Blue Copper Protein Azurin Research Articles

Photoinduced Electron Transfer from the Tryptophan Triplet State in Zn-Azurin.

Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu2+ center with Zn2+ in the blue-copper protein azurin, a long-lived tryptophan neutral radical can be photogenerated. We report the following quantum yield values for Zn-substituted azurin in the presence of the electron acceptor Cu(II)-azurin: formation of the tryptophan neutral radical (Φrad), electron transfer (ΦET), fluorescence (Φfluo), and phosphorescence (Φphos), as well as the efficiency of proton transfer of the cation radical (ΦPT). Increasing the concentration of the electron acceptor increased Φrad and ΦET values and decreased Φphos without affecting Φfluo. At all concentrations of the acceptor, the value of ΦPT was nearly unity. These observations indicate that the phosphorescent triplet state is the parent state of electron transfer and that nearly all electron transfer events lead to proton loss. Similar results regarding the parent state were obtained with a different electron acceptor, [Co(NH3)5Cl]2+; however, Stern-Volmer graphs revealed that [Co(NH3)5Cl]2+ was a more effective phosphorescence quencher (K SV = 230 000 M-1) compared to Cu(II)-azurin (K SV = 88 000 M-1). Competition experiments in the presence of both [Co(NH3)5Cl]2+ and Cu(II)-azurin suggested that [Co(NH3)5Cl]2+ is the preferred electron acceptor. Implications of these results in terms of quenching mechanisms are discussed.

Reorganization free energy of copper proteins in solution, in vacuum, and on metal surfaces.

Metalloproteins, known to efficiently transfer electronic charge in biological systems, recently found their utilization in nanobiotechnological devices where the protein is placed into direct contact with metal surfaces. The feasibility of oxidation/reduction of the protein redox sites is affected by the reorganization free energies, one of the key parameters determining the transfer rates. While their values have been measured and computed for proteins in their native environments, i.e., in aqueous solution, the reorganization free energies of dry proteins or proteins adsorbed to metal surfaces remain unknown. Here, we investigate the redox properties of blue copper protein azurin, a prototypical redox-active metalloprotein previously probed by various experimental techniques both in solution and on metal/vacuum interfaces. We used a hybrid quantum mechanical/molecular mechanical computational technique based on density functional theory to explore protein dynamics, flexibility, and corresponding reorganization free energies in aqueous solution, vacuum, and on vacuum gold interfaces. Surprisingly, the reorganization free energy only slightly decreases when azurin is dried because the loss of the hydration shell leads to larger flexibility of the protein near its redox site. At the vacuum gold surfaces, the energetics of the structure relaxation depends on the adsorption geometry; however, significant reduction of the reorganization free energy was not observed. These findings have important consequences for the charge transport mechanism in vacuum devices, showing that the free energy barriers for protein oxidation remain significant even under ultra-high vacuum conditions.

Protein Binding and Orientation Matter: Bias-Induced Conductance Switching in a Mutated Azurin Junction.

We observe reversible, bias-induced switching of conductance via a blue copper protein azurin mutant, N42C Az, with a nearly 10-fold increase at |V| > 0.8 V than at lower bias. No such switching is found for wild-type azurin, WT Az, up to |1.2 V|, beyond which irreversible changes occur. The N42C Az mutant will, when positioned between electrodes in a solid-state Au–protein–Au junction, have an orientation opposite that of WT Az with respect to the electrodes. Current(s) via both proteins are temperature-independent, consistent with quantum mechanical tunneling as dominant transport mechanism. No noticeable difference is resolved between the two proteins in conductance and inelastic electron tunneling spectra at <|0.5 V| bias voltages. Switching behavior persists from 15 K up to room temperature. The conductance peak is consistent with the system switching in and out of resonance with the changing bias. With further input from UV photoemission measurements on Au–protein systems, these striking differences in conductance are rationalized by having the location of the Cu(II) coordination sphere in the N42C Az mutant, proximal to the (larger) substrate-electrode, to which the protein is chemically bound, while for the WT Az that coordination sphere is closest to the other Au electrode, with which only physical contact is made. Our results establish the key roles that a protein’s orientation and binding nature to the electrodes play in determining the electron transport tunnel barrier.

