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Edward I Solomon - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic and electronic structure studies of the trinuclear cu cluster active site of the Multicopper Oxidase laccase nature of its coordination unsaturation
Journal of the American Chemical Society, 2005Co-Authors: Liliana Quintanar, David R Britt, Jungjoo Yoon, Constantino P Aznar, Amy E Palmer, Kristoffer K Andersson, Edward I SolomonAbstract:Laccase is a Multicopper Oxidase that contains four Cu ions, one type 1 (T1), one type 2 (T2), and a coupled binuclear type 3 Cu pair (T3). The T2 and T3 centers form a trinuclear Cu cluster that is the active site for O2 reduction to H2O. A combination of spectroscopic and DFT studies on a derivative where the T1 Cu has been replaced by a spectroscopically innocent Hg2+ ion has led to a detailed geometric and electronic structure description of the resting trinuclear Cu cluster, complementing crystallographic results. The nature of the T2 Cu ligation has been elucidated; this site is three-coordinate with two histidines and a hydroxide over its functional pH range (stabilized by a large inductive effect, cluster charge, and a hydrogen-bonding network). Both the T2 and T3 Cu centers have open coordination positions oriented toward the center of the cluster. DFT calculations show that the negative protein pocket (four conserved Asp/Glu residues within 12 A) and the dielectric of the protein play important ...
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role of aspartate 94 in the decay of the peroxide intermediate in the Multicopper Oxidase fet3p
Biochemistry, 2005Co-Authors: Liliana Quintanar, Daniel J Kosman, Christopher S Stoj, Tzupin Wang, Edward I SolomonAbstract:Fet3p is a Multicopper Oxidase that contains four Cu ions: one type 1, one type 2, and a coupled binuclear type 3 site. The type 2 and type 3 centers form a trinuclear cluster that is the active site for O(2) reduction to H(2)O. When the type 1 Cu is depleted (C484S mutation), the reaction of the reduced trinuclear cluster with O(2) generates a peroxide intermediate. Kinetic studies of the decay of the peroxide intermediate suggest that a carboxyl residue (D94 in Fet3p) assists the reductive cleavage of the O-O bond at low pH. Mutations at the D94 residue (D94A, D94N, and D94E) have been studied to evaluate its role in the decay of the peroxide intermediate. Spectroscopic studies show that the D94 mutations affect the geometric and electronic structure of the trinuclear cluster in a way that is consistent with the hydrogen bond connectivity of D94. While the D94E mutation does not affect the initial reaction of the cluster with O(2), the D94A mutation causes larger structural changes that render the trinuclear cluster unreactive toward O(2), demonstrating a structural role for the D94 residue. The decay of the peroxide intermediate is markedly affected by the D94E mutation, confirming the involvement of D94 in this reaction. The D94 residue appears to activate a proton of the type 2 Cu(+)-bound water for participation in the transition state. These studies provide new insight into the role of D94 and proton involvement in the reductive cleavage of the O-O bond.
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Targeted suppression of the ferrOxidase and iron trafficking activities of the Multicopper Oxidase Fet3p from Saccharomyces cerevisiae
JBIC Journal of Biological Inorganic Chemistry, 2003Co-Authors: Tzupin Wang, Edward I Solomon, Liliana Quintanar, Scott Severance, Daniel J KosmanAbstract:The Fet3 protein in Saccharomyces cerevisiae is a Multicopper Oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe^2+ to Fe^3+; this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe^2+, one of which is essential also to the coupling of the ferrOxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K _M for Fe^2+ exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferrOxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in ^59Fe uptake. For the Fet3p(E185D) mutant, K _M for iron was 300-fold greater than the wild-type K _M, while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe^2+ binding to Fet3p and to the subsequent trafficking of the Fe^3+ produced to Ftr1p.
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spectroscopic characterization and o2 reactivity of the trinuclear cu cluster of mutants of the Multicopper Oxidase fet3p
Biochemistry, 2002Co-Authors: Amy E Palmer, Daniel J Kosman, Liliana Quintanar, Tzupin Wang, Scott Severance, Edward I SolomonAbstract:Fet3p is a Multicopper Oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O 2 to H2O. The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O 2 reduction. This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed. To examine the reaction between the trinuclear Cu cluster and O 2, the type 1 Cu Cys484 was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme. Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity. One of these mutants (His 126Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p). Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His126Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites. The trinuclear clusters in T1D and T1DT3' Fet3p react with O2 to produce peroxide intermediates analogous to that observed in T1Hg laccase. Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate. In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH. In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's. This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond.
