LIGAND SPIN-DENSITIES IN BLUE COPPER PROTEINS BY Q-BAND H-1 AND N-14 ENDOR SPECTROSCOPY

The type 1 or blue copper centers of poplar plastocyanin (Populus nigra var italica), azurin (Pseudomonas aeruginosa), stellacyanin (Rhus vernicifera), and type 2 reduced fungal (Polyporous versicolor) and tree (R. vernicifera) laccase have been studied by Q-band (35 GHz) ENDOR spectroscopy. At this microwave frequency the H-1 and N-14 resonances occur in completely distinct radio-frequency ranges, and this has enabled us to study them individually for the first time. Each protein exhibits strongly coupled methylene protons of cysteine with isotropic hyperfine splittings in the range 16-31 MHz. The measurements indicate that the geometry of the Cu-cys linkage as measured by the H-beta-C-beta-S-Cu dihedral angles is remarkably similar in all these proteins, -58-degrees less-than-or-similar-to theta(H-beta-2) less-than-or-similar-to -50-degrees. With one exception, all the proteins have a similar, large total spin density on sulfur; fungal laccase appears to have a larger value but rather may differ slightly in structure. The Cu-bound nitrogens of the two histidine ligands of plastocyanin give a single N-14 resonance with isotropic coupling (A(N) approximately 22 MHz) and thus the Cu-N bonds appear effectively equivalent although they differ metrically. In contrast, azurin, stellacyanin, and fungal laccase exhibit N-14 signals with isotropic hyperfine interactions from two inequivalent histidyl nitrogen ligands. We estimate the sum of the spin densities on N to be greater-than-or-similar-to 0.1 and the overall spin density on ligands to be over 50%. The N-14 ENDOR of the similar site of tree laccase requires that it be unlike any of the other type 1 centers studied, with at least one N-14 ligand whose hyperfine tensor is highly anisotropic. Together, the H-1 and N-14 data suggest that the single-site proteins and the laccases fall into different subclasses. The advantages of the Q-band ENDOR technique over alternate methods of determining ligand superhyperfine couplings also are discussed.