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.

Interaction of the anticancer p28 peptide with p53-DBD as studied by fluorescence, FRET, docking and MD simulations

BackgroundThe p28 peptide, derived from the blue copper protein Azurin, exerts an anticancer action due to interaction with the tumor suppressor p53, likely interfering with its down-regulators. Knowledge of both the kinetics and topological details of the interaction, could greatly help to understand the peptide anticancer mechanism. MethodsFluorescence and Förster resonance energy transfer (FRET) were used to determine both the binding affinity and the distance between the lone tryptophan (FRET donor) of DNA Binding Domain (DBD) of p53 and the Iaedens dye (FRET acceptor) bound to the p28 peptide. Docking, Molecular Dynamic simulations and free energy binding calculations were used to single out the best complex model, compatible with the distance measured by FRET. ResultsTryptophan fluorescence quenching provided a 105 M−1 binding affinity for the complex. Both FRET donor fluorescence quenching and acceptor enhancement are consistent with a donor-acceptor distance of about 2.6 nm. Docking and molecular dynamics simulations allowed us to select the best complex, enlightening the contact regions between p28 and DBD. Conclusionsp28 binds to DBD partially engaging the L1 loop, at the same region of the p53 down-regulator COP1, leaving however the DNA binding site available for functional interactions. General significanceElucidation of the DBD-p28 complex gets insights into the functional role of p28 in regulating the p53 anticancer activity, also offering new perspectives to design new drugs able to protect the p53 anticancer function.

Fluorescence Correlation Spectroscopy of Labeled Azurin Reveals Photoinduced Electron Transfer between Label and Cu Center.

Fluorescent labeling of biomacromolecules enjoys increasing popularity for structural, mechanistic, and microscopic investigations. Its success hinges on the ability of the dye to alternate between bright and dark states. Förster resonance energy transfer (FRET) is an important source of fluorescence modulation. Photo-induced electron transfer (PET) may occur as well, but is often considered only when donor and acceptor are in van der Waals contact. In this study, PET is shown between a label and redox centers in oxidoreductases, which may occur over large distances. In the small blue copper protein azurin, labeled with ATTO655, PET is observed when the label is at 18.5 Å, but not when it is at 29.1 Å from the Cu. For CuII , PET from label to Cu occurs at a rate of (4.8±0.3)×104 s-1 and back at (0.7±0.1)×103 s-1 . With CuI the numbers are (3.3±0.7)×106 s-1 and (1.0±0.1)×104 s-1 . Reorganization energies and electronic coupling elements are in the range of 0.8-1.2 eV and 0.02-0.5 cm-1 , respectively. These data are compatible with electron transfer (ET) along a through-bond pathway although transient complex formation followed by ET cannot be ruled out. The outcome of this study is a useful guideline for experimental designs in which oxidoreductases are labelled with fluorescent dyes, with particular attention to single molecule investigations. The labelling position for FRET can be optimized to avoid reactions like PET by evaluating the structure and thermodynamics of protein and label.

The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins.

Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

Electron transfer with self-assembled copper ions at Au-deposited biomimetic films: mechanistic ‘anomalies’ disclosed by temperature- and pressure-assisted fast-scan voltammetry