Bradley M Tebo - One of the best experts on this subject based on the ideXlab platform.
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surface induced dissociation coupled with high resolution mass spectrometry unveils heterogeneity of a 211 kda Multicopper Oxidase protein complex
Journal of the American Society for Mass Spectrometry, 2018Co-Authors: Mowei Zhou, Bradley M Tebo, Christine A. Romano, Vicki H. Wysocki, Jing Yan, Ljiljana PasatolicAbstract:Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese Oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this Multicopper Oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein–ligand complexes.
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Biogenic manganese oxide nanoparticle formation by a multimeric Multicopper Oxidase Mnx.
Nature communications, 2017Co-Authors: Christine A. Romano, Mowei Zhou, Yang Song, Vicki H. Wysocki, Alice Dohnalkova, Libor Kovarik, Ljiljana Paša-tolić, Bradley M TeboAbstract:Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn Oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a Multicopper Oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes. Significant challenges exist for structural characterization of enzymes responsible for biomineralization. Here the authors show that native mass spectrometry and high resolution electron microscopy can define the subunit topology and copper binding of a manganese oxidizing complex, and describe early stage formation of its mineral products
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Mn(II) Oxidation by the Multicopper Oxidase Complex Mnx: A Binuclear Activation Mechanism
Journal of the American Chemical Society, 2017Co-Authors: Alexandra V. Soldatova, Bradley M Tebo, Lizhi Tao, Christine A. Romano, Troy A. Stich, William H. Casey, R. David Britt, Thomas G. SpiroAbstract:The bacterial protein complex Mnx contains a Multicopper Oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO2 biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu2+, the electron acceptor. Indeed the type 1 Cu2+ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO2 formation from MnO2 nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activa...
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substrate specificity and copper loading of the manganese oxidizing Multicopper Oxidase mnx from bacillus sp pl 12
Metallomics, 2017Co-Authors: Cristina N Butterfield, Bradley M TeboAbstract:Manganese(II) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn Oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the Multicopper Oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(II) and Mn(III), generating Mn(IV) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(II) appears to be the best substrate in terms of kcat, but its oxidation does not follow Michaelis–Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(II) substrate, but in a Michaelis–Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(II) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.
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Mn(II) Oxidation by the Multicopper Oxidase Complex Mnx: A Binuclear Activation Mechanism
2017Co-Authors: Alexandra V. Soldatova, Bradley M Tebo, Lizhi Tao, Christine A. Romano, Troy A. Stich, William H. Casey, David R Britt, Thomas G. SpiroAbstract:The bacterial protein complex Mnx contains a Multicopper Oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO2 biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu2+, the electron acceptor. Indeed the type 1 Cu2+ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO2 formation from MnO2 nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pKa > 8.6 deprotonation, which is assigned to Mn(II)-bound H2O; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(μ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pKa 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(μ-OH)2Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(μ-O)2Mn(IV) or an oligomer, which subsequently nucleates MnO2 nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper
Liliana Quintanar - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic and electronic structure studies of the trinuclear cu cluster active site of the Multicopper Oxidase laccase nature of its coordination unsaturation
Journal of the American Chemical Society, 2005Co-Authors: Liliana Quintanar, David R Britt, Jungjoo Yoon, Constantino P Aznar, Amy E Palmer, Kristoffer K Andersson, Edward I SolomonAbstract:Laccase is a Multicopper Oxidase that contains four Cu ions, one type 1 (T1), one type 2 (T2), and a coupled binuclear type 3 Cu pair (T3). The T2 and T3 centers form a trinuclear Cu cluster that is the active site for O2 reduction to H2O. A combination of spectroscopic and DFT studies on a derivative where the T1 Cu has been replaced by a spectroscopically innocent Hg2+ ion has led to a detailed geometric and electronic structure description of the resting trinuclear Cu cluster, complementing crystallographic results. The nature of the T2 Cu ligation has been elucidated; this site is three-coordinate with two histidines and a hydroxide over its functional pH range (stabilized by a large inductive effect, cluster charge, and a hydrogen-bonding network). Both the T2 and T3 Cu centers have open coordination positions oriented toward the center of the cluster. DFT calculations show that the negative protein pocket (four conserved Asp/Glu residues within 12 A) and the dielectric of the protein play important ...
