Redox and spectroscopic properties of the Escherichia coli nitric oxide-detoxifying system involving flavorubredoxin and its NADH-oxidizing redox partner.

Under anaerobic conditions, the flavodiiron NO-reductase from Escherichia coli (flavorubredoxin, FlRd) constitutes one of the major protective enzymes against nitric oxide. The redox and spectroscopic properties of the rubredoxin (Rd), non-heme diiron, and FMN sites of flavorubredoxin were determined, which was complemented by the study of truncated versions of FlRd: one consisting only of its rubredoxin module, and another consisting of its flavodiiron structural core (lacking the Rd domain). The studies here reported were performed by a combination of potentiometry with visible and EPR spectroscopies. Moreover, we present the first direct EPR evidence for the presence of the non-heme diiron site in the flavodiiron proteins family. Also, the redox properties of the FlRd physiological partner, the NADH:flavorubredoxin oxidoreductase (FlRd-Red), were determined. It is further shown that the redox properties of this complex electron transfer system are fine-tuned upon interaction of the two enzymes.