It has been suggested that electron transfer (ET) processes occurring in complex environments capable of glass transitions, specifically in biomolecules, under certain conditions may experience the medium’s nonlinear response and nonergodic kinetic patterns. The interiors of self-assembled organic films (SAMs) deposited on solid conducting platforms (electrodes) are known to undergo glassy dynamics as well, hence they may also exhibit the abovementioned ‘irregularities’. We took advantage of Cu2+ ions as redox-active probes trapped in the Au-deposited −COOH-terminated SAMs, either L-cysteine, or 3-mercaptopropionic acid diluted by the inert 2-mercaptoethanol, to systematically study the impact of glassy dynamics on ET using the fast-scan voltammetry technique and its temperature and high-pressure extensions. We found that respective kinetic data can be rationalized within the extended Marcus theory, taking into account the frictionally controlled (adiabatic) mechanism for short-range ET, and complications due to the medium’s nonlinear response and broken ergodicity. This combination shows up in essential deviations from the conventional energy gap (overpotential) dependence and in essentially nonlinear temperature (Arrhenius) and high-pressure patterns, respectively. Biomimetic aspects for these systems are also discussed in the context of recently published results for interfacial ET involving self-assembled blue copper protein (azurin) placed in contact with a glassy environment.

Electron Transfer Proteins as Electronic Conductors: Significance of the Metal and Its Binding Site in the Blue Cu Protein, Azurin.

Electron transfer (ET) proteins are biomolecules with specific functions, selected by evolution. As such they are attractive candidates for use in potential bioelectronic devices. The blue copper protein azurin (Az) is one of the most‐studied ET proteins. Traditional spectroscopic, electrochemical, and kinetic methods employed for studying ET to/from the protein's Cu ion have been complemented more recently by studies of electrical conduction through a monolayer of Az in the solid‐state, sandwiched between electrodes. As the latter type of measurement does not require involvement of a redox process, it also allows monitoring electronic transport (ETp) via redox‐inactive Az‐derivatives. Here, results of macroscopic ETp via redox‐active and ‐inactive Az derivatives, i.e., Cu(II) and Cu(I)‐Az, apo‐Az, Co(II)‐Az, Ni(II)‐Az, and Zn(II)‐Az are reported and compared. It is found that earlier reported temperature independence of ETp via Cu(II)‐Az (from 20 K until denaturation) is unique, as ETp via all other derivatives is thermally activated at temperatures >≈200 K. Conduction via Cu(I)‐Az shows unexpected temperature dependence >≈200 K, with currents decreasing at positive and increasing at negative bias. Taking all the data together we find a clear compensation effect of Az conduction around the Az denaturation temperature. This compensation can be understood by viewing the Az binding site as an electron trap, unless occupied by Cu(II), as in the native protein, with conduction of the native protein setting the upper transport efficiency limit.

Could tyrosine and tryptophan serve multiple roles in biological redox processes?

Single-step electron tunnelling reactions can transport charges over distances of 15-20 Åin proteins. Longer-range transfer requires multi-step tunnelling processes along redox chains, often referred to as hopping. Long-range hopping via oxidized radicals of tryptophan and tyrosine, which has been identified in several natural enzymes, has been demonstrated in artificial constructs of the blue copper protein azurin. Tryptophan and tyrosine serve as hopping way stations in high-potential charge transport processes. It may be no coincidence that these two residues occur with greater-than-average frequency in O(2)- and H(2)O(2)-reactive enzymes. We suggest that appropriately placed tyrosine and/or tryptophan residues prevent damage from high-potential reactive intermediates by reduction followed by transfer of the oxidizing equivalent to less harmful sites or out of the protein altogether.

Marked changes in electron transport through the blue copper protein azurin in the solid state upon deuteration