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role of aspartate 94 in the decay of the peroxide intermediate in the Multicopper Oxidase fet3p
Biochemistry, 2005Co-Authors: Liliana Quintanar, Daniel J Kosman, Christopher S Stoj, Tzupin Wang, Edward I SolomonAbstract:Fet3p is a Multicopper Oxidase that contains four Cu ions: one type 1, one type 2, and a coupled binuclear type 3 site. The type 2 and type 3 centers form a trinuclear cluster that is the active site for O(2) reduction to H(2)O. When the type 1 Cu is depleted (C484S mutation), the reaction of the reduced trinuclear cluster with O(2) generates a peroxide intermediate. Kinetic studies of the decay of the peroxide intermediate suggest that a carboxyl residue (D94 in Fet3p) assists the reductive cleavage of the O-O bond at low pH. Mutations at the D94 residue (D94A, D94N, and D94E) have been studied to evaluate its role in the decay of the peroxide intermediate. Spectroscopic studies show that the D94 mutations affect the geometric and electronic structure of the trinuclear cluster in a way that is consistent with the hydrogen bond connectivity of D94. While the D94E mutation does not affect the initial reaction of the cluster with O(2), the D94A mutation causes larger structural changes that render the trinuclear cluster unreactive toward O(2), demonstrating a structural role for the D94 residue. The decay of the peroxide intermediate is markedly affected by the D94E mutation, confirming the involvement of D94 in this reaction. The D94 residue appears to activate a proton of the type 2 Cu(+)-bound water for participation in the transition state. These studies provide new insight into the role of D94 and proton involvement in the reductive cleavage of the O-O bond.
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Targeted suppression of the ferrOxidase and iron trafficking activities of the Multicopper Oxidase Fet3p from Saccharomyces cerevisiae
JBIC Journal of Biological Inorganic Chemistry, 2003Co-Authors: Tzupin Wang, Edward I Solomon, Liliana Quintanar, Scott Severance, Daniel J KosmanAbstract:The Fet3 protein in Saccharomyces cerevisiae is a Multicopper Oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe^2+ to Fe^3+; this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe^2+, one of which is essential also to the coupling of the ferrOxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K _M for Fe^2+ exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferrOxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in ^59Fe uptake. For the Fet3p(E185D) mutant, K _M for iron was 300-fold greater than the wild-type K _M, while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe^2+ binding to Fet3p and to the subsequent trafficking of the Fe^3+ produced to Ftr1p.
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spectroscopic characterization and o2 reactivity of the trinuclear cu cluster of mutants of the Multicopper Oxidase fet3p
Biochemistry, 2002Co-Authors: Amy E Palmer, Daniel J Kosman, Liliana Quintanar, Tzupin Wang, Scott Severance, Edward I SolomonAbstract:Fet3p is a Multicopper Oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O 2 to H2O. The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O 2 reduction. This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed. To examine the reaction between the trinuclear Cu cluster and O 2, the type 1 Cu Cys484 was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme. Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity. One of these mutants (His 126Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p). Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His126Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites. The trinuclear clusters in T1D and T1DT3' Fet3p react with O2 to produce peroxide intermediates analogous to that observed in T1Hg laccase. Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate. In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH. In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's. This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond.
Daniel J Kosman - One of the best experts on this subject based on the ideXlab platform.
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nmr study of the exchange coupling in the trinuclear cluster of the Multicopper Oxidase fet3p
Journal of the American Chemical Society, 2010Co-Authors: Mariaeugenia Zaballa, Lynn Ziegler, Daniel J Kosman, Alejandro J VilaAbstract:Fet3p from Saccharomyces cerevisiae is a Multicopper Oxidase (MCO) which oxidizes Fe2+ to Fe3+. The electronic structure of the different copper centers in this family of enzymes has been extensively studied and discussed for years with a particular focus on the exchange coupling regime in the trinuclear cluster (TNC). Using NMR spectroscopy we have quantified the exchange coupling constant in the type 3 center in a fully metalated Oxidase; this value in Fet3p is significantly higher than that reported for proteins containing isolated type 3 centers as tyrosinase. We also provide evidence of exchange coupling between the type 2 and the type 3 Cu2+ ions, which supports the crystallographic evidence of dioxygen binding to the TNC. This work provides the foundation for the application of NMR to these complex systems.
