NUCLEAR-MAGNETIC-RESONANCE N-15 AND H-1 RESONANCE ASSIGNMENTS AND GLOBAL FOLD OF RUSTICYANIN - INSIGHTS INTO THE LIGATION AND ACID STABILITY OF THE BLUE COPPER SITE

Nuclear magnetic resonance assignments are reported at pH similar to 3 for a type I (''blue'') copper protein, rusticyanin, obtained from th e acidophilic organism Thiobacillus ferrooxidans. A combination of hom onuclear proton and heteronuclear N-15-edited NMR spectra has been use d to assign most of the H-1 and N-15 resonances of reduced rusticyanin . The copper-binding site is shown by analogy with other blue copper p roteins to contain the side-chains of Cys138, His143 and Met148 at the C-terminal end of the sequence and a fourth ligand that is most likel y a histidine, His85, consistent with the constitution of other type 1 copper sites. The global fold of the molecule is a compact beta-barre l or beta-sandwich, which contains a high proportion of beta-sheet sec ondary structure and a hydrophobic core particularly rich in aromatic residues. The copper-binding active site is surrounded by aromatic res idues, and many of the resonances of the residues flanking the active site are shifted to unusual values, consistent with the effects of rin g currents. The protected nature of the copper site is demonstrated by the large number of amide protons that are persistent in this region in 99% (H2O)-H-2 solution at pH 3.4. We suggest that the unusual acid stability both of the protein itself and of the blue copper active sit e, is a direct result of the protected and highly hydrophobic nature o f the active site sequence and contacting loops and the high proportio n of secondary structure in the protein.