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Abstract
The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PC-PLC_Bc) is a tri-Zn enzyme with two "tight binding" and one "loose binding" sites. The Zn²⁺ ions can be replaced with Co²⁺ and Cu²⁺ to afford metal-substituted derivatives. Two Cu²⁺-substituted derivatives are detected by means of ¹H NMR spectroscopy, a "transient" derivative (Spectrum A, below) and a "stable" derivative (Spectrum B, below). The detection of sharp hyperfine-shifted ¹H NMR signals in the "transient" derivative indicates the formation of a magnetically coupled di-Cu²⁺ center, which concludes that the Zn²⁺ ions in the dinuclear (Zn1 and Zn3) sites are more easily replaced by Cu²⁺ than that in the Zn2 site (See active-site structure). This might possibly be the case for Co²⁺ binding. Complete replacement of the 3 Zn²⁺ ions can be achieved by extensive dialysis of the enzyme against excess Cu²⁺ to yield the final "stable" derivative. This derivative has been determined to have 5 coordinated His residues and an overall S’ =1/2 spin state with NMR and EPR, consistent with the formation of a tri-Cu²⁺ center (i.e., a di-Cu²⁺/mono-Cu²⁺ center) in this enzyme. The binding of substrate to the inert tri-Cu²⁺ center to form an enzyme-substrate (ES) complex is clearly seen in the ¹H NMR spectrum (Spectrum C, below), which is not obtainable in the case of the native enzyme. The change in the spectral features indicates that the substrate binds directly to the trinuclear metal center. The studies reported here suggest that ¹H NMR spectroscopy can be a valuable tool for the characterization of di- and multi-nuclear metalloproteins using the "NMR friendly" magnetically coupled Cu²⁺ as a probe.