Obafluorin is a Pseudomonas fluorescens antibacterial natural product that inhibits threonyl-tRNA synthetase (ThrRS). It acts as a broad-spectrum antibiotic against a range of clinically relevant pathogens and comprises a strained ß-lactone ring decorated with catechol and 4-nitro-benzyl moieties. The catechol moiety is widespread in nature and its role in the coordination of ferric iron has been well-characterised in siderophores and Trojan horse antibiotics. Here we use a combination of mutasynthesis, bioassays, enzyme assays and metal binding studies to delineate the role of the catechol moiety in the bioactivity of obafluorin. We use P. fluorescens biosynthetic mutants to generate obafluorin analogues with modified catechol moieties. We demonstrate that an intact catechol is required for both antibacterial activity and inhibition of the ThrRS molecular target. Although recent work showed that the obafluorin catechol coordinates Zn2+ in the ThrRS active site, we find that obafluorin is a weak Zn2+ binder in vitro, contrasting with a strong, specific 1?:?1 interaction with Fe3+. We use bioassays with siderophore transporter mutants to probe the role of the obafluorin catechol in Fe3+-mediated uptake. Surprisingly, obafluorin does not behave as a Trojan horse antibiotic but instead exhibits increased antibacterial activity in the presence of Fe3+. We further demonstrate that Fe3+ binding prevents the hydrolytic breakdown of the ß-lactone ring, revealing a hitherto unreported function for the catechol moiety in natural product bioactivity.