The secondary structure of Na + /K + -ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na + /K + -ATPase additionally to α-helix also β-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na + /K + -ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na + /K + -ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H 2 O) 4 ATP and Co(NH 3 ) 4 ATP, on the other hand, affected the secondary structure of Na + /K + -ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.