NMR Studies of Proteins Containing Polynuclear Copper Centers

NMR Studies of Proteins Containing Polynuclear Copper Centers 

Cua centers exist in two redox states [Cu(II)Cu(I)] and [Cu(I)Cu(I)]. The oxidized species is a fully delocalized mixed-valence pair (formally two Cu+ 1.5 ions), as revealed by EPR spectroscopy (Kroneck et al., 1988, 1990). Despite the similar coordination geometry around copper, these systems display sharper NMR lines than do the BCP due to a shorter electron relaxation time of the paramagnetic center (wlO s) (dementi and Luchinat, 1998). NMR studies are available for the native Cua centers from the soluble fragments of the The. thermophilus, Paracoc-cus denitrificans, Paracoccus versutus, and Bacillus subtilis oxidases (Bertini et al., 1996 Dennison et al., 1995 Luchinat et al., 1997 Salgado et al., 1998a) and Pseudomonas stutzeri N2O reductase (Holz et al., 1999), as well as for engineered Cua sites in amicyanin (Dennison et al., 1997) and Escherichia coli quinol oxidase (Kolczak et al., 1999). 

NMR spectra of H2O solutions of Cua domains from (A) Paracoccus denitrificans, 800 MHz, pH 5.6, and (B) Thermus thermophilus, 600 MHz, pH 4.5. Asterisks denote exchangeable signals. The 100- to 500-ppm region of spectrum (A) is recorded as a H NMR spectrum. Signals a and c are not visible in the H spectrum. NOE connectivities are also shown. Adapted with permission from Luchinat et al. (1997 Copyright 1997 American Chemical Society). 

The overall picture describing the electron delocalization in Cua resembles that found in Type I sites most of the delocalized unpaired spin density is found on the Cys ligands. The electron spin density on each P-CH2 Cys proton is about half of that observed on the equivalent protons in blue copper proteins (Bertini etal., 1996, 1999). The unpaired electron is distributed over the two copper ions and the two Cys ligands. The observed values for the hyperfine shifts are consistent with the fact that the hyperfine couplings found in the P-CH2 Cys protons in Type I sites are twice as large as those observed in Cua centers. However, as already discussed, the Cu(H)-Cys covalency in Type I sites can be severely altered by the strength of the Cu(H)-axial ligand interaction. This 

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VI. NMR Studies of Proteins Containing Polynuclear Copper Centers. 434... 

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