Spectroscopic studies and characterization of a novel electron-transfer chain from Escherichia coli involving a flavorubredoxin and its flavoprotein reductase partner

A novel two-component enzyme system from Escherichia coli involving a flavorubredoxin (FIRd) and its reductase was studied in terms of spectroscopic, redox, and biochemical properties of its constituents. FlRd contains one FMN and one rubredoxin (Rd) center per monomer. To assess the role of the Rd domain, FlRd and a truncated form lacking the Rd domain (FlRd Delta Rd), were characterized. FIRd contains 2.9 +/- 0.5 iron atoms/subunit, whereas FlRd Delta Rd contains 2.1 +/- 0.6 iron atoms/subunit. While for FlRd one iron atom corresponds to the Rd center, the other two irons, also present in FlRd Delta Rd, are most probably due to a di-iron site. Redox titrations of FIRd using EPR and visible spectroscopies allowed us to determine that the Rd site has a reduction potential of - 140 +/- 15 mV, whereas the FMN undergoes reduction via a red-semiquinone, at -140 +/- 15 mV (Fl(ox) /Fl(sq)) and -180 +/- 15 mV (Fl(sq)/Fl(red)), at pH 7.6. The Rd site has the lowest potential ever reported for a Rd center, which may be correlated with specific amino acid substitutions close to both cysteine clusters. The gene adjacent to that encoding FlRd was found to code for an FAD-containing protein, (flavo)rubredoxin reductase (FlRd-reductase), which is capable of mediating electron transfer from NADH to Desulfovibrio gigas Rd as well as to E. coli FlRd. Furthermore, electron donation was found to proceed through the Rd domain of FIRd as the Rd-truncated protein does not react with FlRd-reductase. In vitro, this pathway links NADH oxidation with dioxygen reduction. The possible function of this chain is discussed considering the presence of FlRd homologues in all known genomes of anaerobes and facultative aerobes.