CuA Center Research Articles

New Dielis species and structural dichotomy of the mitochondrial cox2 gene in Scoliidae wasps

Some mitochondrial protein-coding genes of protists and land plants have split over the course of evolution into complementary genes whose products can form heteromeric complexes that likely substitute for the undivided proteins. One of these genes, cox2, has also been found to have split in animals, specifically in Scoliidae wasps (Hymenoptera: Apocrita) of the genus Dielis (Campsomerini), while maintaining the conventional structure in related Scolia (Scoliini). Here, a hitherto unrecognized Nearctic species of Dielis, D. tejensis, is described based on its phenotype and mtDNA. The mitogenome of D. tejensis sp. nov. differs from that of the sympatric sibling species Dielis plumipes fossulana by the reduced size of the cox2-dividing insert, which, however, still constitutes the fifth part of the mtDNA; an enlarged nad2-trnW intergenic region; the presence of two trnKttt paralogues; and other features. Both species of Dielis have a unique insertion of a threonine in COXIIA, predicted to be involved in COXIIA-COXIIB docking, and substitutions of two hydrophobic residues with redox-active cysteines around the CuA centre in COXIIB. Importantly, the analysis of mtDNA from another Campsomerini genus, Megacampsomeris, shows that its cox2 gene is also split. The presented data highlight evolutionary processes taking place in hymenopteran mitogenomes that do not fall within the mainstream of animal mitochondrion evolution.

Spectroscopic and electrochemical studies of the pH-Induced transition in the CuA centre from Thermus thermophilus

A detailed study of the pH induced conformational changes at the Cu2S2 coordination site of the CuA centre of cytochrome oxidase from Thermus thermophilus has been carried out to unravel the role of the pH-induced changes in the coordination geometry on the redox potential and electron transfer kinetics of the metal centre. The pH dependence of the UV–visible absorption bands as well as of the visible circular dichroism spectra corresponding to the metal centre of the protein could be analyzed by the model involving two protonation/deprotonation steps associated with the pKa values of 3.5 and 9.6. The redox potential of the protein determined by direct electrochemistry also showed analogous pH dependence. The results suggested that the metal centre undergoes equilibrium conformational transition involving three forms, viz., ‘low pH’ form, ‘neutral pH’ form and ‘high-pH’ form characterized by different redox potentials of the metal centre. The rates of heterogeneous electron transfer were found to decrease drastically at low pH as well as at high pH. The electron transfer from cytochrome c to the CuA site of the cytochrome oxidase is proposed to be gated by conformation changes at the CuA site through a protonation-induced conformation change, which may act as a sensor for optimum proton transfer across the enzyme. The results also have been discussed in the light of understanding fast electron transfer from the CuA to the heme a in the intact enzyme.

Identification of a copper ion recognition peptide sequence in the subunit II of cytochrome c oxidase: a combined theoretical and experimental study

The role of the pentapeptide, NHSFM, derived from the surface exposed part of the metal ion binding loop of the subunit II of cytochrome c oxidase on the maturation of the binuclear purple CuA center of the enzyme has been investigated using several experimental and computational methods. The copper ion was found to form 1:1 complex of the pentapeptide with a binding constant ~ 104M-1 to 105M-1,where a 4 ligand coordination from the peptide in a type 2 copper center was revealed. The pH dependence of the metal-peptide was associated with a [Formula: see text] of ~ 10 suggesting deprotonation of the N-terminal amine. EXAFS studies as well as DFT calculations of the metal-peptide complexes revealed pH dependent changes in the metal-ligand bond distances. Spectroscopic properties of the metal peptides calculated from TDDFT studies agreed with the experimental results. Restrained molecular dynamics (RMD) simulations indicated coordination of a carbonyl oxygen from the asparagine (N) side chain and of water molecules apart from histidine (H), methionine (M)and terminal amine of asparagine (N) in a distorted square planar geometry of Cu-NHSFM. Analyses of the backbone distances as well as B-factors for the metal peptide suggested that the peptide backbone becomes more compact and rigid on binding of the metal ion. This indicated that binding of copper ion to this pentapeptide in the protein possibly cause movement of the protein backbone bringing other coordinating residues closer to the copper ion, and thus helping in sequential uptake of copper ions to the protein.

YtkA (CtaK) and YozB (CtaM) function in the biogenesis of cytochrome c oxidase in Bacillus subtilis.

