A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus-revealed by EPR spectroscopy

As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu2+. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2Cu(2+). The copper may be removed by extensive washing with EDTA. Titration with Ni2+ then induces spin-spin interactions with nitroxyl spin labels that are attached either to the unique Cys(54), or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni2+ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni2+-binding site on the protein is situated around 14-18 angstrom from the spin label on Cys(54), and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains. (c) 2006 Elsevier B.V. All rights reserved.