The twin-arginine translocation (Tat) system is a protein targeting pathway utilised by prokaryotes and chloroplasts. Tat substrates are produced with distinctive N-terminal signal peptides and are translocated as fully folded proteins. In Escherichia coli Tat-dependent proteins often contain redox cofactors that must be loaded before translocation. Trimethylamine N-oxide reductase (TorA) is a model bacterial Tat substrate and is a molybdenum cofactor-dependent enzyme. Co-ordination of cofactor loading and translocation of TorA is directed by the TorD protein, which is a cytoplasmic chaperone known to physically interact with the TorA signal peptide. In this work, a pre-export TorAD complex has been characterised using biochemical and biophysical techniques, including SAXS. A stable, cofactor-free TorAD complex was isolated, which revealed a 1:1 binding stoichiometry. Surprisingly, a TorAD complex with similar architecture can be isolated in the complete absence of the 39-residue TorA signal peptide. This work demonstrates that two high-affinity binding sites for TorD are present on TorA, and that a single TorD protein binds both of those simultaneously. Further characterisation suggested that the C-terminal 'Domain IV' of TorA remained solvent-exposed in the cofactor-free pre-export TorAD complex. It is possible that correct folding of Domain IV upon cofactor loading is the trigger for TorD release and subsequent export of TorA.