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

Abstract

Abstract65 Cu central-transition NMR spectroscopy of the blue copper protein azurin in the reduced Cu(I) state, conducted at 18.8 Tesla 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. Keywords65 Cu NMR; azurin; blue copper protein; nuclear quadrupole couplingCopper is a ubiquitous component of living systems. It fulfills a vital role in many enzymes, particularly redox enzymes. As might be anticipated from the chemistry of the element, the primary redox couple involved in biological redox processes is Cu + /Cu 2+ . However, multinuclear copper centers 1,2 and mixed metal centers 3 give it the ability to participate in multielectron processes. Among its most important redox roles are in cytochrome c oxidase, which was proved to be a copper enzyme by nutritional, enzymological and spectroscopic studies 4 , and which contains three coppers per molecule 5 , two in a dinuclear center. Cytochrome c oxidase acts as the terminal enzyme in the respiratory chain, reducing molecular oxygen in a four-electron process to water. Other copper-containing redox enzymes are tyrosinase 6 and various other oxidases 7 . The copper, zinc superoxide dismutase of mammalian erythrocytes performs the rather different role of eliminating superoxide in an alternating sequence of single-electron oxidation and reduction steps 8 . Some organisms use copper hemocyanins, which contain a binuclear copper site 9 , as oxygen transporters. In addition to these metabolic functions, organisms use copper chaperones 10 to transport the toxic metal. In terms of generalities, it has been noted 11 that copper is usually associated with the metabolism of O 2 or oxides of nitrogen, and copper proteins often also contain organic radicals.CORRESPONDING AUTHOR: gerry@setanta.unl.edu. † Pacific Northwest National Laboratory § University of Nebraska at Lincoln SUPPORTING MATERIALS Supporting materials contain the complete reference 43 and 45 , as well as Tables 5 and 6, which give the optimized coordinates of the model complex bis(imidazole)copper(I)methylsulfide and the quantum region of azurin used in QM/MM calculations. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCopper sites in proteins have been classified into several types: type-1 12 , type-2 13 , and type-3 1 copper, as well as the dinuclear CuA center 14 . Among the best-studied are the type-1 sites, characterized by a strong absorption in the orange region of the visible spectrum. The blue copper proteins, a class of relatively ...

Ab initio electronic structure calculations of entire blue copper azurins

“…1 Blue copper azurins have been extensively characterized by means of multiple techniques which include X-ray diffraction, 3,4 electron paramagnetic resonance 5,6 or nuclear magnetic resonance. 7,8 The pioneering work of E. I. Solomon's group was particularly successful in unveiling the electronic structure of Cu sites in various copper proteins by using a combination of spectroscopic techniques and quantum calculations. [9][10][11][12][13][14] Initially, these seminal works mostly aimed at understanding the main spectroscopic features, especially the investigation of the excited d-states, which are responsible for the peculiar absorption spectra of copper proteins, giving rise, in most cases, to their characteristic intense blue coloration.…”

One hundred fold overall sensitivity enhancements for Silicon-29 NMR spectroscopy of surfaces by dynamic nuclear polarization with CPMG acquisition

“…In this regard Ellis and Lipton have shown that it is possible to apply CP/CPMG pulse sequences to acquire spectra of quadrupolar nuclei contained in metalloproteins which are dissolved in frozen solutions doped with paramagnetic metal ions (that act to enhance 1 H longitudinal relaxation rates). [52][53][54][55][56] Kervern et al have also demonstrated that the CPMG pulse sequence can be utilized to extend 1 H coherence lifetimes of paramagnetic solids under ultra fast MAS. 57 These findings suggest that T 2 0 may remain long enough such that CPMG experiments remain feasible on systems doped with radicals.…”

Solid-State 63Cu, 65Cu, and 31P NMR Spectroscopy of Photoluminescent Copper(I) Triazole Phosphine Complexes

“…15−19 The moderate Q values for 63 Cu and 65 Cu usually result in nuclear quadrupolar coupling constants C Q on the order of megahertz and thus in broad 63/65 Cu NMR powder line shapes for the central transition (i.e., m S = +1/2 ↔ m S = −1/2) that are challenging to acquire experimentally. 15 Early investigations therefore focused on Cu(I) sites with high symmetry (e.g., tetrahedral) and hence with small quadrupolar interactions. 16 However, in the past decade, high-field NMR magnets have become more widely available, and several wideline solid-state NMR techniques 20−26 have permitted the acquisition of NMR spectra with breadths that sometimes are several megahertz.…”