[2Fe-2S] to [4Fe-4S] cluster conversion in Escherichia coli biotin synthase

Area of Science:

• Biochemistry and enzymology
• Bioinorganic chemistry
• Structural biology

Background:

• Iron-sulfur (Fe-S) clusters are vital cofactors in numerous enzymes.
• Biotin synthase, an Fe-S enzyme, is essential for biotin biosynthesis.
• Understanding Fe-S cluster properties is key to elucidating enzyme mechanisms.

Purpose of the Study:

• To investigate the type and properties of Fe-S clusters in recombinant E. coli biotin synthase.
• To characterize the structural and electronic changes of Fe-S clusters during reductive conversion.
• To propose the role of Fe-S clusters in the radical mechanism of biotin synthase.

Main Methods:

• Spectroscopic techniques including UV-visible absorption, VTMCD, EPR, and resonance Raman spectroscopy.
• Analysis of as-prepared and dithionite-reduced samples of recombinant E. coli biotin synthase.
• Investigating cluster conversion under varying reduction conditions and cryoprotectants.

Main Results:

• Aerobically purified samples contain S=0 [2Fe-2S]2+ clusters with incomplete cysteinyl coordination.
• Anaerobic reduction converts [2Fe-2S]2+ clusters to S=0 [4Fe-4S]2+ clusters with complete coordination.
• EPR and VTMCD reveal [4Fe-4S]+ clusters with mixed spin states (S=1/2 and S=3/2) after prolonged reduction.

Conclusions:

• The [4Fe-4S]2+ cluster forms at the subunit interface via reductive dimerization of [2Fe-2S]2+ clusters.
• Subunit-bridging [4Fe-4S] clusters are proposed for radical enzymes utilizing S-adenosylmethionine.
• Oxidative conversion to [2Fe-2S]2+ may regulate enzyme activity under oxidative stress.