Blue Copper Site Research Articles

The Impact of Second Coordination Sphere Methionine-Aromatic Interactions in Copper Proteins.

The interplay between the primary and secondary coordination spheres in biological metal sites plays an essential role in controlling their properties. Some of the clearest examples of this are from copper sites in blue and purple copper proteins. Many such proteins contain methionine (Met) in the primary coordination sphere as a weakly bound ligand to Cu. While the effects of replacing the coordinated Met are understood, less so is the importance of its second-sphere interactions. In this combined informatics and experimental study, we first present a bioinformatics investigation of the second-sphere environments in biological Met-Cu motifs. The most common interaction is between the Met-CH3 and the π-face of a phenylalanine (Phe) (81% of surveyed structures), tyrosine (Tyr) (11%), and tryptophan (Trp) (8%). In most cases, the Met-CH3 also forms a contact with a π-face of one of a Cu-ligating histidine-imidazole. Such interactions are widely distributed in different Cu proteins. Second, to explore the impact of the second-sphere interactions of Met, a series of artificial Pseudomonas aeruginosa azurin proteins were produced where the native Phe15 was replaced with Tyr or Trp. The proteins were characterized using optical and magnetic resonance spectroscopies, X-ray diffraction, electrochemistry, and an investigation of the time-resolved electron-transfer kinetics of photosensitizer-modified proteins. The influence of the Cu-Met-Aro interaction on azurin's physical properties is subtle, and the hallmarks of the azurin blue copper site are maintained. In the Phe15Trp variant, the mutation to Phe15 induces changes in Cu properties that are comparable to replacement of the weak Met ligand. The broader impacts of these widely distributed interactions are discussed.

Electrostatic regulation of blue copper sites.

In the last 50 years, the blue copper proteins became central targets of investigation. Extensive experiments focused on the Cu coordination to probe the effect of local perturbations on its properties. We found that local electric fields, generated by charged residues evolutionarily placed throughout the protein edifice, mainly second sphere, but also more remotely, constitute an additional significant factor regulating blue copper proteins. These fields are not random, but exhibit a highly specific directionality, negative with respect to the and vectors in the Cu first shell. The field magnitude contributes to fine-tuning of the geometric and electronic properties of Cu sites in individual blue copper proteins. Specifically, the local electric fields evidently control the Cu–SMet bond distance, Cu(ii)–SCys bond covalency, and the energies of the frontier molecular orbitals, which, in turn, govern the Cu(ii/i) reduction potential and the relative absorption intensities at 450 nm and 600 nm.

Traversing the Red-Green-Blue Color Spectrum in Rationally Designed Cupredoxins.

Blue copper proteins have a constrained Cu(II) geometry that has proven difficult to recapitulate outside native cupredoxin folds. Previous work has successfully designed green copper proteins which could be tuned blue using exogenous ligands, but the question of how one can create a self-contained blue copper site within a de novo scaffold, especially one removed from a cupredoxin fold, remained. We have recently reported a red copper protein site within a three helical bundle scaffold which we later revisited and determined to be a nitrosocyanin mimic, with a CuHis2CysGlu binding site. We now report efforts to rationally design this construct toward either green or blue copper chromophores using mutation strategies that have proven successful in native cupredoxins. By rotating the metal binding site, we created a de novo green copper protein. This in turn was converted to a blue copper protein by removing an axial methionine. Following this rational sequence, we have successfully created red, green, and blue copper proteins within an alpha helical fold, enabling comparisons for the first time of their structure and function disconnected from the overall cupredoxin fold.

The Type 1 Blue Copper Site: From Electron Transfer to Biological Function.