Measuring solid-state electron transport (ETp) across proteins allows studying electron transfer (ET) mechanism(s), while minimizing solvation effects on the process. ETp is, however, sensitive to any static (conformational) or dynamic (vibrational) changes in the protein. Our macroscopic measurements allow extending ETp studies to low temperatures, with the concomitant resolution of lower current densities, because of the larger electrode contact areas. Thus, earlier we reported temperature-independent ETp via the copper protein azurin (Az), from 80 K until denaturation, whereas for apo-Az ETp was temperature dependent above 180 K. Deuteration (H/D substitution) may provide mechanistic information on the question of whether the ETp involves H-bonds in the solid state. Here we report results of kinetic deuterium isotope effect (KIE) measurements on ETp through holo-Az as a function of temperature (30-340 K). Strikingly, deuteration changed ETp from temperature independent to temperature dependent above 180 K. This H/D effect is expressed in KIE values between 1.8 (340 K) and 9.1 (≤ 180 K). These values are remarkable in light of the previously reported inverse KIE on ET in Az in solution. We ascribe the difference between our KIE results and those observed in solution to the dominance of solvent effects in the latter (larger thermal expansion in H(2)O than in D(2)O), whereas in our case the KIE is primarily due to intramolecular changes, mainly in the low-frequency structural modes of the protein caused by H/D exchange. The observed high KIE values are consistent with a transport mechanism that involves through-H-bonds of the β-sheet structure of Az, likely also those in the Cu coordination sphere.

Immobilization of azurin with retention of its native electrochemical properties at alkylsilane self-assembled monolayer modified indium tin oxide

Indium tin oxide (ITO) is a promising material for developing spectroelectrochemical methods due to its combination of excellent transparency in the visible region and high conductivity over a broad range of potential. However, relatively few examples of immobilization of redox proteins at ITO with retention of the ability to transfer electrons with the underlying material with native characteristics have been reported. In this work, we utilize an alkylsilane functionalized ITO surface as a biocompatible interface for immobilization of the blue copper protein azurin. Adsorption of azurin at ITO as well as ITO coated with self-assembled monolayers of (3-mercaptopropyl)trimethoxysilane (MPTMS) and n-decyltrimethoxysilane (DTMS) was achieved, and immobilized protein probed using protein film electrochemistry. The native redox properties of the protein were perturbed by adsorption directly to ITO or to the MPTMS layer on an ITO surface. However, azurin adsorbed at a DTMS covered ITO surface retained native electrochemical properties (E1/2=122±5mV vs. Ag/AgCl) and could exchange electrons directly with the underlying ITO layer without need for an intervening chemical mediator. These results open new opportunities for immobilizing functional redox proteins at ITO and developing spectroelectrochemical methods for investigating them.

An Unusual Subtilisin-like Serine Protease Is Essential for Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH), an αβγ heterotrimer present in the periplasm of several Gram-negative bacteria, catalyzes the oxidative deamination of various aliphatic amines such as n-butylamine for assimilation as carbon and energy sources. The γ subunit of mature QHNDH contains a protein-derived quinone cofactor, cysteine tryptophylquinone, and three intrapeptidyl thioether cross-links between Cys and Asp or Glu residues. In its cytoplasmic nascent form, the γ subunit has a 28-residue N-terminal leader peptide that is necessary for the production of active QHNDH but must be removed in the following maturation process. Here, we describe the role of a subtilisin-like serine protease encoded in the fifth ORF of the n-butylamine-utilizing operon of Paracoccus denitrificans (termed ORF5) in QHNDH biogenesis. ORF5 disruption caused bacterial cell growth inhibition in n-butylamine-containing medium and production of inactive QHNDH, in which the γ subunit retained the leader peptide. Supply of plasmid-encoded ORF5 restored the cell growth and production of active QHNDH, containing the correctly processed γ subunit. ORF5 expressed in Escherichia coli but not its catalytic triad mutant cleaved synthetic peptides surrogating for the γ subunit leader peptide, although extremely slowly. The cleaved leader peptide remained unstably bound to ORF5, most likely as an acyl enzyme intermediate attached to the active-site Ser residue. These results demonstrate that ORF5 is essential for QHNDH biogenesis, serving as a processing protease to cleave the γ subunit leader peptide nearly in a disposable manner.

