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Titolo:
EPR STUDIES ON THE MONOCOBALT(II)-SUBSTITUTED AND DICOBALT(II)-SUBSTITUTED FORMS OF THE AMINOPEPTIDASE FROM AEROMONAS-PROTEOLYTICA - INSIGHT INTO THE CATALYTIC MECHANISM OF DINUCLEAR HYDROLASES
Autore:
BENNETT B; HOLZ RC;
Indirizzi:
UTAH STATE UNIV,DEPT CHEM & BIOCHEM LOGAN UT 84322 UTAH STATE UNIV,DEPT CHEM & BIOCHEM LOGAN UT 84322
Titolo Testata:
Journal of the American Chemical Society
fascicolo: 8, volume: 119, anno: 1997,
pagine: 1923 - 1933
SICI:
0002-7863(1997)119:8<1923:ESOTMA>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELECTRON-PARAMAGNETIC-RESONANCE; PERIPLASMIC NITRATE REDUCTASE; PURPLE ACID-PHOSPHATASE; BINUCLEAR METAL CENTER; ESCHERICHIA-COLI; CRYSTAL-STRUCTURE; ACTIVE-SITE; SPECTROSCOPIC CHARACTERIZATION; METHIONINE AMINOPEPTIDASE; LEUCINE AMINOPEPTIDASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
75
Recensione:
Indirizzi per estratti:
Citazione:
B. Bennett e R.C. Holz, "EPR STUDIES ON THE MONOCOBALT(II)-SUBSTITUTED AND DICOBALT(II)-SUBSTITUTED FORMS OF THE AMINOPEPTIDASE FROM AEROMONAS-PROTEOLYTICA - INSIGHT INTO THE CATALYTIC MECHANISM OF DINUCLEAR HYDROLASES", Journal of the American Chemical Society, 119(8), 1997, pp. 1923-1933

Abstract

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 allowsthe simulation of line shape using theoretically allowed g(eff) values corresponding to an isotropic g(real) 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 dueto Co-59 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 specieswere, in fact, present in samples of [Co-(AAP)]. The first species was a relatively featureless axial signal with g(eff) values of 5.75, 4.50, and 2.50. These values correspond to an M(s) = \+/-1/2) ground-state transition with g(real) = 2.57 and E/D = 0.08. The second species had g(eff) values of 6.83, 2.95, and 1.96 and exhibited a characteristic eight-line Co-59 hyperfine pattern with A(z) = 7.2 mT. The observed Co-59 hyperfine lines were simulated in both line width as well as signal intensity for the first time. These parameters correspond to the M(s) = \+/-1/2) ground-state transition with g(real) = 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 M(s) = \+/-1/2] and M(s) = \+/-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 thefeature 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 g(eff(1,2,3)) values of 5.10, 3.85, and 2.19; Ms = \+/-1/2]; g(real) = 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. Perpendicularmode EPR titration of apo-AAP with Co(II) revealed a low-field signalextending 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 similar to 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.

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Documento generato il 01/12/20 alle ore 13:09:11