Mechanism of cyanobacterial ferredoxin-dependent flavin thioredoxin reductase reveals thiolate-FAD charge transfer and catalytic asymmetry in a homodimer.

Ferredoxin-dependent flavin thioredoxin reductases (FFTRs) catalyze the reduction of the disulfide bond in thioredoxins using electrons transferred from ferredoxin, and therefore play a pivotal role in cellular disulfide relay reactions. FFTRs are essential in cyanobacteria such as Gloeobacter and Prochlorococcus, in which they serve as the sole thioredoxin reduction system, as well as in certain Clostridium species, where they are implicated in processes such as sporulation. Despite the well-established role of ferredoxin in reducing FFTRs, the underlying mechanistic details remain poorly understood. This study examines the catalytic cycle of FFTR from Gloeobacter violaceus, focusing on the role of its redox-active disulfide in electron transfer. We demonstrate that FFTR has a highly negative flavin adenine dinucleotide (FAD) midpoint reduction potential, which explains its preference for ferredoxin over nicotinamide adenine dinucleotide phosphate (NADPH) as an electron source. Spectroscopic detection of a thiolate-flavin charge transfer complex along the enzyme reduction pathway provides the first experimental evidence of a previously elusive FFTR catalytic conformation. Our results further reveal sequential FAD reduction within the enzyme homodimer that strongly suggests monomer asymmetry. Moreover, the impaired flavin reduction observed in an enzyme variant lacking the disulfide highlights the essential role of this redox group in efficient electron transfer. These findings deepen our understanding of FFTR's unique functional adaptations and evolutionary significance. More broadly, they provide a framework for exploring similar electron transfer mechanisms in other flavoproteins with a view to expanding our understanding of their redox biochemistry.