The aminopeptidase from Aeromonas proteolytica (AAP) was titrated with copper, which bound sequentially at two distinct sites. Both the mono- and disubstituted forms of AAP exhibited catalytic hyperactivity relative to the native dizinc enzyme. Monosubstituted AAP exhibited an axial Cu(II) EPR spectrum with slight pH dependence: at pH 6.0 g(\\) = 2.249, g(perpendicular to) = 2.055, and A(\\)((CU)-C-63/65) = 1.77 x 10(-2) cm(-1), whereas at pH 9.65 g(\\) = 2.245, g(perpendicular to) = 2.056, and A(\\)(Cu-63/65) = 1.77 x 10-2 cm-1. These data indicate oxygen and nitrogen ligation of Cu. AAP further substituted with copper exhibited a complex signal with features around g similar to 2 and 4. The features at g similar to 4 were relatively weak in the B-0 perpendicular to B-1 (perpendicular) mode EPR spectrum but were intense in the B-0 \\ B-1 (parallel) mode spectrum. The g similar to 2 region of the perpendicular mode spectrum exhibited two components, one corresponding to mononuclear Cu(II) with g(\\) = 2.218, g(perpendicular to) = 2.023, and A(\\)(Cu-63/65) = 1.55 x 10(-2) cm(-1) and likely due to adventitious binding of Cu(II) to a site distant from the active site. Excellent simulations were obtained for the second component of the spectrum assuming that two Cu(II) ions experience dipolar coupling corresponding to an inter-copper distance of 5 A with the two Cu(II) g, directions parallel to each other and at an angle of similar to17degrees to the inter-copper vector (H = betaB.g(CuA).S-CuA + betaB.g(CuB).S-CuB + [S.A.I](CuA) + [S.A.I.](CuB) + [S-CuA.J.S-CuB]; g(\\(CuA,CuB)) = 2.218 g(perpendicular to(CuA,CuB)) = 2.060; A(\\(CuA),(CuB)) ((CU)-C-63/65) = 1.59 x 10(-2) cm(-1), J(isotropic) = 50 cm-1, r(Cu-Cu) = 4.93 Angstrom, and chi = 17degrees). The exchange coupling between the two copper ions was found to be ferromagnetic as the signals exhibited Curie law temperature dependence. The Cu-Cu distance of similar to5 Angstrom indicated by EPR was significantly higher than the inter-zinc distance of 3.5 Angstrom in the native enzyme, and the dicopper species therefore represents a novel dinuclear site capable of catalysis of hydrolysis. In contrast to AAP, the related methionyl aminopeptidase from Escherichia Coll (EcMetAP) was found to bind only one Cu(II) ion despite possessing a dinuclear binding site motif. A further difference was the marked pH dependence of the signal in EcMetAP, suggestive of a change in ligation. The structural motifs of these two Cu(II)-substituted aminopeptidases provide important insight into the observed catalytic activity.