4 research outputs found
Investigation of Ion Binding in Chlorite Dismutases by Means of Molecular Dynamics Simulations
Chlorite
dismutases are prokaryotic heme <i>b</i> oxidoreductases
that convert chlorite to chloride and dioxygen. It has been postulated
that during turnover hypochlorite is formed transiently, which might
be responsible for the observed irreversible inactivation of these
iron proteins. The only charged distal residue in the heme cavity
is a conserved and mobile arginine, but its role in catalysis and
inactivation is not fully understood. In the present study, the pentameric
chlorite dismutase (Cld) from the bacterium <i>Candidatus Nitrospira
defluvii</i> was probed for binding of the low spin ligand cyanide,
the substrate chlorite, and the intermediate hypochlorite. Simulations
were performed with the enzyme in the ferrous, ferric, and compound
I state. Additionally, the variant R173A was studied. We report the
parametrization for the GROMOS force field of the anions ClO<sup>–</sup>, ClO<sub>2</sub><sup>–</sup>, ClO<sub>3</sub><sup>–</sup>, and ClO<sub>4</sub><sup>–</sup> and describe spontaneous
binding, unbinding, and rebinding events of chlorite and hypochlorite,
as well as the dynamics of the conformations of Arg173 during simulations.
The findings suggest that (i) chlorite binding to ferric NdCld occurs
spontaneously and (ii) that Arg173 is important for recognition and
to impair hypochlorite leakage from the reaction sphere. The simulation
data is discussed in comparison with experimental data on catalysis
and inhibition of chlorite dismutase
Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase
Chlorite dismutases (Clds) are heme <i>b</i>-containing
prokaryotic oxidoreductases that catalyze the reduction of chlorite
to chloride with the concomitant release of molecular oxygen. Over
time, they are irreversibly inactivated. To elucidate the mechanism
of inactivation and investigate the role of the postulated intermediate
hypochlorite, the pentameric chlorite dismutase of “Candidatus Nitrospira defluvii” (NdCld) and
two variants (having the conserved distal arginine 173 exchanged with
alanine and lysine) were recombinantly produced in <i>Escherichia
coli</i>. Exchange of the distal arginine boosts the extent of
irreversible inactivation. In the presence of the hypochlorite traps
methionine, monochlorodimedone, and 2-[6-(4-aminophenoxy)-3-oxo-3<i>H</i>-xanthen-9-yl]benzoic acid, the extent of chlorite degradation
and release of molecular oxygen is significantly increased, whereas
heme bleaching and oxidative modifications of the protein are suppressed.
Among other modifications, hypochlorite-mediated formation of chlorinated
tyrosines is demonstrated by mass spectrometry. The data obtained
were analyzed with respect to the proposed reaction mechanism for
chlorite degradation and its dependence on pH. We discuss the role
of distal Arg173 by keeping hypochlorite in the reaction sphere for
O–O bond formation
Eukaryotic Catalase-Peroxidase: The Role of the Trp-Tyr-Met Adduct in Protein Stability, Substrate Accessibility, and Catalysis of Hydrogen Peroxide Dismutation
Recently, it was demonstrated that
bifunctional catalase-peroxidases
(KatGs) are found not only in archaea and bacteria but also in lower
eukaryotes. Structural studies and preliminary biochemical data of
the secreted KatG from the rice pathogen <i>Magnaporthe grisea</i> (<i>Mag</i>KatG2) suggested both similar and novel features
when compared to those of the prokaryotic counterparts studied so
far. In this work, we demonstrate the role of the autocatalytically
formed redox-active Trp140-Tyr273-Met299 adduct of <i>Mag</i>KatG2 in (i) the maintenance of the active site architecture, (ii)
the catalysis of hydrogen peroxide dismutation, and (iii) the protein
stability by comparing wild-type <i>Mag</i>KatG2 with the
single mutants Trp140Phe, Tyr273Phe, and Met299Ala. The impact of
disruption of the covalent bonds between the adduct residues on the
spectral signatures and heme cavity architecture was small. By contrast,
loss of its integrity converts bifunctional <i>Mag</i>KatG2
to a monofunctional peroxidase of significantly reduced thermal stability.
It increases the accessibility of ligands due to the increased flexibility
of the KatG-typical large loop 1 (LL1), which contributes to the substrate
access channel and anchors at the adduct Tyr. We discuss these data
with respect to those known from prokaryotic KatGs and in addition
present a high-resolution structure of an oxoiron compound of <i>Mag</i>KatG2
Redox Thermodynamics of High-Spin and Low-Spin Forms of Chlorite Dismutases with Diverse Subunit and Oligomeric Structures
Chlorite dismutases (Clds) are heme <i>b</i>-containing
oxidoreductases that convert chlorite to chloride and dioxygen. In
this work, the thermodynamics of the one-electron reduction of the
ferric high-spin forms and of the six-coordinate low-spin cyanide
adducts of the enzymes from <i>Nitrobacter winogradskyi</i> (NwCld) and <i>Candidatus</i> “Nitrospira defluvii”
(NdCld) were determined through spectroelectrochemical experiments.
These proteins belong to two phylogenetically separated lineages that
differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric
and pentameric, respectively) structure but exhibit similar chlorite
degradation activity. The <i>E</i>°′ values
for free and cyanide-bound proteins were determined to be −119
and −397 mV for NwCld and −113 and −404 mV for
NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical
experiments revealed that the oxidized state of both proteins is enthalpically
stabilized. Molecular dynamics simulations suggest that changes in
the protein structure are negligible, whereas solvent reorganization
is mainly responsible for the increase in entropy during the redox
reaction. Obtained data are discussed with respect to the known structures
of the two Clds and the proposed reaction mechanism