Pulsed EPR studies of the type 2 copper binding site in the mercury derivative of laccase.
The nuclear modulation effect in pulsed EPR spectroscopy was used to study the type 2 copper binding site in the mercury derivative of laccase (MDL) in which the type 1 copper is substituted by Hg(II). By comparing the three-pulse electron spin-echo modulations and Fourier transform spectra of MDL and several model compounds, we conclude that the imidazole groups of two histidyl amino acid residues are equatorially coordinated to Cu(II) in the type 2 site. Computer simulations of these data suggest that the remote nonbonding nitrogens of the two imidazoles possess nuclear quadrupole parameters e2qQ = 1.47 MHz and eta = 0.83. A(iso) values of these two nitrogens are not identical, being 1.5 and 2.0 MHz. We have also used samples of the enzyme exchanged with D2O to examine the coordination of the water to the type 2 copper site. The deuterium modulation that is resolved by taking the ratio of the time domain ESEEM data from native and D2O-exchanged enzyme indicates that there is an equatorial water ligand, and further data show that this water is displaced by azide.
Lu
J
J
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461.
Bender
C J
CJ
McCracken
J
J
Peisach
J
J
Severns
J C
JC
McMillin
D R
DR
D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003903:Deuterium; D017666:Deuterium Oxide; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D042845:Laccase; D008628:Mercury / Q000494:pharmacology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D011487:Protein Conformation; D014867:Water
D014867:Water; D003300:Copper; D003903:Deuterium; D010088:Oxidoreductases; D042845:Laccase; D008628:Mercury; D017666:Deuterium Oxide