Structural roles of the active site iron(III) ions in catechol 1,2-dioxygenases and differential secondary structure changes in isoenzymes A and B from Acinetobacter radioresistens S13 (original) (raw)
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Journal of protein chemistry, 2000
Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been purified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homodimers composed of a single type of subunit with molecular mass of 38,600 and 37,700, Da respectively. In conditions of low ionic strength, IsoA can aggregate as a trimer, in contrast to IsoB, which maintains the dimeric structure, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown on phenol, whereas the IsoB is selectively expressed using benzoate as carbon source. This is the first evidence of the presence of differently expressed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB contain approximately 1 iron(III) ion per subunit and both show electronic absorbance and EPR feature...
An Iron Reservoir to the Catalytic Metal: The Rubredoxin Iron in an Extradiol Dioxygenase
The Journal of biological chemistry, 2015
The rubredoxin motif is present in over 74,000 protein sequences and 2,000 structures, but few have known functions. A secondary, non-catalytic, rubredoxin-like iron site is conserved in 3-hydroxyanthranilate 3,4-dioxygenase (HAO), from single-cellular sources but not multi-cellular sources. Through the population of the two metal binding sites with various metals in bacterial HAO, the structural and functional relationship of the rubredoxin-like site was investigated using kinetic, spectroscopic, crystallographic, and computational approaches. It is shown that the first metal presented preferentially binds to the catalytic site rather than the rubredoxin-like site, which selectively binds iron when the catalytic site is occupied. Furthermore, an iron ion bound to the rubredoxin-like site is readily delivered to an empty catalytic site of metal-free HAO via an intermolecular transfer mechanism. Through the use of metal analysis and catalytic activity measurements, we show that a dow...
Proceedings of the National Academy of Sciences, 2008
Biological O2 activation often occurs after binding to a reduced metal [e.g., M(II)] in an enzyme active site. Subsequent M(II)-toO 2 electron transfer results in a reactive M(III)-superoxo species. For the extradiol aromatic ring-cleaving dioxygenases, we have proposed a different model where an electron is transferred from substrate to O 2 via the M(II) center to which they are both bound, thereby obviating the need for an integral change in metal redox state. This model is tested by using homoprotocatechuate 2,3dioxygenases from Brevibacterium fuscum (Fe-HPCD) and Arthrobacter globiformis (Mn-MndD) that share high sequence identity and very similar structures. Despite these similarities, Fe-HPCD binds Fe(II) whereas Mn-MndD incorporates Mn(II). Methods are described to incorporate the nonphysiological metal into each enzyme (Mn-HPCD and Fe-MndD). The x-ray crystal structure of Mn-HPCD at 1.7 Å is found to be indistinguishable from that of Fe-HPCD, while EPR studies show that the Mn(II) sites of Mn-MndD and Mn-HPCD, and the Fe(II) sites of the NO complexes of Fe-HPCD and Fe-MndD, are very similar. The uniform metal site structures of these enzymes suggest that extradiol dioxygenases cannot differentially compensate for the 0.7-V gap in the redox potentials of free iron and manganese. Nonetheless, all four enzymes exhibit nearly the same K M and Vmax values. These enzymes constitute an unusual pair of metallo-oxygenases that remain fully active after a metal swap, implicating a different way by which metals are used to promote oxygen activation without an integral change in metal redox state. bioinorganic chemistry ͉ extradiol dioxygenase ͉ nonheme iron ͉ manganese E xtradiol catecholic dioxygenases catalyze the cleavage of dihydroxybenzene rings with incorporation of both atoms from O 2 to yield muconic semialdehyde products (1-3). As such, these enzymes play key roles in the ability of nature to reclaim the vast quantities of organic carbon sequestered in aromatic compounds in the environment. The metal in these enzymes is coordinated by two His residues and one Glu/Asp residue that occupy one face of a (pseudo)octahedron, representing the first examples of what has become recognized as the ''2-His-1carboxylate facial triad'' (2H1C triad) motif in many nonheme iron enzymes that activate dioxygen (4, 5). Indeed, a truly remarkable array of oxidative reactions is catalyzed by 2H1C triad enzymes, including C-H hydroxylation, ring expansion, and COC bond cleavage (5). Studies from our laboratories and others have resulted in a mechanistic proposal shown in Fig. 1 for the extradiol dioxygenases that takes advantage of the 2H1C triad motif (3, 6-8). Crystal structures show that the catecholic substrate binds in a bidentate fashion to the reduced metal center trans from the histidines, displacing two or three water molecules (9-11). This primes the metal center for O 2 binding in the coordination site trans to the carboxylate ligand after substrate is in place. We have proposed that electron density is transferred from the aromatic substrate to bound dioxygen via the iron, thereby giving them both radical character and activating them for reaction with each other (6, 12, 13). The attack of the bound superoxide on the oxidized catecholate ring would yield an intermediate alkylperoxy species. This intermediate would then rearrange with OOO bond cleavage and ring insertion to produce an intermediate lactone that is hydrolyzed to complete the reaction. These mechanistic proposals have recently received strong support in
