A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Terpredoxin (Tdx) is a bacterial ferredoxin identified in Pseudomonas sp. utilizing alpha-terpineol.
• The Tdx gene is part of the terp operon involved in terpineol catabolism.
• Understanding Tdx structure is crucial for elucidating its role in microbial metabolism.

Purpose of the Study:

• To clone, express, and characterize the holoprotein of Terpredoxin (Tdx) in E. coli.
• To determine the solution structure of oxidized Terpredoxin (Tdxo) using NMR spectroscopy.
• To compare the structure and dynamics of Tdx with homologous ferredoxins like Adrenodoxin (Adx) and Putidaredoxin (Pdx).

Main Methods:

• Subcloning and expression of the Tdx gene in E. coli.
• High-resolution homo- and heteronuclear NMR spectroscopy for structure determination.
• Modeling of the metal-binding site using restraints from homologous ferredoxin crystal structures and NMR paramagnetic broadening.

Main Results:

• The solution structure of oxidized Tdx (Tdxo) was determined, revealing a five-stranded beta-sheet, a two-stranded beta-sheet, and three alpha-helices.
• Significant structural homology was observed between Tdx, Adx, and Pdx.
• NMR analysis identified key residues modulating protein dynamics and revealed differences in the C-terminal region and UV-visible spectra compared to Pdx, with Tdx showing only 2% of Pdx activity.

Conclusions:

• The study provides the first structural model for Terpredoxin (Tdx), highlighting its homology to other ferredoxins.
• Specific residues, particularly Arg49, are implicated in oxidation-state-dependent protein dynamics.
• Functional differences exist between Tdx and Pdx, suggesting distinct roles in their respective metabolic pathways.