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P. Sarti - Nitric oxide detoxification in pathogenic protozoa: role of flavodiiron proteins
upper part of the small intestine. Thus, it may be
expected that survival of the parasite in the host
requires an efficient O 2 -scavenging function. Such a
function is currently attributed to a H 2 O-producing
NADH oxidase identified and characterized a few
years ago, but based on our results, FDP may also
contribute significantly, if not predominantly, to O 2 -
detoxification in Giardia cells, a hypothesis that
needs to be validated yet. These findings may acquire
clinical relevance too, because if the FDP function
were shown to be essential for parasite survival in
the human host, the enzyme (not expressed in
humans) may become a suitable target for innovative
anti-giardial pharmacological treatments.
In a comparative study, we investigated also the
flavodiiron proteins from Escherichia coli. This
enzyme, named flavorubredoxin (FlRd) from a rubredoxin-type
domain fused to each monomer, was previously
shown to be an efficient NO-reductase under
anaerobic conditions, while being essentially inactive
towards O 2 . By stopped-flow spectroscopy, we found
that E. coli NADH:flavorubredoxin oxidoreductase
(FlRd-reductase) shuttles electrons between NADH
and FlRd very rapidly; at 5 °C, the protein is reduced
by NADH at k = 5.5 ± 2.2 x 10 6 M -1 s -1 and it
reduces in turn FlRd at k~1 x 10 7 M -1 s -1 , the reaction
being highly dependent on pH and ionic
strength. We established that electrons are first
donated by FlRd-reductase to the FeS center in the
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rubredoxin-domain of FlRd and then rapidly equilibrate
intramolecularly with the FMN and Fe-Fe site,
where the NO chemistry occurs.
In summary, our data support the idea that NADH
→ FlRd-reductase → FlRd is an efficient electron
transfer pathway that ensures a fast detoxification
of NO in E. coli. Since in the genome of G. intestinalis,
recently completely sequenced, apparently
there are no genes coding for rubredoxins, the
physiological redox partner protein of the FDP has
to be identified yet.
Selected publications
Vicente JB, Scandurra FM, Rodrigues JV, Brunori
M, Sarti P, Teixeira M, Giuffrè A. Kinetics of electron
transfer from NADH to the Escherichia coli
nitric oxide reductase flavorubredoxin. FEBS J.
2007, 274:677-86.
Di Matteo A, Scandurra FM, Testa F, Forte E, Sarti
P, Brunori M, Giuffrè A. The O 2 -scavenging flavodiiron
protein in the human parasite Giardia intestinalis.
J Biol Chem. 2008, 283:4061-8.
Vicente JB, Scandurra FM, Forte E, Brunori M,
Sarti P, Teixeira M, Giuffrè A. Kinetic characterization
of the Escherichia coli nitric oxide reductase flavorubredoxin.
Methods in Enzymol. 2008, 437:47-62.