Biochemistry, 2008
The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities towards their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M +2 metal ion bound in the active site. The Ni +2 -bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate and carbon monoxide, while the Fe +2 -bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS) and isothermal calorimetry measurements, it is concluded that the same four residues, His 96, His 98, Glu 102 and His 140, provide the protein-based ligands for the metal in both the Ni-and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a His ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved non-ligand acidic residues, Glu 95 and Glu 100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu 100 mutant reconstitutes readily but has low activity. Mutation of Asp 101 results in an active enzyme that incorporates metal in vivo but shows evidence of mixed forms.
Analytical Biochemistry, 2010
The addition of divalent metal ions or substrate taurine to TauD, an a-ketoglutarate-dependent dioxygenase, alters its UV absorption, as clearly observed by monitoring the protein's difference spectra. Binding of metal ions leads to a decrease in absorption at 297nmandmodulationofotherfeatures.Aseparatesignaturewithenhancedabsorptionat297 nm and modulation of other features. A separate signature with enhanced absorption at 297nmandmodulationofotherfeatures.Aseparatesignaturewithenhancedabsorptionat295 nm is identified for binding of taurine. These narrow ($700 cm À1 ) and intense ($0.5 mM À1 cm À1 ) spectral changes are attributed to ligand-induced protein conformational changes affecting the environment of aromatic residues. The changes in the UV difference spectra were exploited to assess directly the thermodynamics and kinetics of ligand interactions in wildtype TauD and selected variants. This approach holds promise as a new tool to probe ligand-induced conformational changes in a wide range of other proteins. Experimental and quantification approaches for a reliable analysis of protein absorption below 320 nm are presented.
Spectroscopic Studies of the Anaerobic Enzyme−Substrate Complex of Catechol 1,2-Dioxygenase
Journal of the American Chemical Society, 2005
The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/ substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm -1 in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm -1 in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution. (1) Bugg, T. D. H.; Lin, G. Chem. Commun. 2001, 941-952. (2) Costas, M.; Mehn, M. P.; Jensen, M. P.; Que, L. Chem. ReV. 2004, 104, 939-986. (3) Bugg, T. D. H. Tetrahedron 2003, 59, 7075-7101. (4) Solomon, E. I.; Brunold, T. C.; Davis, M. I.; Kemsley, J. N.; Lee, S. K.; Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y. S.; Zhou, J.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011
Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Inorganic Chemistry, 1999
Chemical reduction of the mononuclear ferric active site in the bacterial intradiol cleaving catecholic dioxygenase protocatechuate 3,4-dioxygenase (3,4-PCD, BreVibacterium fuscum) produces a high-spin ferrous center. We have applied circular dichroism (CD), magnetic circular dichroism (MCD), variable-temperature-variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies to investigate the geometric and electronic structure of the reduced iron center. Excited-state ligand field CD and MCD data indicate that the site is six-coordinate where the 5 E g excited-state splitting is 2033 cm-1. VTVH MCD analysis of the ground state indicates that the site has negative zero-field splitting with a small rhombic splitting of the lowest doublet (δ) 1.6 (0.3 cm-1). XAS pre-edge analysis also indicates a six-coordinate site while EXAFS analysis provides accurate bond lengths. Since previous spectroscopic analysis and the crystal structure of oxidized 3,4-PCD indicate a five-coordinate ferric active site, the results presented here show that the coordination number increases upon reduction. This is attributed to the coordination of a second solvent ligand. The coordination number increase relative to the oxidized site also appears to be associated with a large decrease in the ligand donor strength in the reduced enzyme due to protonation of the original hydroxide ligand.