Mutations at Asp112 adjacent to the trinuclear Cu center in CueO as the proton donor in the four-electron reduction of dioxygen

Asp112 adjacent to the trinuclear Cu center of a multicopper oxidase, CueO was mutated for Glu, Ala and Asn. Mutations on Asp112 affected not only spectroscopic and magnetic properties derived from the trinuclear Cu center but also enzyme activities. The uncoordinated Asp112 was found to play multiple roles to promote the binding of dioxygen at the trinuclear Cu center and to accelerate the conversion of dioxygen to water molecules by facilitating the supply of H+ to the reaction intermediates.     

On the lability and functional significance of the type 1 copper pool in ceruloplasmin

 The possibility that ceruloplasmin (CP) functions as a copper transferase has fueled a continuing interest in studies of the copper release process. The principal goal of the current investigation has been to identify the most labile copper centers in sheep protein. In fact, subjecting the enzyme to a slow flux of cyanide at pH 5.2 under nitrogen in the presence of ascorbate and a phenanthroline ligand produces partially demetalated forms of the protein. By standard chromatographic techniques it is possible to isolate protein with a Cu/CP ratio of ∼4 or ∼5 as opposed to the native protein which has Cu/CP=5.8. In contrast to other blue oxidases, analysis suggests that CP preferentially loses its type 1 coppers under these conditions. Thus, the spectroscopic signals from the type 1 centers exhibit a loss of intensity while the EPR signal of the type 2 copper becomes stronger. Furthermore, the Cu/CP≈4 and Cu/CP≈5 components retain about 50% of the activity of the native protein, consistent with an intact type 2/type 3 cluster. All three type 1 copper sites appear to suffer copper loss. Reconstitution with a copper(I) reagent restores the spectroscopic properties of the native protein and 90% of the original activity. The results suggest a possible functional significance for the presence of three type 1 coppers in CP. By employing a pool of redox-active but relatively labile type 1 copper centers, the enzyme can serve as a copper donor, if necessary, without completely sacrificing its oxidase activity. Received: 15 February 1999 / Accepted: 22 April 1999     

Spectroscopic analysis of the trinuclear cluster in the Fet3 protein from yeast, a multinuclear copper oxidase

The Fet3 protein (Fet3p) is a multinuclear copper oxidase essential for high-affinity iron uptake in yeast. Fet3p contains one type 1, one type 2, and a strongly antiferromagnetically coupled binuclear Cu(II)-Cu(II) type 3 copper. The type 2 and type 3 sites constitute a structurally distinct trinuclear cluster at which dioxygen is reduced to water. In Fet3p, as in ceruloplasmin, Fe(II) is oxidized to Fe(III) at the type 1 copper; this is the ferroxidase reaction that is fundamental to the physiologic function of these two enzymes. Using site-directed mutagenesis, we have generated type 1-depleted (T1D), type 2-depleted (T2D), and T1D/T2D mutants. None were active in the essential ferroxidase reaction catalyzed by Fet3p. However, the spectroscopic signatures of the remaining Cu(II) sites in any one of the three mutants were indistinguishable from those exhibited by the wild type. Although the native protein and the T1D mutant were isolated in the completely oxidized Cu(II) form, the T2D and T1D/T2D mutants were found to be completely reduced. This result is consistent with the essential role of the type 2 copper in dioxygen turnover, and with the suggestions that cuprous ion is the valence state of intracellular copper. Although stable to dioxygen, the Cu(I) sites in both proteins were readily oxidized by hydrogen peroxide. The double mutant was extensively analyzed by X-ray absorption spectroscopy. Edge and near-edge features clearly distinguished the oxidized from the reduced form of the binuclear cluster. EXAFS was strongly consistent with the expected coordination of each type 3 copper by three histidine imidazoles. Also, copper scattering was observed in the oxidized cluster along with scattering from a ligand corresponding to a bridging oxygen. The data derived from the reduced cluster indicated that the bridge was absent in this redox state. In the reduced form of the double mutant, an N/O ligand was apparent that was not seen in the reduced form of the T1D protein. This ligand in T1D/T2D could be either the remaining type 2 copper imidazole ligand (from His416) or a water molecule that could be stabilized at the type 3 cluster by H-bonding to this side chain. If present in the native protein, this H(2)O could provide acid catalysis of dioxygen reduction at the reduced trinuclear center.     

