Designing Peptide Fossils That Model the Evolution of the Bacterial Ferredoxin Fold

Area of Science:

• Biochemistry and Molecular Evolution
• Origin of Life Studies
• Protein Engineering

Background:

• Electron transfer is fundamental to metabolism, relying on protein electron carriers that likely emerged early in life's history.
• Small iron-sulfur-binding bacterial ferredoxins are ancient proteins, with their current structure suggesting an ancient gene duplication event.
• The evolutionary history of ferredoxins predates phylogenetic analysis of amino acid sequences, necessitating alternative reconstruction methods.

Purpose of the Study:

• To reconstruct the deep-time molecular history of ferredoxins by modeling ancestral protein stages.
• To investigate the functional and structural properties of hypothetical ancestral ferredoxin forms, termed semidoxins and symdoxins.

Main Methods:

• Utilized structure-guided protein design to create computational models of ancestral ferredoxin forms (semidoxins and symdoxins).
• Assessed the structural, thermodynamic, and electrochemical behaviors of designed semidoxins and symdoxins.
• Employed an in vivo electron transfer complementation assay to evaluate protein function in bacterial growth under varying oxygen conditions.

Main Results:

• Designed semidoxins exhibited structural, thermodynamic, and electrochemical properties similar to their symdoxin counterparts.
• In vivo assays revealed that semidoxins conferred greater oxygen sensitivity to bacterial growth compared to symdoxins.
• Identification of naturally occurring semidoxins in anaerobic microorganisms, consistent with the oxygen sensitivity observed in designed semidoxins.

Conclusions:

• The modeled semidoxins represent a plausible ancestral stage in ferredoxin evolution, offering insights into early protein adaptation.
• The differential oxygen sensitivity between semidoxins and symdoxins suggests evolutionary pressures related to environmental oxygen levels.
• The existence of natural semidoxins and their monomer-dimer equilibrium hints at a potential link to even simpler iron-sulfur cluster-binding peptides.