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Spectroscopic Studies and Characterization of a Novel Electron-Transfer Chain
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A novel two-component enzyme system from Escherichia coli involving a flavorubredoxin
(FlRd) 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¢Rd), were characterized. FlRd
contains 2.9 ( 0.5 iron atoms/subunit, whereas FlRd¢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¢Rd, are
most probably due to a di-iron site. Redox titrations of FlRd 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 (Flox/Flsq) and -180 ( 15 mV (Flsq/Flred), 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 FlRd 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.
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American Chemical Society