Cytochrome c oxidase in the respiratory chain of bacteria and mitochondria couples the reduction of molecular oxygen to form water with the generation of a transmembrane proton gradient. Bacillus subtilis has two heme A-containing heme-copper oxidases: the menaquinol oxidase cytochrome aa3 and the cytochrome c oxidase cytochrome caa3 . By screening three collections of mutants for defective cytochrome c oxidase, we found the genes for two, new membrane-bound assembly factors in B. subtilis: ytkA and yozB (renamed ctaK and ctaM, respectively). CtaK is a lipoprotein without sequence similarity to any protein of known function. We show that CtaK functions together with Sco1 (YpmQ) in a pathway, leading to the assembly of the CuA center in cytochrome caa3 and seems to be a functional analogue to proteins of the periplasmic CuA chaperone family (PCuA C). CtaM is required for the activity of both cytochrome caa3 and cytochrome aa3 and dispensable for the insertion of heme A into these oxidases. The orthologous Bacillus anthracis protein and the distantly related Staphylococcus aureus CtaM complemented CtaM deficiency in B. subtilis, establishing a common function of CtaM in these bacteria. As the overall result of our work, 12 different proteins are known to function in the biosynthesis of cytochrome c oxidase in B. subtilis.

A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold.

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here, we present a fully functional CuA center designed in a structurally nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.

Purple Mixed-Valent Copper A.

CuA is a binuclear copper center acting as an electron transfer hub in terminal oxidases such as cytochrome c oxidase and nitrous oxide reductase. Its unique electronic structure is intimately linked to its function and has puzzled the community of biological inorganic chemistry for decades. Here we review the insights provided by different spectroscopic techniques of CuA centers, and the different experimental approaches to tackle its study, that encompass the synthesis of model compounds as well as protein engineering efforts. The contribution of the electronic structure to the thermodynamic and kinetic of electron transfer is extensively discussed. We also describe the proposed mechanism of CuAassembly in different organisms. The recent discovery of a novel CuA site opens new perspectives to this field.

Modulation of Functional Features in Electron Transferring Metalloproteins

Electron transferring metalloproteins are typically implicated in shuttling electrons between energy transduction chains membrane complexes, such as in (aerobic and anaerobic) respiration and photosynthesis, among other functions. The thermodynamic and kinetic electron transfer parameters of the different metalloproteins need to be adjusted in each case to the specific demands, which can be quite diverse among organisms. Notably, biology utilizes very few metals, essentially iron and copper, to cover this broad range of redox needs imposed by biodiversity. Here, I will describe some crucial structural and dynamical characteristics that modulate the electron transfer parameters (and alternative functions) of two prototypical metalloproteins: the iron protein cytochrome c and its redox partner, the CuA center of the terminal respiratory enzyme cytochrome c oxidase. Specifically, I will focus on summarizing results obtained in recent years in my laboratory.

COA6 Facilitates Cytochrome c Oxidase Biogenesis as Thiol-reductase for Copper Metallochaperones in Mitochondria

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2–SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis.

PH-Induced Binding of the Axial Ligand in an Engineered CuA Site Favors the πu State.

CuA centers perform efficient long-range electron transfer. The electronic structure of native CuA sites can be described by a double-potential well with a dominant σu* ground state in fast equilibrium with a less populated πu ground state. Here, we report a CuA mutant in which a lysine was introduced in the axial position. This results in a highly unstable protein with a pH-dependent population of the two ground states. Deep analysis of the high-pH form of this variant shows the stabilization of the πu ground state due to direct binding of the Lys residue to the copper center that we attribute to deprotonation of this residue.

The phenomenon of UV-Vis spectroscopic changing due the binding of CO to the nitric oxide reductase from Bacillus Azotoformans

Gram-positive bacterium, Bacillus azotoformans, possess a nitric oxide reductase named CuANOR (formerly qCuANOR). The purified enzyme consists of two subunits. On the basis of UV-Vis and EPR spectra, it was elucidated that its small subunit contains the mixed-valence dinuclear CuA center and its large subunit of CuANOR contains a low-spin heme b centre, a high-spin heme b and one nonheme iron centre. The latter two grouped is an EPR-silent binuclear iron centre where NO reduction occurred. The CuANOR revealed itself as a new class of nitric oxide reductase, a new member of cytochrome c oxidase superfamily. The structure of CuANOR is not available yet. Previous studies on Raman resonance and FTIR demonstrated the capability of both heme iron and non heme-iron to bind one CO molecule each in the highly free chloride of dithionite-reduced CuANOR, whereas at high chloride concentration the binding of CO at non-heme iron was lost. Whether the binding of CO to CuANOR is followed by the oxidation of CO to CO2, as found in cytochrome c oxidase, hemoglobin, myoglobin, and free heme, has never been reported. This paper reports the phenomenon of UV-Vis spectroscopic changing due the binding of CO to CuANOR which explain the enzyme reactivity toward CO molecule.

HYSCORE Insights into the Distribution of the Unpaired Spin Density in an Engineered CuA Site in Azurin and Its His120Gly Variant.

