EPR Studies on the Mono- and Dicobalt(II)-Substituted Forms of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e. Insight into the Catalytic Mechanism of Dinuclear Hydrolases

The structure and function of the prototypical dinuclear hydrolase, namely, the aminopeptidase from Aeromonas proteolytica (AAP), was probed by EPR spectroscopy of the mono- and dicobalt(II)-substituted derivatives. A new systematic protocol for the interpretation of Co(II) EPR spectra is described and the S = 3/2 spin states of the Co(II)-substituted forms of the enzyme have been characterized. This protocol allows the simulation of line shape using theoretically allowed geff values corresponding to an isotropic greal value. In addition, the gross distortion of EPR spectra of high-spin S = 3/2 Co(II) ions has been investigated, and the effects of saturation on the line shapes and on simulation-derived spectral parameters are discussed. For [Co-(AAP)], a distinctive EPR signal was observed in which the hyperfine pattern due to 59Co was not centered on the low-field absorption feature, and the signal could not be simulated as a single species. Subtraction of EPR spectra recorded at different temperatures revealed that two species were, in fact, present in samples of [Co-(AAP)]. The first species was a relatively featureless axial signal with geff values of 5.75, 4.50, and 2.50. These values correspond to an Ms = |±1/2〉 ground-state transition with greal = 2.57 and E/D = 0.08. The second species had geff values of 6.83, 2.95, and 1.96 and exhibited a characteristic eight-line 59Co hyperfine pattern with Az = 7.2 mT. The observed 59Co hyperfine lines were simulated in both line width as well as signal intensity for the first time. These parameters correspond to the Ms = |±1/2〉 ground-state transition with greal = 2.57; however, the signal exhibited marked rhombicity (E/D = 0.28), consistent with a highly distorted tetrahedral Co(II) species. The possibility that the spectrum could be due to contributions from the Ms = |±1/2〉 and Ms = |±3/2〉 doublets of a single spin system was investigated, but subtraction of spectra recorded at various temperatures clearly indicated that the features at g = 2.95 and g = 1.96 were correlated with the feature at g = 6.83. In addition, at temperatures above 15 K, the signal intensity rapidly decreases and the signal is lost. The EPR spectrum of [CoCo(AAP)] reveals a relatively featureless signal that was simulated as a single species with geff(1,2,3) values of 5.10, 3.85, and 2.19; Ms = |±1/2〉; greal = 2.25; E/D = 0.095. The intensity of the observed signal for [CoCo(AAP)] corresponded to 0.13 spin/mol of Co(II). These data strongly suggest that the two Co(II) ions in the active site of AAP experience significant spin−spin interaction and are either antiferromagnetically or ferromagnetically coupled. Perpendicular mode EPR titration of apo-AAP with Co(II) revealed a low-field signal extending out of zero-field in samples with more than 1 equiv of Co(II) added. This type of EPR absorption is indicative of an integral spin system. Coincident with the appearance of the low-field perpendicular mode signal was the appearance of a parallel mode EPR signal with g ∼ 12. These data represent the first definitive evidence for ferromagnetic coupling between two high-spin S = 3/2 Co(II) ions in a dinuclear center. The effect of pH, added peroxide, and the coordination of the competitive inhibitor 1-butaneboronic acid (BuBA) on the signal both confirm the origin of the signal and provide important mechanistic information for this novel dicobalt(II) active site cluster. Based on the present study and the available literature data, a detailed mechanism of action is proposed for AAP