Spectroscopic studies and a structural model for blue copper centers in proteins.

Abstract

Distorted tetrahedral Cu(II) should exhibit d-d transitions well below 10,000 cm1, assuming that the ligand field is not unusually strong. We have undertaken, therefore, a detailed investigation of the near-infrared absorption, CD, and magnetic circular dichroism (MCD) spectra of stellacyanin, plastocyanin, and azurin. Detailed analysis of these and analogous visible spectral data has allowed assignments to be made of both the d-d and charge transfer transitions. Interpretation of the charge transfer spectra has been aided further by the identification of the probable ligands of Cu(II) in bean plastocyanin. MATERIALS AND METHODSFrench bean plastocyanin (Phaseolus vulgaris) (18), azurin (Pseudomonas aeruginosa) (19), and stellacyanin (Rhus vernicifera) (20) were purified by standard methods. Near-infrared spectra were obtained on protein films to minimize interference by water absorption and permit a wide variation of temperature. The protein solutions were dialyzed against deionized distilled water and concentrated, first by pressurized membrane ultrafiltration and then, by placing drops on a plexiglas disk in a metal dessicator over Drierite. After three of four drops had been successively concentrated (ten to fifteen for the thick films), the film was prepared'by transferring the disk to a dessicator containing a saturated potassium acetate solution. This controlled humidity allowed the film to form slowly, thereby preventing most of the cracking caused by rapid removal of excess water. The drying process was halted by transfer of the film to a dessicator charged with a saturated sodium hydrogen phosphate solution.The near-infrared CD and MCD spectra were obtained with samples run in deuterated phosphate buffer in order to extend the spectral range to about 1850 nm. Although the infrared overtones of water do not give rise to a measureable CD spectrum, an absorbance of 1.0 or greater diminished the light level to the limit of detector response. The deuterated protein solutions were prepared by evaporating unbuffered, concentrated aqueous solutions in a Drierite dessicator as described above. The films were then transferred to a

Geometric and Electronic Structure Contributions to Function in Bioinorganic Chemistry: Active Sites in Non-Heme Iron Enzymes

“…Thus the blue copper absorption spectrum has historically been assigned as band 6 being the cysteine IT and band 4 the cysteine o+Cu(II) dX2.y2 charge transfer transitions. (27) Significant insight into this possible assignment came from LT MCD data(l9) shown at the bottom of Fig. 4A.…”

Electronic Structure of the Perturbed Blue Copper Site in Nitrite Reductase: Spectroscopic Properties, Bonding, and Implications for the Entatic/Rack State

“…[36][37][38] This orbital exhibits a large and highly anisotropic degree of covalency. X-ray absorption spectroscopy (XAS) at the S K-edge 39 and Cu L-edge 40 has indicated that the HOMO contains 38% S(Cys) character and only ∼40% Cu character, and electronic structure calculations 41 indicate that only ∼4% N(His) character is present in the HOMO (because the Cu-S(Met) bond is oriented approximately perpendicular to the HOMO, no S(Met) character is present in the HOMO).…”

Biological Screening Studies of DNA Relate Metal Complexes from Benzylidene-4-imino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one and 2-aminothiazole

“…The SEM image of the complexes confirms the presence of ligands in the metalchelates. Hence, the metal ion affects the morphology of Schiff base in the metal-chelates [28][29][30].…”