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in vitro unfolding of yeast Multicopper Oxidase fet3p variants reveals unique role of each metal site
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Erik Sedlák, Lynn Ziegler, Daniel J Kosman, Pernilla WittungstafshedeAbstract:Fet3p from Saccharomyces cerevisiae is a Multicopper Oxidase (MCO) that contains 3 cupredoxin-like β-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint (Tm) of 45 °C, 62 °C, and 72 °C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability (Tm of 50 °C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by ≈5 °C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations (Tm 50–55 °C; 1 K/min), followed by unfolding of domains 1 and 3 (≈65–70 °C; 1 K/min). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family.
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role of aspartate 94 in the decay of the peroxide intermediate in the Multicopper Oxidase fet3p
Biochemistry, 2005Co-Authors: Liliana Quintanar, Daniel J Kosman, Christopher S Stoj, Tzupin Wang, Edward I SolomonAbstract:Fet3p is a Multicopper Oxidase that contains four Cu ions: one type 1, one type 2, and a coupled binuclear type 3 site. The type 2 and type 3 centers form a trinuclear cluster that is the active site for O(2) reduction to H(2)O. When the type 1 Cu is depleted (C484S mutation), the reaction of the reduced trinuclear cluster with O(2) generates a peroxide intermediate. Kinetic studies of the decay of the peroxide intermediate suggest that a carboxyl residue (D94 in Fet3p) assists the reductive cleavage of the O-O bond at low pH. Mutations at the D94 residue (D94A, D94N, and D94E) have been studied to evaluate its role in the decay of the peroxide intermediate. Spectroscopic studies show that the D94 mutations affect the geometric and electronic structure of the trinuclear cluster in a way that is consistent with the hydrogen bond connectivity of D94. While the D94E mutation does not affect the initial reaction of the cluster with O(2), the D94A mutation causes larger structural changes that render the trinuclear cluster unreactive toward O(2), demonstrating a structural role for the D94 residue. The decay of the peroxide intermediate is markedly affected by the D94E mutation, confirming the involvement of D94 in this reaction. The D94 residue appears to activate a proton of the type 2 Cu(+)-bound water for participation in the transition state. These studies provide new insight into the role of D94 and proton involvement in the reductive cleavage of the O-O bond.
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Targeted suppression of the ferrOxidase and iron trafficking activities of the Multicopper Oxidase Fet3p from Saccharomyces cerevisiae
JBIC Journal of Biological Inorganic Chemistry, 2003Co-Authors: Tzupin Wang, Edward I Solomon, Liliana Quintanar, Scott Severance, Daniel J KosmanAbstract:The Fet3 protein in Saccharomyces cerevisiae is a Multicopper Oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe^2+ to Fe^3+; this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe^2+, one of which is essential also to the coupling of the ferrOxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K _M for Fe^2+ exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferrOxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in ^59Fe uptake. For the Fet3p(E185D) mutant, K _M for iron was 300-fold greater than the wild-type K _M, while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe^2+ binding to Fet3p and to the subsequent trafficking of the Fe^3+ produced to Ftr1p.
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spectroscopic characterization and o2 reactivity of the trinuclear cu cluster of mutants of the Multicopper Oxidase fet3p
Biochemistry, 2002Co-Authors: Amy E Palmer, Daniel J Kosman, Liliana Quintanar, Tzupin Wang, Scott Severance, Edward I SolomonAbstract:Fet3p is a Multicopper Oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O 2 to H2O. The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O 2 reduction. This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed. To examine the reaction between the trinuclear Cu cluster and O 2, the type 1 Cu Cys484 was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme. Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity. One of these mutants (His 126Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p). Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His126Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites. The trinuclear clusters in T1D and T1DT3' Fet3p react with O2 to produce peroxide intermediates analogous to that observed in T1Hg laccase. Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate. In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH. In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's. This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond.