Cupredoxins host in their scaffold one of the most studied and interesting metal sites in biology: the type 1 (T1) or blue Cu center. Blue Cu proteins have evolved to play key roles in biological electron transfer and have the ability to react with a wide variety of redox partners. The inner coordination sphere of T1 Cu sites conserves two histidines and one cysteine with a short Cu-S(Cys) bond as ligands in a trigonal arrangement, with a variable axial ligand that modulates the electronic structure and reactivity. The structural, electronic and geometric features of T1 Cu centers provide the basis for a site that can be optimized by the protein structure for each biological function. This chapter highlights the properties that make this unique Cu center in biology an efficient and tunable electron transfer site. The contributions of the first coordination shell and the high covalency of the Cu-S(Cys) bond in the T1 Cu site to its distinctive geometric and spectroscopic features are discussed, as well as the role of the protein scaffold in imposing an 'entatic' state with a distorted tetrahedral geometry that minimizes geometric changes upon redox cycling. The analysis of naturally occurring perturbed blue Cu sites provides further insights into how the protein scaffold can tune the properties of the T1 Cu site. Blue Cu sites display a wide range of reduction potentials, as these are tuned to be consistent with their physiologically relevant electron donors and acceptors. The different properties of the protein matrix that play important roles in finetuning the reduction potential of T1 Cu sites are also discussed, including the nature of the axial ligand and outer coordination sphere effects. These concepts are further illustrated by the discussion of examples of biosynthetic blue Cu proteins. Finally, the different features of the T1 Cu site that make it an optimal site for electron transfer (ET) are discussed, in terms of Markus theory for intra- and inter-molecular ET. The active site in multicopper oxidases is used as an example to illustrate the contributions of the anisotropic covalency of the blue Cu site to an efficient ET, while the diverse reactivity of the T1 Cu sites in these enzymes is discussed to dissect the different properties provided by the protein that help tune these unique sites for biological ET.

Shift from Entropic Cu2+ Binding to Enthalpic Cu+ Binding Determines the Reduction Thermodynamics of Blue Copper Proteins.

The enthalpic and entropic components of Cu2+ and Cu+ binding to the blue copper protein azurin have been quantified with isothermal titration calorimetry (ITC) measurements and analysis, providing the first such experimental values for Cu+ binding to a protein. The high affinity of azurin for Cu2+ is entirely due to a very favorable binding entropy, while its even higher affinity for Cu+ is due to a favorable binding enthalpy and entropy. The binding thermodynamics provide insight into bond enthalpies at the blue copper site and entropic contributions from desolvation and proton displacement. These values were used in thermodynamic cycles to determine the enthalpic and entropic contributions to the free energy of reduction and thus the reduction potential. The reduction thermodynamics obtained with this method are in good agreement with previous results from temperature-dependent electrochemical measurements. The calorimetry method, however, provides new insight into contributions from the initial (oxidized) and final (reduced) states of the reduction. Since ITC measurements quantify the protons that are displaced upon metal binding, the proton transfer that is coupled with electron transfer is also determined with this method. Preliminary results for Cu2+ and Cu+ binding to the Phe114Pro variant of azurin demonstrate the insight about protein tuning of the reduction potential that is provided by the binding thermodynamics of each metal oxidation state.

New molecular packing in a crystal of pseudoazurin from Alcaligenes faecalis: a double-helical arrangement of blue copper.

Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis (AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P61, with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz.

Site-directed mutagenesis of the highly perturbed copper site of auracyanin D

Type-1 copper proteins participate in redox reactions and biological catalysis. Significant variation exists within the electronic structure of type-1 copper sites, producing both blue and green proteins. Classical, “blue” sites have been extensively studied, but “green” sites have been poorly characterized. We recently discovered a green copper protein, called auracyanin D. Here, we report a series of axial ligand mutations in auracyanin D, and characterize the resulting spectral and redox changes. The resulting mutants appear blue, green, and red and vary in redox potential from +56mV to +786mV. This is the largest change in redox potential to date for any type-1 center. We found that in this green protein, modifications of the axial ligand produce significantly larger changes than similar mutations in blue type-1 copper sites.

Multicopper oxidases: intramolecular electron transfer and O2 reduction

The multicopper oxidases are an intriguing, widespread family of enzymes that catalyze the reduction of O2 to water by a variety of single-electron and multiple-electron reducing agents. The structure and properties of the copper binding sites responsible for the latter chemical transformations have been studied for over 40years and a detailed picture is emerging. This review focuses particularly on the kinetics of internal electron transfer between the type1 (blue) copper site and the trinuclear center, as well as on the nature of the intermediates formed in the oxygen reduction process.

