A five-coordinate metal center in Co(II)-substituted VanX

Area of Science:

• Biochemistry
• Biophysical Chemistry
• Enzymology

Background:

• VanX is a metalloenzyme implicated in bacterial cell wall synthesis.
• Understanding the metal binding site is crucial for enzyme mechanism elucidation.
• Previous crystallographic studies suggested a different metal coordination number.

Purpose of the Study:

• To structurally characterize the metal binding site of VanX using spectroscopic methods.
• To determine the coordination number and environment of the metal ion in VanX.
• To reconcile discrepancies between crystallographic and spectroscopic data.

Main Methods:

• Cobalt(II)-substituted VanX was prepared for spectroscopic analysis.
• Electronic absorption spectroscopy was used to assess Co(II) ligand field transitions.
• Electron Paramagnetic Resonance (EPR) spectroscopy provided insights into the electronic structure.
• Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy determined metal-ligand distances and coordination numbers.

Main Results:

• Electronic spectroscopy indicated five-coordinate Co(II) with molar absorptivities around 100 m⁻¹cm⁻¹.
• EPR spectral simulations suggested a five-coordinate Co(II) with potential solvent ligands.
• EXAFS data revealed a coordination number of 5 for metal ions (Zn(II) and Co(II)) at ~2.0 Å.
• Spectroscopic findings consistently showed five-coordinate metal ions, contrasting with prior crystallographic data.
• The metal ion remained five-coordinate even when complexed with a phosphinate substrate analog.

Conclusions:

• Spectroscopic evidence strongly supports a five-coordinate metal binding site in VanX.
• This five-coordination is independent of the metal ion (Zn(II) or Co(II)) and substrate analog binding.
• The findings necessitate a re-evaluation of the VanX active site structure based on crystallographic data.