Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

Abstract

The biological function of metalloproteins is closely tied to the geometric and electronic structures of the metal sites. Here, we show that the geometric structure of the metal site of a metalloprotein in solution can be determined from experimentally measured electron-nuclear spin-spin interactions obtained by NMR. Thus, the geometric metal site structure of plastocyanin from Anabaena variabilis was determined by including the paramagnetic relaxation enhancement of protons close to the copper site as restraints in a conventional NMR structure determination, together with the distribution of the unpaired electron onto the ligand atoms. Also, the interproton distances (nuclear Overhauser enhancements) and dihedral angles (scalar nuclear spin-spin couplings) normally used in NMR structure determinations were included as restraints. The structure calculations were carried out with the program X-PLOR and a module that takes into account the specific characteristics of the paramagnetic restraints. A well defined metal site structure was obtained with the structural characteristics of the blue copper site, including a distorted tetrahedral geometry, a short Cu-Cys S ␥ bond, and a long Cu-Met S ␦ bond. Overall, the agreement of the obtained metal site structure of Anabaena variabilis plastocyanin with those of other plastocyanins obtained by x-ray crystallography confirms the reliability of the approach.metal site structure ͉ metalloproteins ͉ paramagnetic nuclear relaxation T he biological function of many metalloproteins stems from the geometric and electronic structures of the metal sites imposed by the protein environment. In the blue copper proteins, such as plastocyanins, amicyanins, and azurins, the geometry of the copper site is unusual as compared with smallmolecule copper complexes (1-15). In particular, the metal sites of blue copper proteins are characterized by a short coppersulfur bond. This unusual geometry is believed to be the main reason for the strong covalency of the metal site (10,16,17) and, thus, responsible for the rapid and long-range electron transfer reactivity (18-22) that characterizes the blue copper proteins. Detailed knowledge of the geometric and electronic metal site structures of the blue copper proteins is, therefore, imperative for understanding the function of the proteins at the molecular level.So far, the geometric structure of the metal site in blue copper proteins (12-14) has been determined primarily by x-ray crystallography and extended x-ray absorption fine structure (3,23,24), whereas the electronic structure of the blue copper site has been determined theoretically from quantum chemical calculations (5, 25-27) and experimentally by x-ray absorption spectroscopy (16, 17), and more recently by nuclear paramagnetic relaxation (28-31). These structures have formed the basis for a detailed understanding of the biological function of the proteins by elucidating the interplay between the electronic and geometric structure of the metal site (8,15,(32)(33)(34). However, the geomet...

Predicting the NMR Spectra of Paramagnetic Molecules by DFT: Application to Organic Free Radicals and Transition‐Metal Complexes

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Investigating metal-binding in proteins by nuclear magnetic resonance

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