Rieske-type Cluster Research Articles

Spectroscopic Characterisation of the Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038.

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O2-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the “peroxide shunt”, an alternative mechanism for the oxidation of substrate in the presence of H2O2, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.

Bacterial Genes of Non-Heme Iron Oxygenases, Which Have a Rieske-Type Cluster, Catalyzing Initial Stages of Degradation of Chlorophenoxyacetic Acids

Hydroxylation of the benzoic ring by non-heme iron oxygenases having a Rieske-type cluster is the key step in the aerobic degradation of chloroaromatic compounds by bacteria. Rieske oxygenases (RO) catalyze the oxidative decarboxylation reaction unique to the enzymes of this family with the formation of corresponding phenolic compounds. This review discusses the general structure, function, and classification of ROs that catalyze the oxidation of chlorophenoxyacetic acids; genes encoding the ROs with their phylogenetic classes are also reviewed.

Two-dimensional pulsed electron spin resonance characterization of 15N-labeled archaeal Rieske-type ferredoxin

Two-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly 15N-labeled archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 has been conducted in comparison with the previously characterized high-potential protein homologs. Major differences among these proteins were found in the hyperfine sublevel correlation (HYSCORE) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinated) peptide nitrogens near the reduced clusters. They are less pronounced in the HYSCORE spectra of ARF than those of the high-potential protein homologs, and may account for the tuning of Rieske-type clusters in various redox systems.

Arsenite oxidation by a chemoautotrophic moderately acidophilic Thiomonas sp.: from the strain isolation to the gene study

An autotrophic bacterium able to gain energy from the oxidation of arsenite was isolated from arsenite-containing acid mine drainage waters. It belongs to the genus Thiomonas as shown by DNA-DNA hybridization experiments, 16S rRNA gene sequence, quinone and fatty acid content analyses. Carboxysomes were observed and the cbbSL genes encoding the ribulose 1,5-bisphosphate carboxylase/oxygenase were detected, confirming that this bacterium is able to fix CO(2). Arsenite oxidation was catalysed by a membrane-bound enzyme, and this activity was detected essentially in cells grown in the presence of arsenite. The genes encoding the two subunits of the arsenite oxidase of the Thiomonas isolate have been sequenced. The small subunit has a characteristic Tat signal sequence and contains the residues binding the [2Fe-2S] Rieske-type cluster. The large subunit has the [3Fe-4S] cluster-binding motif as well as the residues proposed to bind arsenite. In addition, most of the residues interacting with the molybdenum cofactor are conserved. The genes encoding both subunits belong to an operon, likely with a gene encoding a cytochrome c. The expression of this operon is greater in cells grown in the presence than in the absence of arsenite, in agreement with a transcriptional regulation in the presence of this metalloid.

Tolerance of the Rieske-type [2Fe-2S] cluster in recombinant ferredoxin BphA3 from Pseudomonas sp. KKS102 to histidine ligand mutations.

BphA3 from Pseudomonas sp. KKS102 is a Rieske-type [2Fe-2S] ferredoxin that transfers electrons from an NADH-dependent oxidoreductase, BphA4, to a biphenyl dioxygenase complex. A high-level expression and purification system for the recombinant BphA3 in Escherichia coli was constructed. Two histidine ligands of the Rieske-type cluster in BphA3, were each replaced with serine, cysteine, asparagine and tyrosine. The single mutants, in which either His44 or His65 was replaced with a cysteine residue (CH and HC mutants respectively), and the double mutant, in which both histidine residues were replaced with cysteine residue (CC mutant), accumulated to high levels in the E. coli cells, while the other single mutants did not. The purified WT (wild-type) protein showed characteristic near-UV and visible absorption and CD spectra of Rieske-type clusters. The X-ray absorption spectra were suggestive of the existence of [2Fe-2S] clusters, with one histidine and three cysteine ligands in the CH and HC mutants, and an [2Fe-2S] cluster with four cysteine ligands in the CC mutant. The BphA4-dependent cytochrome c reductase activities of the mutants were less than 0.3% of that of the WT protein. The redox potential of the WT protein determined by cyclic voltammetry was -180+/-5 mV compared with the standard hydrogen electrode, and that of the CH mutant was approx. 175 mV lower. The changes in the near-UV and visible absorption spectra of the mutants showed that the reduced iron-sulphur clusters in the mutants were unstable. His44 and His65 in BphA3 can be replaced with cysteine residues, but are required for the stabilization of the reduced form of the cluster.

Redox-dependent structural changes in archaeal and bacterial Rieske-type [2Fe-2S] clusters.

Proteins containing Rieske-type [2Fe-2S] clusters play important roles in many biological electron transfer reactions. Typically, [2Fe-2S] clusters are not directly involved in the catalytic transformation of substrate, but rather supply electrons to the active site. We report herein X-ray absorption spectroscopic (XAS) data that directly demonstrate an average increase in the iron-histidine bond length of at least 0.1 A upon reduction of two distantly related Rieske-type clusters in archaeal Rieske ferredoxin from Sulfolobus solfataricus strain P-1 and bacterial anthranilate dioxygenases from Acinetobacter sp. strain ADP1. This localized redox-dependent structural change may fine tune the protein-protein interaction (in the case of ARF) or the interdomain interaction (in AntDO) to facilitate rapid electron transfer between a lower potential Rieske-type cluster and its redox partners, thereby regulating overall oxygenase reactions in the cells.

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys)3 ligands.

