Paramagnetic NMR studies of blue and purple copper proteins

H-1- and C-13-NMR spectroscopy is applied to investigate the Cu, and type 1 active sites of copper proteins in solution. The analysis of hyperfine shi fted H-1 resonances allows the comparison of the electron spin density delo calization in the Cu-A site of the wild-type soluble domains of various cyt ochrome c oxidases (Thermus thermophilus, Paracoccus denitrificans, and Par acoccus versutus) and genetically engineered constructs (soluble domain of quinol oxidase from Escherichia coli and Thiobacillus versutus amicyanin). Comparable spin densities are found on the two terminal His ligands for the wild-type constructs as opposed to the engineered proteins where the spin is more unevenly distributed on the two His residues. A reevaluation of the Cys H-beta chemical shifts that is in agreement with the data published fo r both the P. denitrificans and the P. versutus Cu-A soluble domains confir ms the thermal accessibility of the B-2(3u) electronic excited state and in dicates the existence of slightly different spin densities on the two bridg ing Cys ligands. The C-13-NMR spectrum of isotopically enriched oxidized az urin from Pseudomonas aeruginosa reveals six fast relaxing signals, which c an be partially identified by 1- and 2-dimensional (1-D, 2-D) direct detect ion techniques combined with 3-D triple resonance experiments. The observed contact shifts suggest the presence of direct spin density transfer and sp in polarization mechanisms for the delocalization of the unpaired electron. (C) 1999 John Wiley & Sons, Inc.