Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Towards understanding the electronic structure of alkane monooxygenase’s diiron active site (#475)

Stella Bailey 1 , Rachel N Austin 1
  1. Barnard College, New York, NY, United States

Alkane monooxygenase (AlkB) is an integral membrane protein containing a non-heme diiron active site which selectively oxidizes liquid alkanes to terminal alcohols in the environment,1 as well as defluorinates fluoroalkanes to form their respective alcohols, aldehydes, and fluoroaldehydes.2 Recent cryo-EM and EXAFS studies have shown that the diiron active site is coordinated by nine histidine residues with a distance of at least 5 Å separating the iron ions.3,4 Despite the knowledge surrounding the active site’s molecular structure, much is still unknown about the electronic relationship between its two iron ions. While previous literature characterized AlkB as containing an antiferromagetically coupled pair of high-spin Fe(III) ions,5 the recent structural studies revealed a new mystery surrounding how the iron ions in AlkB’s active site can be electronically coupled at such a large internuclear distance without a bridging ligand. A key component in elucidating the electronic structure of the active site is ensuring proper iron occupation. The occupation of the diiron site was probed using Helium collision ICP-MS, in combination with colorimetric protein quantification assays. The ICP-MS method was developed based on EPA Method 6020 to facilitate isotopic quantification of iron. Recombinant Fontimonas thermophila AlkB was purified from E. coli with a poly-histidine tag via detergent solubilization and metal affinity chromatography. Samples for preliminary Mössbauer studies were prepared by growing AlkB expressing E. coli using isotopically enriched 57Fe. Further purification of AlkB was afforded using a histidine-cleavage protocol with the TEV enzyme. The development of a Helium collision ICP-MS method, in combination with optimizing histidine-tag cleavage and protein purification, enabled progress to be made towards spectroscopic analysis of AlkB. Future steps include optimizing the growth method for isotopically enriched 57Fe AlkB in preparation for Mössbauer spectroscopy, to analyze the oxidation state and electronic coupling of AlkB’s diiron active site.

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