A comparative study of the 1H and 14N hyperfine interactions between the CuA site in an engineered CuA center in azurin (WT-CuAAz) and its His120Gly variant (H120G-CuAAz) using the two-dimensional ESEEM technique, HYSCORE, is reported. HYSCORE spectroscopy has clarified conflicting results in previous electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies and found clear differences between the two CuA azurins. Specifically, a hyperfine coupling AN⊥ of 15.3 MHz was determined for the first time from the frequencies of double-quantum transitions of 14N histidine nitrogens coordinated to CuA in WT-CuAAz. In contrast, such coupling was not observed in the spectra of H120G-CuAAz, indicating at least a several megahertz increase in AN⊥ for the coordinated nitrogen in this variant. In addition, 14N HYSCORE spectra of WT-CuAAz show interaction with only one type of weakly coupled nitrogen assigned to the remote Nε atom of coordinated imidazole residues based on the quadrupole coupling constant ( e2 Qq/4 h) of ∼0.4 MHz. The spectrum of H120G-CuAAz resolves additional features typical for backbone peptide nitrogens with larger e2 Qq/4 h values of ∼0.7 MHz. Hyperfine couplings with these nitrogens vary between ∼0.4 and 0.7 MHz. In addition, the two resolved cross-peaks from Cβ protons in H120G-CuAAz display only ∼1 MHz shifts relative to the corresponding peaks in WT-CuAAz. These new findings have provided the first experimental evidence of the previous density functional theory analysis that predicted changes in the delocalized electron spin population of ∼0.02-0.03 (i.e., ∼10%) on copper and sulfur atoms of the CuA center in H120 variants relative to WT-CuAAz and resolved contradicting results between EPR and ENDOR studies of the valence distribution in CuAAz and its variants.

Formation and Electronic Structure of an Atypical CuA Site.

PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper CuA center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD CuA bear similarities to those of canonical CuA sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric CuA site to two mononuclear Cu2+ sites, as well as its electronic and geometric structure, using stopped-flow optical and advanced paramagnetic resonance spectroscopies. PmoD CuA formation occurs rapidly and involves a short-lived intermediate with a λmax of 360 nm. Unlike other CuA sites, the PmoD CuA is unstable, decaying to two type 2 Cu2+ centers. Surprisingly, NMR data indicate that the PmoD CuA has a pure σu* ground state rather than the typical equilibrium between σu* and πu of all other CuA proteins. EPR, ENDOR, ESEEM, and HYSCORE data indicate the presence of two histidine and two cysteine ligands coordinating the CuA core in a highly symmetrical fashion. This report significantly expands the diversity and understanding of known CuA sites.

Connecting CuA with metal centers of heme a, heme a3, CuB and Zn by pathways with hydrogen bond as the bridging element in cytochrome c oxidase

Pathways formed of delocalized π-electron systems and polar groups of polypeptide chains bridged by hydrogen bonds are referred as π-H pathways. Suitable for electron transfer, these pathways in cytochrome c oxidase connect CuA, the source of electrons distributed in cytochrome c oxidase, with the metal centers, heme a, heme a3, CuB, the constituents of the catalytic binuclear center. The unusually rapid electron transfer between heme a and heme a3 would have been facilitated by the link pathway of a long sequence of alternate peptide unit and hydrogen bond spanning Pro336-Val374, referred as suprahelix, between these hemes. Two pathways between CuA center and zinc center, share some portions with purported proton-translocating channels, designated “K” and “D”.

Dramatic Electronic Perturbations of CuA Centers via Subtle Geometric Changes.

CuA is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, CuA is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima, σu* and πu, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states. A series of designed mutants span σu*/πu energy gaps ranging from 900 to 13 cm-1. The smallest gap corresponds to a variant with an effectively degenerate ground state. All engineered sites preserve the mixed-valence character of this metal center and the electron transfer functionality. An increase of the Cu-Cu distance less than 0.06 Å modifies the σu*/πu energy gap by almost 2 orders of magnitude, with longer distances eliciting a larger population of the πu state. This scenario offers a stark contrast to synthetic systems, as model compounds require a lengthening of 0.5 Å in the Cu-Cu distance to stabilize the πu state. These findings show that the tight control of the protein environment allows drastic perturbations in the electronic structure of CuA sites with minor geometric changes.

DEPC modification of the CuA protein from Thermus thermophilus.

The CuA center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the CuA site (the TtCuA protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0-9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCuA increased the reduction potential by over 70mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the CuA site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCuA through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines.

Walking the seven lines: binuclear copper A in cytochrome c oxidase and nitrous oxide reductase.

