Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• The MbnBC enzyme complex is essential for methanobactin (Mbn) biosynthesis, modifying cysteine residues in MbnA.
• MbnBC is a mixed-valent diiron oxygenase (MVDO) that utilizes an Fe(II)Fe(III) cofactor.
• Previous crystal structures captured the inactive Fe(III)Fe(III) state, limiting mechanistic understanding.

Purpose of the Study:

• To determine the oxidation states of iron ions in the active Fe(II)Fe(III) state of MbnBC.
• To elucidate the substrate binding mode of MbnA to the MbnBC active site.
• To gain mechanistic insights into the initial steps of Mbn biosynthesis.

Main Methods:

• Electron nuclear double resonance (ENDOR) spectroscopy was employed to study the active site.
• ¹⁵N ENDOR was used to determine the oxidation states of the iron ions.
• ¹H and ²H ENDOR were utilized to identify solvent ligands and substrate binding interactions.

Main Results:

• The active Fe(II)Fe(III) state of the MbnBC enzyme was characterized.
• ¹⁵N ENDOR confirmed the oxidation states of the iron ions.
• ²H ENDOR revealed that MbnA binds via its N-terminal cysteine sulfur to the Fe(III) ion, displacing a solvent ligand.

Conclusions:

• ENDOR spectroscopy provides crucial information on MVDO active sites, complementing X-ray crystallography.
• The study reveals the molecular details of substrate binding in the active Fe(II)Fe(III) state of MbnBC.
• This work advances the understanding of methanobactin biosynthesis mechanisms.