Structural characterization of mononuclear Cu(II) and its nitrite complex in the active site of Carcinus maenas hemocyanin.
The preparation of a mononuclear Cu(II) derivative of Carcinus maenas hemocyanin (Cu(II)-Hc) and a nitrite complex of the derivative (Cu(II)-Hc-NO2-) are described. Several techniques have been used in their characterization, including X-ray absorption, continuous wave (cw) EPR, and electron spin-echo envelope modulation (ESEEM) spectroscopies. EXAFS results for Cu(II)-Hc indicate the presence of three ligands at 1.99 +/- 0.01 A and a fourth one at 2.26 +/- 0.01 A from the copper. The same coordination number and very similar bond lengths were obtained for Cu(II)-Hc-NO2-. On the basis of simulations of three-pulse ESEEM spectra, three equivalent imidazole nitrogens coupled to Cu(II) were identified in Cu(II)-Hc. Upon the binding of nitrite, a decrease in the hyperfine interaction for two of the three imidazole nitrogens was observed by ESSEM. Further, the results of a two-pulse ESEEM experiment are consistent with the assignment of the protons of a water ligand to Cu(II), which is displaced when nitrite is added. An analysis of X-ray absorption K-edge spectra suggests a coordination geometry intermediate between square-planar and tetrahedral for the metal centers in Cu(II)-Hc and Cu(II)-Hc-NO2-, in agreement with the g and ACu values determined by cw-EPR. On the basis of these results, an equivalent structure is suggested for Cu(II)-Hc-NO2- and the Cu(II) site in green half-methemocyanin, a partially oxidized binuclear derivative formed in the reaction of the native protein with nitrite.
Bubacco
L
L
Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461.
Magliozzo
R S
RS
Wirt
M D
MD
Beltramini
M
M
Salvato
B
B
Peisach
J
J
D000818:Animals; D003386:Brachyura / Q000737:chemistry*; D003198:Computer Simulation; D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006433:Hemocyanins / Q000737:chemistry*; D006860:Hydrogen Bonding; D009572:Nitrite Reductases / Q000737:chemistry; D009573:Nitrites / Q000737:chemistry*; D017433:Protein Structure, Secondary
D009573:Nitrites; D003300:Copper; D006433:Hemocyanins; D009572:Nitrite Reductases