The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PC-PLCBc; EC 188.8.131.52; Mr = 28,520) catalyzes the hydrolysis of phospholipids to yield a diacylglycerol and a phosphate ester with high specificity toward a positive polar group, such as choline, ethanolamine, and serine [1,2] Unlike the mammalian phosphatidylinositiol-PLC that plays an important role in Ca2+ regulation and signal transduction [3–6] the function of PC-PLCBc in vivo is not fully known other than its involvement in membrane metabolism. It has been suggested that this enzyme may replenish lost supplies of phosphate in the bacterial cell via a phosphate retrieval mechanism . Since PC-PLCBc is structurally related to mammalian PC-PLC that plays a role in cell growth and tumor formation , it can thus serve as a good model system for the poorly characterized mammalian enzymes [9–11]. Moreover, PC-PLCBc is also a widely used tool in phospholipid research and thus contributes significantly to the fields of membrane biochemistry, lipid metabolism, and blood coagulation [12–14].
The X-ray crystal structures of PC-PLCBc and its inhibitor complexes have been solved [15–17]. The active site was revealed to contain three Zn2+ ions (structure), wherein Zn1 and Zn3 in the active site form a dinuclear center (3.3 Å) bridged by Asp122 and a water (or hydroxide) molecule as observed in several other Zn proteins [18,19], including aminopeptidases and phosphotriesterase. Zn2 is not bridged to either Zn1 or Zn3 (with distances 6.0 and 4.7 Å, respectively), and has a coordination similar to carboxypeptidase A . Although the crystal structure showed three bound Zn2+ ions in PC-PLCBc , all previous physical studies of the enzyme in solution indicated the presence of only two metal ions per molecule, including atomic absorption , EPR , EXAFS , and 113Cd NMR  studies. One explanation for the discrepancy is attributed to the removal of loosely bound metal ions in previous experiments. Since all of the previous studies used an incorrect molecular weight of 23 kDa, a correction changes the metal content from 2 to 2.3 Zn2+/molecule.
Several other metal ions have been shown to bind to PC-PLCBc, including Mn2+, Co2+, Ni2+, Cu2+, Cd2+, Hg2+, and Ag+ [25,26]. Metal substitution was found to decrease the activity of PC-PLCBc with the effectiveness that follows the trend Cu2+ > Ni2+ > Cd2+ > Co2+ > Hg2+ > Ag+ > Mn2+ toward lecithin substrates which may also reflect the trend of metal binding affinity with PLC . A Co2+ derivative of PC-PLCBc was prepared by dialyzing the native enzyme against low concentrations of Co2+ (1–5 mM), resulting in exchange of one high-affinity Zn2+ with Co2+. The Zn site that did not exchange with Co2+ was referred to as the "structural site" while the exchangeable site was considered the "catalytic site", possibly Zn2 . However, a recent mutagenesis study indicates that Glu146 in the Zn2 site is not involved in the activation of the nucleophilic water . Furthermore, a molecular dynamic study suggests that Asp55 may act as a general base to deprotonate a water molecule for nucleophilic attack instead of Zn2 since there are no coordinated water molecules directly bound to the three Zn2+ when a substrate inhibitor is bound . Co2+-substituted derivatives were also prepared by adding 1 or 2 equivalents of Co2+ to apo protein . Electronic absorption and EPR studies suggest that the Co2+ in PC-PLCBc has a distorted octahedral coordination sphere in both high-affinity metal binding sites as well as the presence of a magnetic coupling between two paramagnetic Co2+ centers in Co2-PLCBc ; however, X-ray crystal structure shows 5-coordinate geometries . We have reinvestigated Co2+- and Cu2+-substituted derivatives of PC-PLCBc by the use of 1H NMR spectroscopy. The binding patterns of these two metal ions are suggested based on the spectral features. A stable tri-Cu2+-substituted PC-PLCBc has been prepared and characterized with NMR and EPR spectroscopies (Spectra), which represents the first trinuclear Cu2+ center in proteins studied by the use of NMR spectroscopy.