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

## TL;DR

Scientists designed ancient-like proteins called semidoxins to model early evolutionary stages of bacterial ferredoxins, revealing insights into how these electron-carrying proteins may have evolved.

## Contribution

The study introduces semidoxins and symdoxins as designed peptide fossils to model the evolutionary history of ferredoxins.

## Key findings

- Semidoxins homodimerize and behave similarly to symdoxins in structural and electrochemical properties.
- Semidoxins show greater oxygen sensitivity in bacterial growth compared to symdoxins.
- A naturally occurring semidoxin was found to be folded and redox active, suggesting evolutionary links to simpler peptides.

## Abstract

Electron transfer
coupled to redox chemistry is at the heart of
metabolism. The proteins responsible for moving electrons (protein
electron carriers) must have emerged at the origin of life. The small
iron–sulfur-binding bacterial ferredoxins were likely among
these first proteins. Embedded within the ferredoxin sequence and
structure is a symmetry that points to an ancient gene duplication
event. Little is understood about the nature of ferredoxins prior
to this duplication event or what environmental factors may have driven
the selection for more complex forms. The deep-time molecular history
of ferredoxins goes back billions of years and cannot be reconstructed
by phylogenetic analyses based on amino acid sequences. Here, we use
structure-guided protein design to model a fossil half-ferredoxin
stage in the evolution of this fold, the semidoxins, and their symmetric
full-length counterparts, the symdoxins. Semidoxin designs homodimerize,
exhibiting structural, thermodynamic, and electrochemical behaviors
in most cases identical to cognate symdoxins. However, the semi- and
symdoxin fossil stages behave differently when incorporated into an
in vivo electron transfer complementation assay. Both can support
bacterial growth dependent on protein expression. Growth rates of
bacteria expressing the semidoxins are much more sensitive to oxygen
than those of bacteria expressing symdoxins. Motivated by the in vivo
functionality of designed semidoxins, we identified putative naturally
occurring semidoxins in extant anaerobic microorganisms. This is consistent
with the observed in vivo oxygen sensitivity of the semidoxin designs.
One natural semidoxin is shown to be folded and redox active. However,
it exists as a mixture of monomers and dimers, suggesting a potential
connection between semidoxins and even simpler single iron–sulfur
cluster-binding peptides.