Characterization of the blue copper site in oxidized azurin by extended x-ray absorption fine structure: Determination of a short Cu—S distance

Abstract

The primary coordination environment of the "blue" copper ion in oxidized azurin has been elucidated by x-ray absorption spectroscopy. The most striking-feature is the unambiguous presence of a very short copper-sulfur, distance at 2.10 + 0.02 A. Nitrogen ligands, presumed to be from imidazoles, are found at 1.97 A. There is some evidence that the copper coordination sphere may be completed by a second sulfur, the distance of which is determined with much less certainty.Proteins that contain the "blue" (or type 1) copper site (1) have long offered a challenge to chemists to explain their unique spectroscopic and redox properties. Some of the basic aspects of the structure of such blue sites have already become clear, as revealed by interpretation of several types of spectroscopic data. The model for the blue site of bean plastocyanin put forth by Gray and coworkers (2) is probably the best distillation of current spectroscopic and chemical information on blue copper. The ligand field spectrum implies a distorted tetrahedral coordination of the copper, which is also reasonable when the high redox potential of the copper is considered. A thiolate sulfur from cysteine is proposed as one of the metal ligands. Two imidazole nitrogens from histidines are also implicated as ligands, from NMR studies (3-5). The fourth ligand of copper is less certain. A deprotonated peptide nitrogen from the protein has been proposed as this ligand (6), but methionine sulfur now seems to be the most likely possibility. The x-ray protein structure of poplar plastocyanin has recently been solved to 2.7 A resolution (7). The copper in this plastocyanin is coordinated by cysteinate, two imidazoles, and methionine.The availability of even a high-resolution x-ray crystallographic structure of a blue protein would not necessarily explain all of the unusual spectroscopic and redox properties of blue copper. Subtle changes in bond distance and coordination geometry, which the limited accuracy of even a good protein crystal structure might render difficult to resolve, can be expected to have major effects on these properties.A technique that has been recently used to examine the environment of a selected element in a complex macromolecular system is x-ray absorption spectroscopy. The fine structure that occurs at energies above the x-ray absorption edge (called EXAFS) provides a way of determining distances, numbers, and h-ypes of ligands coordinating a metal. Since the EXAFS method does not require crystalline samples, it can be applied to the study of metalloproteins under noncrystalline or physiological conditions (8)(9)(10)(11). In contrast to macromolecular crystallographic results, where it is difficult to obtain high accuracy except in the few cases of very well refined structures, EXAFS can provide accurate metal-to-ligand distances (12). Coupled with the element-selective nature of the measurements, the EXAFS technique is ideal for investigating the environment of a specific metal ion in a protein.In order to further elucidate t...

Copper Proteins with Type 1 SitesBased in part on the article Copper Proteins with Type 1 Sites by Edward N. Baker which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

“…EXAFS results also provided the first measure of the short Cu-S(thiolate) distance in blue copper proteins 135 and have been used extensively to measure structural changes under varied conditions.…”

Early Work with Synchrotron Radiation at Stanford

“…Applications to cytochrome P-450 (Cramer, Dawson et aL, 1978) quantified the presence of an axial sulfur ligand. The 'blue copper protein' azufin was found to have an unusually short Cu-S ligand which defined the electronic structure and function of the active site (Tullius et al, 1978).…”

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

“…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)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(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).…”

“…So far, the geometric structure of the metal site in blue copper proteins (12)(13)(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)(26)(27) and experimentally by x-ray absorption spectroscopy (16,17), and more recently by nuclear paramagnetic relaxation (28)(29)(30)(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).…”