Molecular Modeling of the Ternary Complex of Rusticyanin-Cytochrome c4-Cytochrome Oxidase: An Insight to Possible H-Bond Mediated Recognition and Electron Transfer Reaction in T.ferrooxidans

Abstract

The metabolism of Thiobacillus ferrooxidans involves electron transfer from the Fe⁺² ions in the extracellular environment to the terminal oxygen in the bacterial cytoplasm through a series of periplasmic proteins like Rusticyanin (RCy), Cytochrome (Cyt c₄), and Cytochrome oxidase (CcO). The energy minimization and MD studies reveal the stabilization of the three redox proteins in their ternary complex through the direct and water mediated H-bonds and electrostatic interaction. The surface exposed polar residues of the three proteins, i.e., RCy (His 143, Thr 146, Lys 81, Glu 20), Cyt c₄ (Asp 5, 15, 52, Ser 14, Glu 61), and CcO (Asp 135, Glu 126, 140, 142, Thr 177) formed the intermolecular hydrogen bonds and stabilized the ternary complex. The oxygen (O^εl) of Glu 126, 140, and 142 on subunit II of the CcO interact to the exposed side-chain and O^b atoms of the Asp 52 of Cyt c₄ and Glu 20 and Leu 12 of RCy. The Asp 135 of subunit II also forms H-bond with the N^ε atom of Lys 81 of RCy. The O^εl of Glu 61 of Cyt c₄ is also H-bonded to O^γ atom of Thr 177 of CcO. Solvation followed by MD studies of the ternary protein complex revealed the presence of seven water molecules in the interfacial region of the interacting proteins. Three of the seven water molecules (W 79, W 437, and W 606) bridged the three proteins by forming the hydrogen bonded network (with the distances ～ 2.10–2.95 Å) between the Lys 81 (RCy), Glu 61 (Cyt c₄), and Asp 135 (CcO). Another water molecule W 603 was H-bonded to Tyr 122 (CcO) and interconnected the Lys 81 (RCy) and Asp 135 (CcO) through the water molecules W 606 and W 437. The other two water molecules (W 21 and W 455) bridged the RCy to Cyt c₄ through H-bonds, whereas the remaining W 76 interconnected the His 53 (Cytc₄) to Glu 126 (CcO) with distances ～ 2.95–3.0 Å.     

Spectroscopic characterization and O2 reactivity of the trinuclear Cu cluster of mutants of the multicopper oxidase Fet3p

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 H(2)O. 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 Cys(484) 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(126)Gln) 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 His(126)Gln 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 O(2) 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.     

Molecular modeling of the ternary complex of Rusticyanin-cytochrome c4-cytochrome oxidase: an insight to possible H-bond mediated recognition and electron transfer reaction in T. ferrooxidans

The metabolism of Thiobacillus ferrooxidans involves electron transfer from the Fe+2 ions in the extracellular environment to the terminal oxygen in the bacterial cytoplasm through a series of periplasmic proteins like Rusticyanin (RCy), Cytochrome (Cyt c4), and Cytochrome oxidase (CcO). The energy minimization and MD studies reveal the stabilization of the three redox proteins in their ternary complex through the direct and water mediated H-bonds and electrostatic interaction. The surface exposed polar residues of the three proteins, i.e., RCy (His 143, Thr 146, Lys 81, Glu 20), Cyt c4 (Asp 5, 15, 52, Ser 14, Glu 61), and CcO (Asp 135, Glu 126, 140, 142, Thr 177) formed the intermolecular hydrogen bonds and stabilized the ternary complex. The oxygen (Oepsilon1) of Glu 126, 140, and 142 on subunit II of the CcO interact to the exposed side-chain and Ob atoms of the Asp 52 of Cyt c4 and Glu 20 and Leu 12 of RCy. The Asp 135 of subunit II also forms H-bond with the Nepsilon atom of Lys 81 of RCy. The Oepsilon1 of Glu 61 of Cyt c4 is also H-bonded to Ogamma atom of Thr 177 of CcO. Solvation followed by MD studies of the ternary protein complex revealed the presence of seven water molecules in the interfacial region of the interacting proteins. Three of the seven water molecules (W 79, W 437, and W 606) bridged the three proteins by forming the hydrogen bonded network (with the distances approximately 2.10-2.95 A) between the Lys 81 (RCy), Glu 61 (Cyt c4), and Asp 135 (CcO). Another water molecule W 603 was H-bonded to Tyr 122 (CcO) and interconnected the Lys 81 (RCy) and Asp 135 (CcO) through the water molecules W 606 and W 437. The other two water molecules (W 21 and W 455) bridged the RCy to Cyt c4 through H-bonds, whereas the remaining W 76 interconnected the His 53 (Cytc4) to Glu 126 (CcO) with distances approximately 2.95-3.0 A.     

