Charge Movement in Recombinant Copper ATPase is Investigated on a Solid Supported Membrane

The ATP7B copper ATPase is included in the P1-type ATPase subfamily, which is selective for soft and transition metals. Its function is to deliver copper to nascent metalloproteins and export excessive copper from the cell (1). Biochemical characterization of this enzyme is hindered by its very low native abundance and difficult detection of copper signal within the catalytic time frame. We recently reported high yield heterologous expression of ATP7B in COS-1 cells infected with adenovirus vector, and functional characterization of membrane-bound ATPase obtained with the microsomal fraction of infected cells (2). Here we show that the membrane-bound copper ATPase obtained under the above-mentioned conditions is suited for adsorption on a solid supported membrane and measurements of charge transfer. Using this method, we detected charge movement within a single catalytic cycle upon addition of ATP. We suggest that the observed charge movement is due to displacement of bound copper, and it is related to formation of phosphoenzyme intermediate. ATP dependent charge movements, as well as phosphoenzyme formation, are totally prevented by single mutations of copper binding sites. This work was supported by the U.S. National Institutes of Health (RO301-69830 from the NHBLI), the Italian Ministry of Education, University and Research (PRIN 2008), the Ente Cassa di Risparmio di Firenze and the Italian Ministry of Foreign Affairs (Joint Mobility Project n.22 for the Exchange of Researchers between Italy and USA, 2008-10). 1. Lutsenko S., N.L. Barnes, M.Y. Bartee, and O.Y. Dmitriev. 2007. Physiol. Rev. 87:1011-1046. 2. Pilankatta R., D. Lewis, C.M. Adams, and G. Inesi. 2009. J. Biol. Chem. 284:21307-21316.