Paramagnetic NMR studies of blue and purple copper proteins

¹H- and ¹³C-NMR spectroscopy is applied to investigate the Cu_A and type 1 active sites of copper proteins in solution. The analysis of hyperfine shifted ¹H resonances allows the comparison of the electron spin density delocalization in the Cu_A site of the wild-type soluble domains of various cytochrome c oxidases (Thermus thermophilus, Paracoccus denitrificans, and Paracoccus 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^β chemical shifts that is in agreement with the data published for both the P. denitrificans and the P. versutus Cu_A soluble domains confirms the thermal accessibility of the ²B_3u electronic excited state and indicates the existence of slightly different spin densities on the two bridging Cys ligands. The ¹³C-NMR spectrum of isotopically enriched oxidized azurin from Pseudomonas aeruginosa reveals six fast relaxing signals, which can be partially identified by 1- and 2-dimensional (1-D, 2-D) direct detection techniques combined with 3-D triple resonance experiments. The observed contact shifts suggest the presence of direct spin density transfer and spin polarization mechanisms for the delocalization of the unpaired electron. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: S19–S32, 1999