalene dioxygenase is essential for activity

The naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The crystal structure of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571-586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske [2Fe-2S] center of one alpha subunit and mononuclear iron in the adjacent alpha subunit. In this study, we constructed four site-directed mutations that changed aspartate 205 to alanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity. The mutant proteins were very inefficient in oxidizing naphthalene to cis-naphthalene dihydrodiol, and oxygen uptake in the presence of naphthalene was below detectable levels. The purified mutant protein with glutamine in place of aspartate 205 had identical spectral properties to wild-type naphthalene dioxygenase and was reduced by NADH in the presence of catalytic amounts of ferredoxinNAP and reductaseNAP. Benzene, an effective uncoupler of oxygen consumption in purified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant protein. These results indicate that electron transfer from NADH to the Rieske center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site.     

Benzoate 1,2-dioxygenase from Pseudomonas putida: single turnover kinetics and regulation of a two-component Rieske dioxygenase

The benzoate 1,2-dioxygenase system (BZDOS) from Pseudomonas putida mt-2 catalyzes the NADH-dependent oxidation of benzoate to 1-carboxy-1,2-cis-dihydroxycyclohexa-3,5-diene. Both the oxygenase (BZDO) and reductase (BZDR) components of BZDOS have been purified and characterized kinetically and by optical, EPR, and M?ssbauer spectroscopies. BZDO has an (alpha beta)(3) subunit structure in which each alpha subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron site. Two different purification protocols were developed for BZDO allowing the mononuclear iron to be stabilized in either the Fe(III) or the Fe(II) state for spectroscopic characterization. Using single turnover reactions, it is shown that fully reduced BZDO alone is capable of yielding the cis-diol product in high yield at rates that exceed the BZDOS turnover number. At the conclusion of turnover, quantification of each oxidation state of the metal sites by EPR and M?ssbauer spectroscopies shows that the Rieske cluster and mononuclear iron are each oxidized in amounts equal to the product yield, suggesting that the two electrons required for catalysis derive from the two metal centers. These results are in agreement with our previous study of naphthalene 1,2-dioxygenase [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953], which belongs to a different Rieske dioxygenase subclass, suggesting that it is a universal characteristic of Rieske dioxygenases that oxygen activation and substrate oxidation are catalyzed by the oxygenase component alone. The EPR spectrum of the Fe(III) center after a single turnover is distinct from either of those of substrate-free or substrate-bound enzyme. The complex with this spectrum is not formed by addition of cis-diol product to the resting Fe(III) form of the enzyme but is observed when the Fe(II) form is oxidized in the presence of product. Together, these results suggest that product exchange occurs only when the mononuclear iron is reduced. Stopped-flow and rapid scan analyses monitoring the oxidation of the Rieske cluster during the single turnover reaction show that it occurs in three phases that are kinetically competent for catalysis. The rate of each phase was found to be dependent on the type of substrate present, suggesting that the substrate influences the rate of electron transfer between the metal clusters. The participation of substrate in the oxygen activation reaction suggests a new aspect of the mechanism of this process by the Rieske dioxygenase class.     

Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.     

Structural characterization of the mononuclear iron site in Pseudomonas cepacia phthalate DB01 dioxygenase using X-ray absorption spectroscopy

Phthalate dioxygenase (PDO) from Pseudomonas cepacia contains a Rieske-like [2Fe-2S] cluster and a mononuclear non-heme Fe(II) site. The mononuclear iron can be replaced by a variety of divalent metal ions, although only Fe(II) permits catalytic activity. We used X-ray absorption spectroscopy to characterize the structural properties of the mononuclear iron site and to follow the structural changes in this site as a function both of Rieske site oxidation state and of phthalate binding. Data for the mononuclear site have been measured directly for PDO substituted with Co or Zn in the mononuclear site, and by difference for the native 3-Fe protein. The mononuclear site was modeled well by low Z-ligation (oxygen or nitrogen) and showed no evidence for high-Z ligands (e.g., sulfur). The relatively short average first shell bond lengths and the absence of significant outer shell scattering suggest that the mononuclear site has several oxygen ligands. With Zn in the mononuclear site, the average bond length (2.00?Å) suggests a 5-coordinate site under all conditions. In contrast, the Co- or Fe-containing mononuclear site appeared to be 6-coordinate and changed to 5-coordinate when substrate was bound, since the first shell bond length changed from 2.08 to 2.02?Å (Co) or 2.10 to 2.06?Å (Fe). The implications of these findings for the PDO mechanism are discussed.     