Spectroscopic and DFT studies of second-sphere variants of the type 1 copper site in azurin: covalent and nonlocal electrostatic contributions to reduction potentials.

The reduction potentials (E(0)) of type 1 (T1) or blue copper (BC) sites in proteins and enzymes with identical first coordination spheres around the redox active copper ion can vary by ~400 mV. Here, we use a combination of low-temperature electronic absorption and magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, and S K-edge X-ray absorption spectroscopies to investigate a series of second-sphere variants--F114P, N47S, and F114N in Pseudomonas aeruginosa azurin--which modulate hydrogen bonding to and protein-derived dipoles nearby the Cu-S(Cys) bond. Density functional theory calculations correlated to the experimental data allow for the fractionation of the contributions to tuning E(0) into covalent and nonlocal electrostatic components. These are found to be significant, comparable in magnitude, and additive for active H-bonds, while passive H-bonds are mostly nonlocal electrostatic in nature. For dipoles, these terms can be additive to or oppose one another. This study provides a methodology for uncoupling covalency from nonlocal electrostatics, which, when coupled to X-ray crystallographic data, distinguishes specific local interactions from more long-range protein/active interactions, while affording further insight into the second-sphere mechanisms available to the protein to tune the E(0) of electron-transfer sites in biology.

Analysis of resonance Raman data on the blue copper site in pseudoazurin: Excited state π and σ charge transfer distortions and their relation to ground state reorganization energy

The short Cu2+–S(Met) bond in pseudoazurin (PAz) results in the presence of two relatively intense Sp(π) and Sp(σ) charge transfer (CT) transitions. This has enabled resonance Raman (rR) data to be obtained for each excited state. The rR data show very different intensity distribution patterns for the vibrations in the 300–500cm−1 region. Time-dependent density functional theory (TDDFT) calculations have been used to determine that the change in intensity distribution between the Sp(π) and Sp(σ) excited states reflects the differential enhancement of S(Cys) backbone modes with Cu–S(Cys)–Cβ out-of-plane (oop) and in-plane (ip) bend character in their respective potential energy distributions (PEDs). The rR excited state distortions have been related to ground state reorganization energies (λs) and predict that, in addition to M–L stretches, the Cu–S(Cys)–Cβ oop bend needs to be considered. DFT calculations predict a large distortion in the Cu–S(Cys)–Cβ oop bending coordinate upon reduction of a blue copper (BC) site; however, this distortion is not present in the X-ray crystal structures of reduced BC sites. The lack of Cu–S(Cys)–Cβ oop distortion upon reduction corresponds to a previously unconsidered constraint on the thiolate ligand orientation in the reduced state of BC proteins and can be considered as a contribution to the entatic/rack nature of BC sites.

Creation of a Type 1 Blue Copper Site within a de Novo Coiled-Coil Protein Scaffold

Type 1 blue copper proteins uniquely coordinate Cu(2+) in a trigonal planar geometry, formed by three strong equatorial ligands, His, His, and Cys, in the protein. We designed a stable Cu(2+) coordination scaffold composed of a four-stranded α-helical coiled-coil structure. Two His residues and one Cys residue were situated to form the trigonal planar geometry and to coordinate the Cu(2+) in the hydrophobic core of the scaffold. The protein bound Cu(2+), displayed a blue color, and exhibited UV-vis spectra with a maximum of 602-616 nm, arising from the thiolate-Cu(2+) ligand to metal charge transfer, depending on the exogenous axial ligand, Cl(-) or HPO(4)(2-). The protein-Cu(2+) complex also showed unresolved small A(∥) values in the electron paramagnetic resonance (EPR) spectral analysis and a 328 mV (vs normal hydrogen electrode, NHE) redox potential with a fast electron reaction rate. The X-ray absorption spectrum revealed that the Cu(2+) coordination environment was identical to that found in natural type 1 blue copper proteins. The extended X-ray absorption fine structure (EXAFS) analysis of the protein showed two typical Cu-N(His) at around 1.9-2.0 Å, Cu-S(Cys) at 2.3 Å, and a long Cu-Cl at a 2.66 Å, which are also characteristic of the natural type 1 blue copper proteins.

