# Adaptation of Fe-S Cluster Assembly to Rising O2 Levels over Geological Time

## TL;DR

This study explores how Fe-S cluster assembly adapted to rising oxygen levels during Earth's history, focusing on the SUF pathway and its role in microbial survival.

## Contribution

The study reveals the molecular adaptation of SufE and SufS in the SUF pathway to rising O2 levels, providing insights into coevolution of geosphere and biosphere.

## Key findings

- Ancestral SufSLCA/SufELCA is active at up to ~2% O2, while SufSGOE/SufEGOE is active at up to ~10% O2.
- Overproduction of SufEGOE or SufSGOE/SufEGOE in E. coli mutants better restores growth compared to LCA counterparts.
- Adaptation involved replacement of amino acids in key catalytic sites and conformational changes in enzymes.

## Abstract

One of the most important events in Earth history is the Great Oxidation Event (GOE). While O2 killed most anaerobic microorganisms, some survived. Fe-S clusters are cofactors essential for cellular processes in all life forms, but how they adapt to rising O2 remains unclear. Sulfur utilization factor (SUF) pathway is one of the most common Fe-S assembly pathways. We hypothesize that within the SUF pathway, SufE, as a sulfur-transfer partner of cysteine desulfurase SufS, maintains its functions under oxidative stress through molecular adaptation. Molecular clock dating showed SufE originated ~2.67 Ga (i.e., last common ancestor, LCA) and diversified considerably around the GOE (~2.14 Ga). The corresponding ancestral SufS was also reconstructed for these two times. Biochemical assays reveal that SufSLCA/SufELCA is active at up to ~2% O2, higher than Archaean atmospheric O2, whereas SufSGOE/SufEGOE is active at up to ~10% O2, higher than the level during the GOE. These advanced evolutions may have provided resilience to redox fluctuations through Earth history. Growth experiments showed that overproduction of either SufEGOE or SufSGOE/SufEGOE in Escherichia coli mutants lacking SufE or SufSE better restores its growth than overproduction of their LCA counterparts, consistent with the in vitro results. Enzyme structure prediction revealed that such adaptation was achieved through replacement of a few amino acids in key catalytic sites and consequent conformational changes of key enzymes. Our results reveal the molecular mechanism of adaptation of Fe-S cluster assembly to rising O2 and significantly contributes to the coevolution of the geosphere and biosphere.