Rieske business: structure-function of Rieske non-heme oxygenases

Rieske non-heme iron oxygenases (RO) catalyze stereo- and regiospecific reactions. Recently, an explosion of structural information on this class of enzymes has occurred in the literature. ROs are two/three component systems: a reductase component that obtains electrons from NAD(P)H, often a Rieske ferredoxin component that shuttles the electrons and an oxygenase component that performs catalysis. The oxygenase component structures have all shown to be of the alpha3 or alpha3beta3 types. The transfer of electrons happens from the Rieske center to the mononuclear iron of the neighboring subunit via a conserved aspartate, which is shown to be involved in gating electron transport. Molecular oxygen has been shown to bind side-on in naphthalene dioxygenase and a concerted mechanism of oxygen activation and hydroxylation of the ring has been proposed. The orientation of binding of the substrate to the enzyme is hypothesized to control the substrate selectivity and regio-specificity of product formation.     

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske Fe-S centers of the cytochrome b6f complex of spinach and the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria.

Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b6f complex of spinach chloroplasts and of the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of 14N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b6f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A1 = 4.6 MHz and A2 = 3.8 MHz. Similar couplings are observed for the Rieske centers in the three cytochrome bc1 complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic coupling to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferredoxin ESEEM patterns.     

High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli

Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II)-mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at approximately 30mg of crude PDO and approximately 1mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to E. coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1g/L for PDO and about 0.2g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20h) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was <8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein.     

Single turnover chemistry and regulation of O2 activation by the oxygenase component of naphthalene 1,2-dioxygenase

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.     

The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays

Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.     

Diversity of 16S rRNA and dioxygenase genes detected in coal‐tar‐contaminated site undergoing active bioremediation

Aims: In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation‐independent communities is available. Methods and Results: Coal‐tar‐contaminated soil was collected, which consisted of 122·5 mg g^?1 total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal‐tar‐contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (α‐subunit) common to all PAH dioxygenase enzymes and (iii) β‐subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha‐, Epsilon‐ and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of α‐subunit and β‐subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe‐2S] cluster binding site suggested that these gene fragments encode for α‐subunit of dioxygenase gene. Conclusions: Sequencing of the cloned libraries representing α‐subunit gene fragments (Rf1) and β‐subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal‐tar‐contaminated soil. Significance and Impact of the Study: The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.     

2-Oxoquinoline 8-monooxygenase oxygenase component: active site modulation by Rieske-[2Fe-2S] center oxidation/reduction

2-Oxoquinoline 8-monooxygenase is a Rieske non-heme iron oxygenase that catalyzes the NADH-dependent oxidation of the N-heterocyclic aromatic compound 2-oxoquinoline to 8-hydroxy-2-oxoquinoline in the soil bacterium Pseudomonas putida 86. The crystal structure of the oxygenase component of 2-oxoquinoline 8-monooxygenase shows a ring-shaped, C3-symmetric arrangement in which the mononuclear Fe(II) ion active site of one monomer is at a distance of 13 A from the Rieske-[2Fe-2S] center of a second monomer. Structural analyses of oxidized, reduced, and substrate bound states reveal the molecular bases for a new function of Fe-S clusters. Reduction of the Rieske center modulates the mononuclear Fe through a chain of conformational changes across the subunit interface, resulting in the displacement of Fe and its histidine ligand away from the substrate binding site. This creates an additional coordination site at the mononuclear Fe(II) ion and can open a pathway for dioxygen to bind in the substrate-containing active site.