• Journal Article

Nuclear Magnetic Resonance 15N and 1H Resonance Assignments and Global Fold of Rusticyanin: Insights into the Ligation and Acid Stability of the Blue Copper Site

Citation

HUNT, A. H., Toy-Palmer, A., ASSA-MUNT, N., CAVANAGH, J., BLAKE, R. C., & DYSON, H. J. (1994). Nuclear Magnetic Resonance 15N and 1H Resonance Assignments and Global Fold of Rusticyanin: Insights into the Ligation and Acid Stability of the Blue Copper Site. Journal of Molecular Biology, 244(4), 370-384.

Abstract

Nuclear magnetic resonance assignments are reported at pH ∼3 for a type 1 ("blue") copper protein, rusticyanin, obtained from the acidophilic organism Thiobacillus ferrooxidans . A combination of homonuclear proton and heteronuclear 16N-edited NMR spectra has been used to assign most of the 1H and 15N resonances of reduced rusticyanin. The copper-binding site is shown by analogy with other blue copper proteins 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 likely a histidine, His85, consistent with the constitution of other type 1 copper sites. The global fold of the molecule is a compact β-barrel or β-sandwich, which contains a high proportion of β-sheet secondary structure and a hydrophobic core particularly rich in aromatic residues. The copper-binding active site is surrounded by aromatic residues, and many of the resonances of the residues flanking the active site are shifted to unusual values, consistent with the effects of ring 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% 2H2O solution at pH 3.4. We suggest that the unusual acid stability, both of the protein itself and of the blue copper active site, is a direct result of the protected and highly hydrophobic nature of the active site sequence and contacting loops and the high proportion of secondary structure in the protein.