Point mutations at the type I Cu ligands, Cys457 and Met467, and at the putative proton donor, Asp105, in Myrothecium verrucaria bilirubin oxidase and reactions with dioxygen

The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.     

Angular trinuclear copper(II) complexes of N4O-donor ligand: Syntheses, crystal structures and magnetic properties

Two trinuclear copper(II) complexes of a Schiff-base type N₄O-donor ligand (LH) derived from 4,4,9,9-tetramethyl-5,8-diazadodecane-2,11-dione and 1,3-diaminopropan-2-ol are reported. Complex [Cu₃L₂(ClO₄)₄] (1) has an angular C₂-symmetric trinuclear core as revealed from single-crystal X-ray diffraction studies. The terminal coppers are in square-pyramidal geometry with an N₃O₂ coordination environment while the central one is in octahedral geometry with an N₂O₄ donor environment. Complex [Cu₃L₂(ClO₄)(N₃)(H₂O)](ClO₄)₂ · H₂O (2) has an unsymmetrical trinuclear core with an intramolecular hydrogen bonding interaction between the water and azide anion coordinated to Cu(1) and Cu(3) center, respectively. All the copper centers in 2 are in square-pyramidal geometry. The average Cu?Cu distance between closest metal ions in both the complexes is 3.897 Å. The coordination environment of coppers in 1 approximately mimics that of multicopper oxidases in the oxidized form and the environment in 2 mimics that of the azide derivative of ascorbate oxidase. Both 1 and 2 exhibit doublet spin ground state due to strong antiferromagnetic coupling operating through the alkoxo-bridged oxygen atoms between the copper centers.     

Modeling study of Rusticyanin-Cytochrome C(4) complex: an insight to possible H-bond mediated recognition and electron--transfer process

Rusticyanin (RCy) mediated transfer of electron to Cytochrome C(4) (Cytc(4)) from the extracellular Fe(+2) ion is primarily involved in the Thiobacillus ferrooxidans induced bio-leaching of pyrite ore and also in the metabolism of this acidophilic bacteria. The modeling studies have revealed the two possible mode of RCy-Cytc(4) complexation involving nearly the same stabilization energy approximately -15 x 10(3) kJ/mol, one through N-terminal Asp 15 and another -C terminal Glu 121 of Cytc(4) with the Cu-bonded His 143 of RCy. The Asp 15:His 143 associated complex (DH) of Cytc(4)-RCy was stabilized by the intermolecular H-bonds of the carboxyl oxygen atoms O(delta1) and O(delta2) of Asp 15 with the Nepsilon-atom of His 143 and O(b) atoms of Ala 8 and Asp 5 (of Cytc(4)) with the Thr 146 and Phe 51 (of RCy). But the other Glu 121:His 143 associated complex (EH) of Cytc(4)-RCy was stabilized by the H-bonding interaction of the oxygen atoms O(epsilon1) and O(epsilon2) of Glu 121 with the Nepsilon and Ogamma atoms of His 143 and Thr 146 of RCy. The six water molecules were present in the binding region of the two proteins in the energy minimized autosolvated DH and EH-complexes. The MD studies also revealed the presence of six interacting water molecules at the binding region between the two proteins in both the complexes. Several residues Gly 82 and 84, His 143 (RCy) were participated through the water mediated (W 389, W 430, W 413, W 431, W 373, and W 478) interaction with the Asp 15, Ile 82, and 62, Tyr 63 (Cytc(4)) in DH complex, whereas in EH complex the Phe 51, Asn 80, Tyr 146 (RCy) residues were observed to interact with Asn 108, Met 120, Glu 121 (of Cytc(4)) through the water molecules W 507, W 445, W 401, W 446, and W 440. The direct water mediated (W 478) interaction of His 143 (RCy) to Asp 15 (of Cytc(4)) was observed only in the DH complex but not in EH. These direct and water mediated H-bonding between the two respective proteins and the binding free energy with higher interacting buried surface area of the DH complex compare to other EH complex have indicated an alternative possibility of the electron transfer route through the interaction of His 143 of RCy and the N-terminal Asp 15 of Cytc(4).     

