Copper, on the beat with NMR

The first NMR spectroscopy study of the copper site in an important blue metalloprotein, azurin, has been undertaken. Copper mediates many biochemical redox reactions and azurin plays an important role in catalysing electron transfer in cellular reactions; understanding its activity could lead to advances in biotechnology and medicine.

Given that copper is a ubiquitous trace element in biochemistry the development of tools to study its chemistry and in particular its role in redox enzymes is imperative in studies of a wide-range of metabolic processes, several diseases, and the biochemistry of many organisms other than humans. The primary redox couple is the shuttling between Cu(I) and Cu(II) but the existence of multinuclear copper centres, and mixed metal centres endow copper with the ability to conduct multielectron reactions. As such, it is found in the well-known respiratory enzyme cytochrome c oxidase, which contains three copper ions per molecule, and reduces molecular oxygen in a four-electron process to water. Other copper enzymes include tyrosinase, various oxidases, copper, zinc superoxide dismutase, copper hemocyanins oxygen transporters.

Researchers have studied the Cu(II) state of blue copper proteins extensively using X-ray crystallography and spectroscopy, electron paramagnetic resonance spectroscopy, and NMR. Quantum calculations lend support to many of these studies. However, Cu(I) state is largely "spectroscopically silent" being colourless and diamagnetic.

Researchers at the Pacific Northwest National Laboratory (Andrew Lipton, Robert Heck, Wibe de Jong, Amy Gao, and Paul Ellis) and the University of Nebraska at Lincoln (Xiongjian Wu and Adrienne Roehrich), in the USA, have now obtained the first high-field NMR spectrum of azurin's copper centre with a view to accurately characterizing the bonding - covalent, ionic, or hybrid - around the metal.

Until now, NMR of copper-containing proteins was not technically feasible as the metal gives rise to broad NMR lines. By combining the power of 800 MHz NMR spectroscopy with the Department of Energy's EMSL Chinook supercomputer, the team has now turned the presence of copper to their advantage. They have gained what they describe as an "exquisitely sensitive measure of the electron density around the nuclear site." This has revealed details about the electronic structure and environment of azurin's copper centre that had remained invisible to investigators. The strategy could be used to study any diamagnetic metalloprotein.

The team obtained copper-65 central-transition NMR spectra for azurin with copper in the reduced Cu(I) state at 18.8 T and 10 K. This they explained gave a strongly second-order quadrupole perturbed spectrum, with a 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. They then carried out quantum chemical calculations on the Chinook using second-order Møller?Plesset perturbation theory and large basis sets to successfully reproduce the experimental data. The new study helps resolve some of the conflicting evidence from earlier studies of azurin in which implausible ligand-copper distances were revealed. "Sensitivity and relaxation times were quite favourable, suggesting that NMR may be a useful probe of the electronic state of copper sites in proteins," the team says.

The team explains that because their approach is relatively facile at cryogenic temperatures that it "opens up new vistas for the NMR of metalloenzymes?of this size or even larger." They conclude that, "the successful computation of electric field gradients using basis sets of moderate size and electron correlation corrections by MP2 methods suggest that electronic structure theory combined with NMR experiments should allow the electrostatic environment of copper nuclei in metalloproteins to be probed in detail and with high precision."

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