The enzymes nitrous oxide reductase (N2OR) and cytochrome c oxidase (COX) are constituents of important biological processes. N2OR is the terminal reductase in a respiratory chain converting N2O to N2 in denitrifying bacteria; COX is the terminal oxidase of the aerobic respiratory chain of certain bacteria and eukaryotic organisms transforming O2 to H2O accompanied by proton pumping. Different spectroscopies including magnetic resonance techniques, were applied to show that N2OR has a mixed-valent Cys-bridged [Cu1.5+(CyS)2Cu1.5+] copper site, and that such a binuclear center, called CuA, does also exist in COX. A sequence motif shared between the CuA center of N2OR and the subunit II of COX raises the issue of a putative evolutionary relationship of the two enzymes. The suggestion of a binuclear CuA in COX, with one unpaired electron delocalized between two equivalent Cu nuclei, was difficult to accept originally, even though regarded as a clever solution to many experimental observations. This minireview in honor of Helmut Sigel traces several of the critical steps forward in understanding the nature of CuA in N2OR and COX, and discusses its unique electronic features to some extent including the contributions made by the development of methodology and the discovery of a novel multi-copper enzyme. Left: X-band (9.130 GHz) and C-band (4.530 GHz, 1st harmonic display of experimental spectrum) EPR spectra of bovine heart cytochrome c oxidase, recorded at 20K. Right: Ribbon presentation of the CuA domain in cytochrome c oxidase and nitrous oxide reductase.

The catalytic cycle of nitrous oxide reductase — The enzyme that catalyzes the last step of denitrification

The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper “CuZ center”. This enzyme has been isolated with its catalytic center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), proven to be spectroscopic and structurally different. In the last decades, it has been a challenge to characterize the properties of this complex enzyme, due to the different oxidation states observed for each of its centers and the heterogeneity of its preparations. The substrate binding site in those two “CuZ center” forms and which is the active form of the enzyme is still a matter of debate. However, in the last years the application of different spectroscopies, together with theoretical calculations have been useful in answering these questions and in identifying intermediate species of the catalytic cycle. An overview of the spectroscopic, kinetics and structural properties of the two forms of the catalytic “CuZ center” is given here, together with the current knowledge on nitrous oxide reduction mechanism by nitrous oxide reductase and its intermediate species.

Loss of Human Tyrosinase DOPA Oxidase Activity in Artificial M374 Arg and M 374 Lys Mutants

Tyrosinase (Ec: 1.14.18.1) is a copper - containing enzyme which is distributed in all domains of life such as prokaryote, eukaryote, mammals, invertebrates and plants. Tyrosinase catalyzes the oxidation of monophenols to diphenols and diphenols to o-quinones . The tyrosinase crystallographic data shows two histidine -rich regions named CuA and CuB. A loop containing residues M374, S375 and V377 connects the CuA and CuB Centers. This loop is essential for stability of the enzyme. In this study, site directed mutagenesis was used for the replacement of M374 by Arginine and Lysine.in synthesized cDNA cloned in pET 28b (+) . These mutations don't affect the orientation of the Histidin 367(H367) side chain, resulting in loss of activity.

Copper(I) Complexes with Thiourea Derivatives as Ligands: Revealing Secrets of Their Bonding Scheme

We have synthesized and characterized five copper(I) complexes with unsymmetrically substituted thiourea ligands. These include two coordination polymers, [Cu(StuNH)Br]nand [Cu(Stu(Py)NH)Br]n, two dinuclear complexes, [Cu2(StuNH)4Br2] and [Cu2(Stu(Py)NH)2Br2(MeCN)2], and one hexanuclear complex, [Cu6(StuN)6]. The Cu atoms of the coordination polymers are connected through alternating thione and bromide bridges and the dimeric structures through thione bridges. The thione‐bridged structures show similarities with the biologically highly relevant CuAcenter in its fully reduced oxidation state. Three of the complexes were further investigated by using DFT, including a full orbital analysis utilizing fragment orbitals (FOs) and overlap populations (OPs). The bonding motifs of the dinuclear complexes are similar to that of the CuAcomplex, whereas the analysis of the hexanuclear species showed the influence of the ligands on the core and hence on its electronic structure. Moreover, a comparative analysis of related dinuclear and hexanuclear copper–sulfur systems is presented.

Probing the role of the backbone carbonyl interaction with the CuA center in azurin by replacing the peptide bond with an ester linkage.

The role of a backbone carbonyl interaction with an engineered CuA center in azurin was investigated by developing a method of synthesis and incorporation of a depsipeptide where one of the amide bonds in azurin is replaced by an ester bond using expressed protein ligation. Studies by electronic absorption and electron paramagnetic resonance spectroscopic techniques indicate that, while the substitution does not significantly alter the geometry of the site, it weakens the axial interaction to the CuA center and strengthens the Cu-Cu bond, as evidenced by the blue shift of the near-IR absorption that has been assigned to the Cu-Cu ψ → ψ* transition. Interestingly, the changes in the electronic structure from the replacement did not result in a change in the reduction potential of the CuA center, suggesting that the diamond core structure of Cu2SCys2 is resistant to variations in axial interactions.