A Magnetic and Electronic Circular Dichroism Study of Azurin, Plastocyanin, Cucumber Basic Protein, and Nitrite Reductase Based on Time-Dependent Density Functional Theory Calculations

The excitation, circular dichroism, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra of small models of four blue copper proteins are simulated on the TDDFT/BP86 level. X-Ray diffraction geometries are used for the modeling of the blue copper sites in azurin, plastocyanin, cucumber basic protein, and nitrite reductase. Comparison with experimental data reveals that the calculations reproduce most of the qualitative trends of the observed experimental spectra with some discrepancies in the orbital decompositions and the values of the excitation energies, the g( parallel) components of the g tensor, and the components of the A tensor. These discrepancies are discussed relative to deficiencies in the time-dependent density functional theory (TDDFT) methodology, as opposed to previous studies which address them as a result of insufficient model size or poor performance of the BP86 functional. In addition, attempts are made to elucidate the correlation between the MCD and EPR signals.

Evaluation of Electronic and Geometrical Properties of the Blue Copper Site in Fully Solvated Azurin by QM/MM Hybrid Calculations Using a New Interface Program Connecting QM and MM Engines

Evaluation of Electronic and Geometrical Properties of the Blue Copper Site in Fully Solvated Azurin by QM/MM Hybrid Calculations Using a New Interface Program Connecting QM and MM Engines

A Proton ENDOR Study of Azurin

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cβ atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein.

Thermodynamic equilibrium between blue and green copper sites and the role of the protein in controlling function

A combination of spectroscopies and density functional theory calculations indicate that there are large temperature-dependent absorption spectral changes present in green nitrite reductases (NiRs) due to a thermodynamic equilibrium between a green and a blue type 1 (T1) copper site. The axial methionine (Met) ligand is unconstrained in the oxidized NiRs, which results in an enthalpically favored (DeltaH approximately 4.6 kcal/mol) Met-bound green copper site at low temperatures, and an entropically favored (TDeltaS approximately 4.5 kcal/mol, at room temperature) Met-elongated blue copper site at elevated temperatures. In contrast to the NiRs, the classic blue copper sites in plastocyanin and azurin show no temperature-dependent behavior, indicating that a single species is present at all temperatures. For these blue copper proteins, the polypeptide matrix opposes the gain in entropy that would be associated with the loss of the weak axial Met ligand at physiological temperatures by constraining its coordination to copper. The potential energy surfaces of Met binding indicate that it stabilizes the oxidized state more than the reduced state. This provides a mechanism to tune down the reduction potential of blue copper sites by >200 mV.

Electronic and geometric structures of the blue copper site of azurin investigated byQM/MM hybrid calculations

The electronic and geometric structures of the copper-binding site in a fully solvated azurinwere investigated using quantum mechanics (QM) and molecular mechanics (MM) hybridcalculations. Two types of computational models were applied to evaluate theeffects of the environment surrounding the active site. In model I, long-distanceelectrostatic interactions between QM region atoms and partial point chargesof the surrounding protein moieties and solvent water were calculated in a QMHamiltonian, for which the spin-unrestricted Hartree–Fock (UHF)/density functionaltheory (DFT) hybrid all-electron calculation with the B3LYP functional wasadopted. In model II, the QM Hamiltonian was not allowed to be polarized by thosepartial point charges. Models I and II provided different descriptions of the coppercoordination structure, particularly for the coordinative bonds including a largedipole. In fact, the Cu–O(Gly45) and Cu–S(Cys112) bonds are sensitive to thetreatment of long-distance electrostatic interactions in the QM Hamiltonian. Thissuggests that biological processes occurring in the active site are regulated by thesurrounding structures of protein and solvent, and therefore the effects of long-rangeelectrostatic interactions involved in the QM Hamiltonian are crucial for accuratedescriptions of electronic structures of the copper active site of metalloenzymes.

Spectroscopic and density functional theory studies of the blue-copper site in M121SeM and C112SeC azurin: Cu-Se versus Cu-S bonding.

