High-yield heterologous SERCA1 (Ca(2+) ATPase) expression was obtained in COS-1 cells infected with recombinant adenovirus vector (rAdSERCA). Higher transcription and expression were obtained in the presence of a His(6) tag at the amino terminus, as compared with a His(6) tag at the carboxyl SERCA terminus, or no tag. The expressed protein was targeted extensively to intracellular membranes. Optimal yield of functional Ca(2+) ATPase corresponded to 10% of total protein, with phosphoenzyme levels, catalytic turnover and Ca(2+) transport identical with those of native SERCA1. This recombinant membrane-bound (detergent-free) enzyme was used for characterization of Ca(2+) binding at the two specific transmembrane sites (ATP-free) by measurements of net charge transfer upon Ca(2+) binding to the protein, yielding cooperative isotherms (K(1)=5.9+/-0.5x10(5) M(-1) and K(2)=5.7+/-0.3x10(6) M(-1)). Non-cooperative binding of only one Ca(2+), and loss of ATPase activation, were observed following E309 mutation at site II. On the other hand, as a consequence of the site II mutation, the affinity of site I for Ca(2+) was increased (K=4.4+/-0.2x10(6) M(-1)). This change was due to a pK(a) shift of site I acidic residues, and to contributions of oxygen functions from empty site II to Ca(2+) binding at site I. No charge movement was observed following E771Q mutation at site I, indicating no Ca(2+) binding to either site. Therefore, calcium occupancy of site I is required to trigger cooperative binding to site II and catalytic activation. In the presence of millimolar Mg(2+), the charge movement upon addition of Ca(2+) to WT ATPase was reduced by 50%, while it was reduced by 90% when Ca(2+) was added to the E309Q/A mutants, demonstrating that competitive Mg(2+) binding can occur at site I but not at site II.