The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon
dioxide, which is present in high concentrations in intra- and extracellular ...compartments. Peroxynitrite anion (ONOO â ) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO 2
â ), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (COÂ·Ì 3 ). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate
radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6â9. The radical was unambiguously
identified by its EPR parameters (g = 2.0113; line width = 5.5 G) and by experiments with bicarbonate labeled with 13 C. In this case, the singlet EPR signal obtained with 12 C bicarbonate splits into the expected doublet because of 13 C (a( 13 C)= 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range
the detected radical is the carbonate radical anion (COÂ·Ì 3 ). Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this
is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct
EPR spectroscopy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this work, we have designed and generated a Fe(
iii
)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a ...model peptide and on the surface of thioredoxin (TRX) from
E. coli
. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(
iii
) and Al(
iii
). The interaction of the grafted TRX with iron(
iii
) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(
iii
)
per
TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(
iii
), a Fe(
iii
) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical.
The iron-binding motif EexxED from the frataxin family was grafted on the surface of
E. coli
thioredoxin. The variant showed native-conformational features and was enzimatically functional in the context of the new protein structure.
Human peroxiredoxin 6 (PRDX6) is the only mammalian 1-Cys based member of the Prx family.1 In the first step of its catalytic cycle a sulfenic acid (CP-SOH) is formed at the peroxidatic cysteine, ...C47, which is then reduced by a thiol-containing agent like glutathione or by ascorbate. The finding of ascorbate as a Prx reducing agent2 revolutionized the thiol-specific antioxidant paradigm, expanding the world of possible 1-Cys Prxs reductants. Rate constants for reduction of CP-SOH by ascorbate have been measured for different 1-Cys based Prxs at L. Netto’s Lab, obtaining values in the 500-2000 M-1s-1 range. Here we address the molecular mechanism of PRDX6 CP-SOH reduction by ascorbate from a theoretical perspective, by combining molecular dynamics and ligand-protein docking with QM/MM methods. Our results show the reaction follows a SN2-like substitution mechanism coupled at the transition state with a proton transfer (PT) from R155 into the leaving OH-. A free-energy activation barrier of 11.5 kcal-1mol-1 is obtained as our best theoretical estimation, which can be transformed into a rate constant of 2.2 x 104M-1 s-1 using the Eyring-Polanyi equation, in very good agreement with reported data3. This kind of mechanism represents a novelty for sulfenic acid reduction and will be discussed in detail, dissecting the role of PRDX6 residues critical in determining the reaction efficiency.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Thiol peroxidase from
Escherichia coli
(
Ec
TPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of
Ec
TPx, the peroxidatic cysteine (C
P
) is ...oxidized to a sulfenic acid by peroxide, then the resolving cysteine (C
R
) condenses with the sulfenic acid of C
P
to form a disulfide bond, which is finally reduced by thioredoxin. Purified
Ec
TPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of
Ec
TPx. To explore this process, the FF → LU and LU → FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues C
P
and C
R
. Our results suggest that the FF → LU transition has a higher associated energy barrier than the refolding LU → FF process in agreement with the relatively low experimental turnover number of
Ec
TPx. Furthermore,
in silico
designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type
Ec
TPx.
Temporal acquisition of the
fully folded
conformational substate of the
Escherichia coli
thiol peroxidase by accelerated molecular dynamics simulations.
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