Azurin Synthesis from Pseudomonas Aeruginosa MTCC 2453, Properties, Induction of Reactive Oxygen Species, and p53 Stimulated Apoptosis in Breast Carcinoma Cells

Breast cancers are usually treated with surgery and radiation excretes adverse effects. Azurin, a potent anticancer redox protein secreted by Pseudomonas aeruginosa (P. aeruginosa) species has been reported to have activity against breast cancer cell lines; this had prompted researchers to search for novel methods to enhance this protein’s production. Researchers previously have reported on the synthesis of blue copper protein azurin from different microbial sources specifically from P. aeruginosa. Our investigation used customized methods to focus on synthesizing azurin from different strains of P. aeruginosa with apparent homogeneity. We screened the growth of different P. aeruginosa strains (1934, 741, 2453, and 1942) for the synthesis of azurin and for enhanced azurin production. We exposed azurin properties using matrix-assisted laser desorption/ionization, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Fourier transform infrared spectroscopy. Additional studies of possible molecular mechanisms and reactive oxygen species (ROS) generation of P. aeruginosa 2453 secreted azurin are needed. We examined which strain among P. aeruginosa strains 1934, 741, 2453, and 1942 best enhanced azurin production. Our current study also revealed which strain of the four had the strongest antiproliferative effect of azurin. P. aeruginosa MTCC (Microbial Type culture collection) 2453 was the strain that secreted the most azurin and showed remarkable apoptosis in breast carcinoma cells like T- 47D and ZR-75-1. This study demonstrates customized methods to synthesize azurin from different strains of P. aeruginosa with apparent homogeneity and their apoptotic effects on breast carcinoma cells with possible molecular mechanisms and ROS.

Incorporation of the red copper nitrosocyanin binding loop into blue copper azurin

Loop-directed mutagenesis was applied to the blue copper protein azurin to replace its copper binding loop with that from the red copper protein nitrosocyanin. A ten amino acid long loop that provides three of the four copper ligands from nitrosocyanin was incorporated into azurin to make a variant called NC-azurin. The chimeric protein displayed a red color, and UV-vis absorption and EPR spectra that closely resembled those of the loop parent, nitrosocyanin. We added the fourth ligand from nitrosocyanin into NC-azurin, a carboxylate-containing amino acid, but the proteins had altered stability and spectroscopic properties that did not resemble those of either parent copper protein. The loop alone, however, was enough to impart red copper site characteristics to the NC-azurin protein. Finally, the reduction potential of the variant was found to be between the reduction potentials of the parent proteins and about 50mV below that of wild-type azurin.

Visualizing and Tuning Thermodynamic Dispersion in Metalloprotein Monolayers

In coupling the redox state of an adsorbed molecule to its spectral characteristics redox profiles can be directly imaged by means of far-field fluorescence. At suitable levels of dilution, on optically transparent electrode surfaces, reversible interfacial electron transfer processes can be followed pixel by pixel down to scales which approach the molecular. In mapping out switching potentials across a surface population, thermodynamic dispersion, related to variance in the orientation, electronic coupling, protein fold, electric field drop, and general surface order, can be quantified. The self-assembled monolayer buffering the protein from the underlying metallic electrode surface not only acts to tune electronic coupling between the two but also potentially provides a variable more easily segmented from other contributions to molecular dispersion. We have, specifically, considered the possibility that the supporting monolayer crystallinity is a significant contributor to the subsequently observed spread in half-wave potentials. We report here that this is indeed the case and that this spread diminishes from 17 to 12 mV for the blue copper protein azurin as the supporting alkanethiol layer crystallinity increases. The work herein, then, presents not only a direct determination of submonolayer scale variance in redox character but also a means of tuning this through gross surface and entirely standard chemical means.

Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung

The long-term persistance of P. aeruginosa in the cystic fibrosis (CF) lung is characterized by the selection of a variety of genotypes and phenotypes that typically descend from one infecting P. aeruginosa clone, a process known as adaptive radiation. This adaptation process of P. aeruginosa includes complex physiological changes that likely confer a selective advantage to better thrive in the diverse niches and microenvironments of the inflamed and hostile CF airways. The occurrence of P. aeruginosa variants is fixed by mutation and selection. Common loss-of-function mutations in genes such as lasR, mucA and mexT lead to a general adaptation pattern and P. aeruginosa variants with increased antimicrobial resistance, alginate overproduction, reduced acute virulence, and improved metabolic fitness. Strikingly, several virulence-associated traits and immunostimulatory components of P. aeruginosa are turned off. In contrast, other cellular factors are positively selected such as the outer membrane protein OprF, the blue copper protein azurin, the cytochrome c peroxidase c551, and the enzymes of the arginine deiminase pathway ArcA-ArcD. These metabolic components probably are required for the optimal anaerobic or microaerobic growth and viability of P. aeruginosa within CF airways. Besides these common adaptations found by the comparison of P. aeruginosa isolates from different CF patients, the overall diversity of isogenic isolates from one CF patient is extended by variable changes in the expression of regulatory-, transport-, metabolic-, and virulence-associated genes. A better understanding of the microevolution of P. aeruginosa towards niche specialists according the selection pressure in the CF lung is a prerequisite to develop new strategies for the detection of P. aeruginosa variants, the antipseudomonal treatment, the prediction of the infectious disease state, and the development of efficient vaccines.

Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations

Recent evidence has shown that the properties of metal binding sites can be tuned by more than the ligands in the primary coordination sphere. We investigated the incorporation of four phenylalanine residues into the secondary coordination sphere of the small soluble blue copper protein azurin. The locations for placement of these residues in azurin were based on the structure of the highly hydrophobic blue copper protein rusticyanin, which is known to have a significantly higher reduction potential than azurin. Using site-directed mutagenesis, these residues in close proximity to the copper binding site were mutated to large hydrophobic phenylalanine residues individually and in combination. We also added the Met121Leu mutation on top of the Phe mutations to construct a total of 13 variants. We found little change in the UV–visible absorption and EPR data for these proteins, however modest increases in reduction potential were observed with increases by as much as 30 mV per Phe residue. Furthermore, we observed the increases in potential to be additive.

Fundamental signatures of short- and long-range electron transfer for the blue copper protein azurin at Au/SAM junctions.

The blue copper protein from Pseudomonas aeruginosa, azurin, immobilized at gold electrodes through hydrophobic interaction with alkanethiol self-assembled monolayers (SAMs) of the general type [-S-(CH(2))(n)-CH(3)] (n = 4, 10, and 15) was employed to gain detailed insight into the physical mechanisms of short- and long-range biomolecular electron transfer (ET). Fast scan cyclic voltammetry and a Marcus equation analysis were used to determine unimolecular standard rate constants and reorganization free energies for variable n, temperature (2-55 degrees C), and pressure (5-150 MPa) conditions. A novel global fitting procedure was found to account for the reduced ET rate constant over almost five orders of magnitude (covering different n, temperature, and pressure) and revealed that electron exchange is a direct ET process and not conformationally gated. All the ET data, addressing SAMs with thickness variable over ca. 12 A, could be described by using a single reorganization energy (0.3 eV), however, the values for the enthalpies and volumes of activation were found to vary with n. These data and their comparison with theory show how to discriminate between the fundamental signatures of short- and long-range biomolecular ET that are theoretically anticipated for the adiabatic and nonadiabatic ET mechanisms, respectively.

Low Temperature 65Cu NMR Spectroscopy of the Cu+ Site in Azurin

(65)Cu central-transition NMR spectroscopy of the blue copper protein azurin in the reduced Cu(I) state, conducted at 18.8 T and 10 K, gave a strongly second order quadrupole perturbed spectrum, which yielded a (65)Cu quadrupole coupling constant of +/-71.2 +/- 1 MHz, corresponding to an electric field gradient of +/-1.49 atomic units at the copper site, and an asymmetry parameter of approximately 0.2. Quantum chemical calculations employing second order Møller-Plesset perturbation theory and large basis sets successfully reproduced these experimental results. Sensitivity and relaxation times were quite favorable, suggesting that NMR may be a useful probe of the electronic state of copper sites in proteins.