An insight to the conserved water mediated dynamics of catalytic His88 and its recognition to thyroxin and RBP binding residues in human transthyretin

Human transthyretin (hTTR) is a multifunctional protein involved in several amyloidogenic diseases. Besides transportation of thyroxin and vitamin-A, its role towards the catalysis of apolipoprotein-A1 and Aβ-peptide are also drawing interest. The role of water molecules in the catalytic mechanism is still unknown. Extensive analyses of 14 high-resolution X-ray structures of human transthyretin and MD simulation studies have revealed the presence of eight conserved hydrophilic centres near its catalytic zone which may be indispensable for the function, dynamics and stability of the protein. Three water molecules (W1, W2 and W3) form a cluster and play an important role in the recognition of the catalytic and RBP-binding residues. They also induce the reorganisation of the His88 for coupling with other catalytic residues (His90, Glu92). Another water molecule (W5) participate in inter-monomer recognition between the catalytic and thyroxin binding sites. The rest four water molecules (W6, W*, W^# and W^?) form a distorted tetrahedral cluster and impart stability to the catalytic core of hTTR. The conserved water mediated recognition dynamics of the different functional sites may provide some rational clues towards the understanding of the activity and mechanism of hTTR.     

Discrete roles of copper ions in chemical unfolding of human ceruloplasmin

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six beta-barrel domains with one type 1 copper in each of domains 2, 4, and 6; the remaining copper ions form a catalytic trinuclear cluster, one type 2 and two type 3 coppers, at the interface between domains 1 and 6. We have characterized urea-induced unfolding of holo- and apo-forms of CP by far-UV circular dichroism, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid binding, visible absorption, copper content, and oxidase activity probes (pH 7, 23 degrees C). We find that holo-CP unfolds in a complex reaction with at least one intermediate. The formation of the intermediate correlates with decreased secondary structure, exposure of aromatics, loss of two coppers, and reduced oxidase activity; this step is reversible, indicating that the trinuclear cluster remains intact. Further additions of urea trigger complete protein unfolding and loss of all coppers. Attempts to refold this species result in an inactive apoprotein with molten-globule characteristics. The apo-form of CP also unfolds in a multistep reaction, albeit the intermediate appears at a slightly lower urea concentration. Again, correct refolding is possible from the intermediate but not the unfolded state. Our study demonstrates that in vitro equilibrium unfolding of CP involves intermediates and that the copper ions are removed in stages. When the catalytic site is finally destroyed, refolding is not possible at neutral pH. This implies a mechanistic role for the trinuclear metal cluster as a nucleation point, aligning domains 1 and 6, during CP folding in vivo.     

Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding

Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.     

Crystal structure of a blue laccase from <Emphasis Type="Italic">Lentinus tigrinus</Emphasis>: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases

Background  

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O₂ reduction to H₂O.     

Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer

Background  

Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction of oxygen to water occurs at the trinuclear centre.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO

CueO, a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present. The 1.7-A resolution structure of a crystal soaked in CuCl2 reveals a Cu(II) ion bound to the protein 7.5 A from the T1 copper site in a region rich in methionine residues. The trigonal bipyramidal coordination sphere is unusual, containing two methionine sulfur atoms, two aspartate carboxylate oxygen atoms, and a water molecule. Asp-439 both ligates the labile copper and hydrogen-bonds to His-443, which ligates the T1 copper. This arrangement may mediate electron transfer from substrates to the T1 copper. Mutation of residues bound to the labile copper results in loss of oxidase activity and of copper tolerance, confirming a regulatory role for this site. The methionine-rich portion of the protein, which is similar to that of other proteins involved in copper homeostasis, does not display additional copper binding. The type 3 copper atoms of the trinuclear cluster in the structure are bridged by a chloride ion that completes a square planar coordination sphere for the T2 copper atom but does not affect oxidase activity.     