Lígia O. Martins - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of the Multicopper Oxidase from the pathogenic bacterium Campylobacter jejuni CGUG11284: characterization of a metallo-Oxidase
Metallomics : integrated biometal science, 2011Co-Authors: Catarina S. Silva, Lígia O. Martins, Paulo Durão, Amanda Fillat, Peter F. Lindley, Isabel BentoAbstract:Multicopper Oxidases are a multi-domain family of enzymes that are able to couple oxidation of substrates with reduction of dioxygen to water. These enzymes are capable of oxidizing a vast range of substrates, varying from aromatic to inorganic compounds such as metals. This metallo-Oxidase activity observed in several members of this family has been linked to mechanisms of homeostasis in different organisms. Recently, a periplasmic Multicopper Oxidase, encoded by Campylobacter jejuni, has been characterised and associated with copper homeostasis and with the protection against oxidative stress as it may scavenge metallic ions into their less toxic form and also inhibit the formation of radical oxygen species. In order to contribute to the understanding of its functional role, the crystal structure of the recombinant McoC (Campylobacter jejuni CGUG11284) has been determined at 1.95 A resolution and its structural and biochemical characterizations undertaken. The results obtained indicate that McoC has the characteristic fold of a laccase having, besides the catalytic centres, another putative binding site for metals. Indeed, its biochemical and enzymatic characterization shows that McoC is essentially a metallo-Oxidase, showing low enzymatic efficiency towards phenolic substrates.
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the Multicopper Oxidase from the archaeon pyrobaculum aerophilum shows nitrous oxide reductase activity
FEBS Journal, 2010Co-Authors: Auguste Fernandes, João M. Damas, Smilja Todorovic, Rebecca Pogni, Robert Huber, Claudio M Soares, Camilla M Baratto, Lígia O. MartinsAbstract:The Multicopper Oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) was overproduced in Escherichia coli and purified to homogeneity. The enzyme consists of a single 49.6 kDa subunit, and the combined results of UV-visible, CD, EPR and resonance Raman spectroscopies showed the characteristic features of the Multicopper Oxidases. Analysis of the McoP sequence allowed its structure to be derived by comparative modeling methods. This model provided a criterion for designing meaningful site-directed mutants of the enzyme. McoP is a hyperthermoactive and thermostable enzyme with an optimum reaction temperature of 85 degrees C, a half-life of inactivation of approximately 6 h at 80 degrees C, and temperature values at the midpoint from 97 to 112 degrees C. McoP is an efficient metallo-Oxidase that catalyzes the oxidation of cuprous and ferrous ions with turnover rate constants of 356 and 128 min(-1), respectively, at 40 degrees C. It is noteworthy that McoP follows a ping-pong mechanism, with three-fold higher catalytic efficiency when using nitrous oxide as electron acceptor than when using dioxygen, the typical oxidizing substrate of Multicopper Oxidases. This finding led us to propose that McoP represents a novel archaeal nitrous oxide reductase that is most probably involved in the final step of the denitrification pathway of P. aerophilum.
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The Multicopper Oxidase from the archaeon Pyrobaculum aerophilum shows nitrous oxide reductase activity
FEBS Journal, 2010Co-Authors: Auguste Fernandes, João M. Damas, M. Camilla Baratto, Smilja Todorovic, Rebecca Pogni, Robert Huber, Claudio M Soares, Lígia O. MartinsAbstract:The Multicopper Oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) was overproduced in Escherichia coli and purified to homogeneity. The enzyme consists of a single 49.6 kDa subunit, and the combined results of UV–visible, CD, EPR and resonance Raman spectroscopies showed the characteristic features of the Multicopper Oxidases. Analysis of the McoP sequence allowed its structure to be derived by comparative modeling methods. This model provided a criterion for designing meaningful site-directed mutants of the enzyme. McoP is a hyperthermoactive and thermostable enzyme with an optimum reaction temperature of 85 °C, a half-life of inactivation of ∼ 6 h at 80 °C, and temperature values at the midpoint from 97 to 112 °C. McoP is an efficient metallo-Oxidase that catalyzes the oxidation of cuprous and ferrous ions with turnover rate constants of 356 and 128 min−1, respectively, at 40 °C. It is noteworthy that McoP follows a ping-pong mechanism, with three-fold higher catalytic efficiency when using nitrous oxide as electron acceptor than when using dioxygen, the typical oxidizing substrate of Multicopper Oxidases. This finding led us to propose that McoP represents a novel archaeal nitrous oxide reductase that is most probably involved in the final step of the denitrification pathway of P. aerophilum.