Structure-function analyses of the O₂-dependent dimer-monomer transition in the FNR transcription factor

About 2,5 billion years ago, oxygenic photosynthesis favored the evolution of gene expression regulatory strategies to respire atmospheric O₂ in many anaerobic organisms. One such strategy was the evolution of the Fumarate Nitrate Reduction (FNR) regulator from a cAMP-binding transcription factor. The activity of FNR is regulated by a [Fe₄S₄] cluster-dependent dimer-monomer transition. The dimeric FNR responds to O₂ through cluster degradation, which causes monomerization and loss of binding to its palindromic DNA receptor sites. Despite extensive structural and functional characterization, the molecular mechanism underlying signal transmission from the cluster to the DNA-binding domain and the dimer interface remains unclear. Here, we used a series of specific variants to explore this process both functionally, through stopped-flow and in vivo activity tests, and structurally, by exposing FNR crystals to O₂ and three-dimensionally determining its effects. The emerging picture consists of three clearly interconnected major steps: cluster degradation, breaking of two intermolecular salt bridges, and bending and separation of the two dimerization α-helices. We also performed a preliminary analysis of FNRs belonging to different bacterial classes and concluded that their variable cysteine-based cluster coordination motifs might influence signal transmission.