Discovery of native metal ion sites located on the ferredoxin docking side of photosystem I

Photosystem I (PSI) is a large membrane protein that catalyzes light-driven electron transfer across the thylakoid membrane from plastocyanin located in the lumen to ferredoxin in the stroma. Metal analysis reveals that PSI isolated from the cyanobacterial membranes of Synechococcus leopoliensishas a near-stoichiometric 1 molar equiv of Zn (2+) per PSI monomer and two additional surface metal ion sites that favor Cu (2+) binding. Two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy reveals coupling to the so-called remote nitrogen of a single histidine coordinated to one of the Cu (2+) centers. EPR and X-ray absorption fine structure (XAFS) studies of 2Cu-PSI complexes reveal the direct interaction of ferredoxin with the Cu (2+) centers on PSI, establishing the location of native metal sites on the ferredoxin docking side of PSI. On the basis of these spectroscopic results and previously reported site-directed mutagenesis studies, inspection of the PSI crystal structure reveals a cluster of three highly conserved residues, His(D95), Glu(D103), and Asp(C23), as a likely Cu (2+) binding site. The discovery of surface metal sites on the acceptor side of PSI provides a unique opportunity to probe the stromal region of PSI and the interactions of PSI with its reaction partner, the soluble electron carrier protein ferredoxin.     

Simulation of the cavity-binding site of three bacterial multicopper oxidases upon complex stabilization: interactional profile and electron transference pathways

Previous studies have shown that multicopper oxidases (MCOs) oxidize organic and inorganic compounds through oxidation–reduction reactions in which three structurally and functionally arranged copper centers coordinate the uptake of an electron from a reduced substrate. Structural comparisons among three bacterial MCOs, with high structural homology and available three-dimensional information, reveal that the primary structural differences between these MCOs are located near the mononuclear copper center (T1Cu), where substrate oxidation occurs, as opposed to where the reduction of oxygen to water occurs at the trinuclear center. Nevertheless, this substrate oxidation is achieved through an outer-sphere electron transfer mechanism that does not generate a stable substrate–enzyme complex. In this study, MCOs from Thermus thermophilus (Tth-MCO), Bacillus subtilis (CotA), and Escherichia coli (CueO), which have been previously determined through X-ray crystallography, were used as models to analyze the binding modes of these MCOs to three organic molecules, with specific interest in the substrate-binding site. The binding mode of the electron-donor molecule to the electron transfer binding site was primarily attributed to hydrophobic contacts, which likely play an important role in the determination of substrate specificity. Some complexes generated in this study showed an electron donor molecule conformation in which an electron could be directly transferred to the histidines coordinating T1Cu, while for others additional electron transference pathways were also possible through the participation of charged residues during electron transfer.     

The structure of Rigidoporus lignosus Laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair

Laccase is a multicopper blue oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols and arylamines. The enzyme is the object of intense biotechnological research, due to its employment in bioremediation of soils and water as well as in other biotechnological applications. We report here the cDNA and protein sequences, the post-translational modifications, the crystallization and X-ray structure determination of a laccase from the white-rot fungus Rigidoporus lignosus. The amino acid residues sequence deduced from cDNA clearly identified a pre-sequence of 21 residues representing the signal for extra-cellular localization. Mass spectrometry analysis performed on the salvage enzyme, confirmed the deduced sequence and precisely mapped two glycosylation sites at Asn337 and Asn435, determining the nature of the bound glycosidic moieties. The crystal structure was determined at 1.7A resolution from perfectly hemihedrally twinned crystals, by molecular replacement technique. While the overall structure closely resembled those reported for other fungal laccases, the analysis of the T2/T3 trinuclear cluster revealed an unprecedented coordination sphere for the T3 copper pair. No bridging oxygen ligand was present between the two T3 copper ions, which were no longer symmetrically coordinated. The observed structure could represent an intermediate along the process of four-electron reduction of oxygen to water taking place at the trinuclear copper cluster.