The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus. The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da. The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a [2Fe-2S]2+,+ prosthetic group. The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands. The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum. The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a [3Fe-4S]+,0 cluster, and a [4Fe-4S]2+,+ cluster. The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV. The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster. The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH. SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase. However, P. furiosus sulfide dehydrogenase does not have glutamate synthase activity. SudB is highly homologous to HydG, the gamma-subunit of P. furiosus NiFe hydrogenase. The latter enzyme also has sulfide dehydrogenase activity. The P. furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme. Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.

Orientationally-Selected Two-Dimensional ESEEM Spectroscopy of the Rieske-Type Iron−Sulfur Cluster in 2,4,5-Trichlorophenoxyacetate Monooxygenase from Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 is able to use the chlorinated compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. CW EPR and one-dimensional ESEEM spectroscopy studies performed earlier indicate the presence of a Rieske-type [2Fe-2S] cluster with two coordinated histidine residues in 2,4,5-T monooxygenase from B. cepacia. This paper describes the application of two-dimensional ESEEM (called HYSCORE) spectroscopy for further characterization of the nitrogens surrounding the reduced Rieske-type cluster. The HYSCORE spectra measured at field positions in the neighborhood of the principal directions of the g tensor contain major contributions from cross-peaks correlating the two double-quantum transitions from each histidine nitrogen. These allow the estimation of the diagonal components of the hyperfine tensors along the principal axes of the g tensor: 4.05, 3.88, and 4.01 MHz (N1) and 4.71, 5.07, and 5.02 MHz (N2). Other spectral features from the histidine nitrogens ...

EPR, Electron Spin Echo Envelope Modulation, and Electron Nuclear Double Resonance Studies of the 2Fe2S Centers of the 2-Halobenzoate 1,2-Dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS

The 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS (Fetzner, S., Müller, R., and Lingens, F. (1992) J. Bacteriol. 174, 279-290) contains both a ferredoxin-type and a Rieske-type 2Fe2S center. These two significantly different 2Fe2S clusters were characterized with respect to their EPR spectra, electrochemical properties (Rieske-type cluster with gz = 2.025, gy = 1.91, gx = 1.79, gav = 1.91, Em = -125 +/- 10 mV; ferredoxin-type center with gz = 2.05, gy = 1.96, gx = 1.89, gav = 1.97, Em = -200 +/- 10 mV) and pH dependence thereof. X band electron spin echo envelope modulation and electron nuclear double resonance spectroscopy was applied to study the interaction of the Rieske-type center of the 2-halobenzoate 1,2-dioxygenase with 14N and 1H nuclei in the vicinity of the 2Fe2S cluster. The results are compared to those obtained on the Rieske protein of the cytochrome b6f complex (Em = +320 mV) and the water-soluble ferredoxin (Em = -430 mV) of spinach chloroplasts, as typical representatives of the gav = 1.91 and gav = 1.96 class of 2Fe2S centers. Properties common to all Rieske-type clusters and those restricted to the respective centers in bacterial oxygenases are discussed.

Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism of 2,4-dichloro- and 2-chlorobenzoate

Cell extracts of Pseudomonas aeruginosa 142, which was previously isolated from a polychlorinated biphenyl-degrading consortium, were shown to degrade 2,4-dichlorobenzoate, 2-chlorobenzoate, and a variety of other substituted ortho-halobenzoates by a reaction that requires oxygen, NADH, Fe(II), and flavin adenine dinucleotide. By using extracts that were chromatographically depleted of chlorocatechol and catechol 1,2-dioxygenase activities, products of the initial reaction with 2,4- or 2,5-dichlorobenzoate and 2-chlorobenzoate were identified by mass spectrometry as 4-chlorocatechol and catechol. In contrast to the well-characterized benzoate dioxygenases or the recently described 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS (S. Fetzner, R. Müller, and F. Lingens, J. Bacteriol. 174:279-290, 1992) that possess two protein components, the P. aeruginosa enzyme was resolved by ion-exchange chromatography into three components, each of which is required for activity. To verify the distinct nature of this enzyme, we purified, characterized, and identified one component as a ferredoxin (M(r), approximately 13,000) containing a single [2Fe-2S] Rieske-type cluster (electron paramagnetic resonance spectroscopic values of gx = 1.82, gy = 1.905, and gz = 2.02 in the reduced state) that is related in sequence to ferredoxins found in the naphthalene and biphenyl three-component dioxygenase systems. By analogy to these enzymes, we propose that the P. aeruginosa ferredoxin serves as an electron carrier between an NADH-dependent ferredoxin reductase and the terminal component of the ortho-halobenzoate 1,2-dioxygenase. The broad specificity and high regiospecificity of the enzyme make it a promising candidate for use in the degradation of mixtures of chlorobenzoates.

Resonance Raman studies of Rieske-type proteins

Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm −1, which consists of components at 266 cm −1 and 274 cm −1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm −1) and deprotonated-hydrogen-bonded imidazolate (274 cm −1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferredoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an S t 2FeS b 2Fe[N(His)] t 2 structure for the Rieske-type cluster.

Q-band ENDOR spectra of the Rieske protein from Rhodobactor capsulatus ubiquinol-cytochrome c oxidoreductase show two histidines coordinated to the [2Fe-2S] cluster.

Electron nuclear double resonance (ENDOR) experiments were performed on 14N (natural abundance) and 15N-enriched iron-sulfur Rieske protein in the ubiquinol-cytochrome c2 oxidoreductase from Rhodobactor capsulatus. The experiments proved that two distinct nitrogenous ligands, histidines, are undoubtedly ligated to the Rieske [2Fe-2S] center. The calculations of hyperfine tensors give values similar but not identical to those of the Rieske-type cluster in phthalate dioxygenase of Pseudomonas cepacia and suggest a slightly different geometry of the iron-sulfur cluster in the two proteins.

Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases

The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.