Abstract
The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide range of organophosphates.l The monomeric enzyme has a molecular weight of 36 OOOand contains two divalent metal ions that are required for catalytic activity.2~3 Zn2+ is present in the native enzyme, but this metal can be replaced with Mn2+, Cd2+, Co2+, or Ni2+ without loss of enzymatic activity.2 The 1Wd-NMR spectrum of the Cd/Cd-substituted enzyme exhibits resonances at 212 and 116 ppm downfield from Cd(C104)~.~ The separation in chemical shift between the two signals indicates that the ligand environment of the two metal ion sites is dissimilara2 Furthermore, the position of the two l13Cd-NMR resonances suggests that the coordination environment of the two metal sites consists of a mixture of nitrogen and oxygen ligands while direct ligation to cysteine is excluded. However, little is known of the roles thesemetal ions play in the catalytic mechanism. To address whether the binding sites for the two required metal ions are structurally independent of each other, we have conducted an EPR investigation of the Mn/Mn-substituted enzyme. In this communication, we report that the two MnZ+ ions are present as an antiferromagnetically-coupled binuclear center. A sample of Mn/Mn-substituted phosphotriesterase4 displayed a complex EPR spectrum (Figure 1A) at the X-band (9.4 GHz). The predominant features were near g = 2 and exhibited what appeared to be more than 26 Mn hyperfine splittings at -45-G intervals. To confirm that these features originated from Mn2+ ions that were enzyme-bound, a sample of the Mn/Mnphosphotriesterase was digested with 2 M perchloric acid for 3 h. The resulting spectrum (Figure 1 B) exhibited six Mn hyperfine lines separated by -90 G, virtually identical to the spectrum of Mn2+ ions in 50 mM HEPES buffer. Thus, the unusual spectral features of Figure 1A undoubtedly arise from Mn2+ ions bound to the enzyme. The large number of hyperfine splittings, separated by approximately half the magnitude expected for a mononuclear Mn2+ center, suggests the presence of two spin-coupled MnZ+
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