Cobalt(II) and copper(II) binding of Bacillus cereus trinuclear phospholipase C: a novel 1H NMR spectrum of a 'Tri-Cu(II)' center in protein.
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 and a 'stable' derivative. 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. This might possibly be the case for Co2+ binding. Complete replacement of the three 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 five-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, 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.
Epperson
J D
JD
Department of Chemistry and Institute for Biomolecular Science, University of South Florida, 4202 Fowler Ave., CHE305, Tampa, FL 33620-5250, USA.
Ming
L J
LJ
D001409:Bacillus cereus / Q000201:enzymology / Q000378:metabolism*; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D003035:Cobalt; D003300:Copper; D010738:Type C Phospholipases