S K-edge X-ray absorption, UV-vis absorption, magnetic circular dichroism (MCD), and resonance Raman spectroscopies are used to investigate the electronic structure differences among WT, M121SeM, and C112SeC Pseudomonas aeruginosa (P.a) azurin. A comparison of S K-edge XAS of WT and M121SeM azurin and a CuII-thioether model complex shows that the 38% S character in the ground state wave function of the blue-copper (BC) sites solely reflects the Cu-SCys bond. Resonance Raman (rR) data on WT and C112SeC azurin give direct evidence for the kinematic coupling between the Cu-SCys stretch and the cysteine deformation modes in WT azurin, which leads to multiple features in the rR spectrum of the BC site. The UV-vis absorption and MCD data on WT, M121SeM, and C112SeC give very similar C0/D0 ratios, indicating that the C-term MCD intensity mechanism involves Cu-centered spin-orbit coupling (SOC). The spectroscopic data combined with density functional theory (DFT) calculations indicate that SCys and SeCys have similar covalent interactions with Cu at their respective bond lengths of 2.1 and 2.3 A. This reflects the similar electronegativites of S and Se in the thiolate/selenolate ligand fragment and explains the strong spectroscopic similarities between WT and C112SeC azurin.

The Met Axial Ligand Determines the Redox Potential in CuA Sites

The replacement of the axial methionine ligand in a native CuA protein rendered a series of stable mutants with spectroscopic features of a mixed valence center. The mutations resulted in minor perturbations of the electronic structure of this site but led to significant changes in the redox potential. This suggests that the axial ligand plays a role in tuning the redox potential of CuA, resembling the behavior observed in blue copper sites.

Density Functional Study of EPR Parameters and Spin-Density Distribution of Azurin and Other Blue Copper Proteins

Modern density functional methods have been used to study spin-density distribution, g tensors, as well as Cu and ligand hyperfine tensors for azurin models, for two more blue copper proteins plastocyanin and stellacyanin, and for small model complexes. The aim was to establish a consistent computational protocol that provides a realistic description of the EPR parameters as probes of the spin-density distribution between metal and coordinated ligands in copper proteins. In agreement with earlier conclusions for plastocyanin, hybrid functionals with appreciable exact-exchange admixtures, roughly around 50%, provide the best overall agreement with all parameters. Then the bulk of the spin density is almost equally shared by the copper atom and the sulfur atom of the equatorial cysteine ligand, and the best values are obtained for copper, histidine nitrogen, and cysteine beta-proton hyperfine couplings, as well as for g(parallel). Spin-orbit effects on the EPR parameters may be appreciable and have to be treated carefully to obtain agreement with experiment. Most notably, spin-orbit effects on the (65)Cu hyperfine coupling tensors in blue copper sites are unusually large compared to more regularly coordinated Cu(II) complexes with similar spin density on copper. In addition to the often emphasized high covalency of the Cu-S(Cys) bond, the characteristically small A(parallel) component of blue copper proteins is shown to derive to a large part from a near-cancellation between negative first-order (Fermi contact and dipolar) and unusually large positive second-order (spin-orbital) contributions. The large spin-orbit effects relate to the distorted tetrahedral structures. Square planar dithiolene complexes with similar spin density on copper exhibit much more negative A(parallel) values, as the cancellation between nonrelativistic and spin-orbit contributions is less complete. Calculations on a selenocysteine-substituted variant of azurin have provided further insight into the relations between bonding and EPR parameters.

π–π interaction between aromatic ring and copper-coordinated His81 imidazole regulates the blue copper active-site structure

Noncovalent weak interactions play important roles in biological systems. In particular, such interactions in the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion site in functionally significant ways. Recently, pi-pi interactions between metal ion coordinated histidine imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties of metal ion sites. In this paper we demonstrate that in pseudoazurin (a blue copper protein) the pi-pi interaction between a coordinated histidine imidazole ring and the side chains of aromatic amino acids in the second coordination sphere, significantly influences the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance spectra indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell pi-pi interaction. We suggest that the pi-pi interaction with the metal ion coordinated histidine imidazole ring modulates the electron delocalization in the active site, and that such interactions may be functionally important in refining the reactivity of blue copper sites.