Changes in the A(3)B(3)CDF-complex of the Methanosarcina mazei Gö1 A(1)-ATPase in response to ligand binding have been studied by small-angle x-ray scattering, protease digestion, fluorescence spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and CuCl(2)-induced disulfide formation. The value of the radius of gyration, R(g), increases slightly when MgATP, MgADP, or MgADP + P(i) (but not MgAMP-PNP) is present. The nucleotide-binding subunits A and B were reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide, and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP-PNP, MgATP, MgADP + P(i), or MgADP. Trypsin treatment of A(1) resulted in cleavage of the stalk subunits C and F, which was rapid in the presence of MgAMP-PNP but slow when MgATP or MgADP were added to the enzyme. When A(1) was supplemented with CuCl(2) a clear nucleotide dependence of an A-A-D cross-linking product was generated in the presence of MgADP and MgATP but not when MgAMP-PNP or MgADP + P(i) was added. The site of cross-link formation was located in the region of the N and C termini of subunit D. The data suggest that the stalk subunits C, D, and F in A(1) undergo conformational changes during ATP hydrolysis.