The Active-Site [4Fe-4S] Cluster in the Isoprenoid Biosynthesis Enzyme IspH Adopts Unexpected Redox States during Ligand Binding and Catalysis

Selamawit Ghebreamlak, Sebastian A. Stoian*, Nicholas S. Lees, Bryan Cronin, Forrest Smith, Matthew O. Ross, Joshua Telser, Brian M. Hoffman, Evert C. Duin*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase, or IspH (formerly known as LytB), catalyzes the terminal step of the bacterial methylerythritol phosphate (MEP) pathway for isoprene synthesis. This step converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into one of two possible isomeric products, either isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This reaction involves the removal of the C4 hydroxyl group of HMBPP and addition of two electrons. IspH contains a [4Fe-4S] cluster in its active site, and multiple cluster-based paramagnetic species of uncertain redox and ligation states can be detected after incubation with reductant, addition of a ligand, or during catalysis. To characterize the clusters in these species, 57Fe-labeled samples of IspH were prepared and studied by electron paramagnetic resonance (EPR), 57Fe electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies. Notably, this ENDOR study provides a rarely reported, complete determination of the 57Fe hyperfine tensors for all four Fe ions in a [4Fe-4S] cluster. The resting state of the enzyme (Ox) has a diamagnetic [4Fe-4S]2+ cluster. Reduction generates [4Fe-4S]+ (Red) with both S = 1/2 and S = 3/2 spin ground states. When the reduced enzyme is incubated with substrate, a transient paramagnetic reaction intermediate is detected (Int) which is thought to contain a cluster-bound substrate-derived species. The EPR properties of Int are indicative of a 3+ iron-sulfur cluster oxidation state, and the Mössbauer spectra presented here confirm this. Incubation of reduced enzyme with the product IPP induced yet another paramagnetic [4Fe-4S]+ species (Red+P) with S = 1/2. However, the g-tensor of this state is commonly associated with a 3+ oxidation state, while Mössbauer parameters show features typical for 2+ clusters. Implications of these complicated results are discussed.

Original languageEnglish (US)
Pages (from-to)3926-3942
Number of pages17
JournalJournal of the American Chemical Society
Volume146
Issue number6
DOIs
StatePublished - Feb 14 2024

Funding

This work was supported by National Science Foundation Grants 0848196 (E.C.D.) and MCB-1908587 (B.M.H.). A portion of this work was performed at the National High Magnetic Field Laboratory, which is supported by National Science Foundation Cooperative Agreement No. DMR-2128556 and the State of Florida. In addition, thanks go out to Dr. Hassam Jomaa and Dr. Michael Groll for providing some of the expression plasmids used in this work.

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry

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