8 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
Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes
Coproheme decarboxylases (ChdC) catalyze
the hydrogen peroxide-mediated
conversion of coproheme to heme <i>b</i>. This work compares
the structure and function of wild-type (WT) coproheme decarboxylase
from Listeria monocytogenes and its
M149A, Q187A, and M149A/Q187A mutants. The UV–vis, resonance
Raman, and electron paramagnetic resonance spectroscopies clearly
show that the ferric form of the WT protein is a pentacoordinate quantum
mechanically mixed-spin state, which is very unusual in biological
systems. Exchange of the Met149 residue to Ala dramatically alters
the heme coordination, which becomes a 6-coordinate low spin species
with the amide nitrogen atom of the Q187 residue bound to the heme
iron. The interaction between M149 and propionyl 2 is found to play
an important role in keeping the Q187 residue correctly positioned
for closure of the distal cavity. This is confirmed by the observation
that in the M149A variant two CO conformers are present corresponding
to open (A<sub>0</sub>) and closed (A<sub>1</sub>) conformations.
The CO of the latter species, the only conformer observed in the WT
protein, is H-bonded to Q187. In the absence of the Q187 residue or
in the adducts of all the heme <i>b</i> forms of ChdC investigated
herein (containing vinyls in positions 2 and 4), only the A<sub>0</sub> conformer has been found. Moreover, M149 is shown to be involved
in the formation of a covalent bond with a vinyl substituent of heme <i>b</i> at excess of hydrogen peroxide
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
Chemistry and Molecular Dynamics Simulations of Heme <i>b</i>‑HemQ and Coproheme-HemQ
Recently,
a novel pathway for heme <i>b</i> biosynthesis
in Gram-positive bacteria has been proposed. The final poorly understood
step is catalyzed by an enzyme called HemQ and includes two decarboxylation
reactions leading from coproheme to heme <i>b</i>. Coproheme
has been suggested to act as both substrate and redox active cofactor
in this reaction. In the study presented here, we focus on HemQs from <i>Listeria monocytogenes</i> (LmHemQ) and <i>Staphylococcus
aureus</i> (SaHemQ) recombinantly produced as apoproteins in <i>Escherichia coli.</i> We demonstrate the rapid and two-phase
uptake of coproheme by both apo forms and the significant differences
in thermal stability of the apo forms, coproheme-HemQ and heme <i>b</i>-HemQ. Reduction of ferric high-spin coproheme-HemQ to
the ferrous form is shown to be enthalpically favored but entropically
disfavored with standard reduction potentials of −205 ±
3 mV for LmHemQ and −207 ± 3 mV for SaHemQ versus the
standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests
the presence of a pronounced H-bonding network and restricted solvent
mobility in the heme cavity. Binding of cyanide to the sixth coproheme
position is monophasic but relatively slow (∼1 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>). On the basis
of the available structures of apo-HemQ and modeling of both loaded
forms, molecular dynamics simulation allowed analysis of the interaction
of coproheme and heme <i>b</i> with the protein as well
as the role of the flexibility at the proximal heme cavity and the
substrate access channel for coproheme binding and heme <i>b</i> release. Obtained data are discussed with respect to the proposed
function of HemQ in monoderm bacteria
Posttranslational Modification of Heme <i>b</i> in a Bacterial Peroxidase: The Role of Heme to Protein Ester Bonds in Ligand Binding and Catalysis
The
existence of covalent heme to protein bonds is the most striking
structural feature of mammalian peroxidases, including myeloperoxidase
and lactoperoxidase (LPO). These autocatalytic posttranslational modifications
(PTMs) were shown to strongly influence the biophysical and biochemical
properties of these oxidoreductases. Recently, we reported the occurrence
of stable LPO-like counterparts with two heme to protein ester linkages
in bacteria. This study focuses on the model wild-type peroxidase
from the cyanobacterium <i>Lyngbya</i> sp. PCC 8106 (LspPOX)
and the mutants D109A, E238A, and D109A/E238A that could be recombinantly
produced as apoproteins in <i>Escherichia coli</i>, fully
reconstituted to the respective heme <i>b</i> proteins,
and posttranslationally modified by hydrogen peroxide. This for the
first time allows not only a direct comparison of the catalytic properties
of the heme <i>b</i> and PTM forms but also a study of the
impact of D109 and E238 on PTM and catalysis, including Compound I
formation and the two-electron reduction of Compound I by bromide,
iodide, and thiocyanate. It is demonstrated that both heme to protein
ester bonds can form independently and that elimination of E238, in
contrast to exchange of D109, does not cause significant structural
rearrangements or changes in the catalytic properties neither in heme <i>b</i> nor in the PTM form. The obtained findings are discussed
with respect to published structural and functional data of human
peroxidases
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
Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry, and Reactivity
Chlorite dismutases (Clds) are heme <i>b</i> containing
oxidoreductases that convert chlorite to chloride and molecular oxygen.
In order to elucidate the role of conserved heme cavity residues in
the catalysis of this reaction comprehensive mutational and biochemical
analyses of Cld from “<i>Candidatus</i> Nitrospira
defluvii” (NdCld) were performed. Particularly, point mutations
of the cavity-forming residues R173, K141, W145, W146, and E210 were
performed. The effect of manipulation in 12 single and double mutants
was probed by UV–vis spectroscopy, spectroelectrochemistry,
pre-steady-state and steady-state kinetics, and X-ray crystallography.
Resulting biochemical data are discussed with respect to the known
crystal structure of wild-type NdCld and the variants R173A and R173K
as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y
solved in this work. The findings allow a critical analysis of the
role of these heme cavity residues in the reaction mechanism of chlorite
degradation that is proposed to involve hypohalous acid as transient
intermediate and formation of an OO bond. The distal R173
is shown to be important (but not fully essential) for the reaction
with chlorite, and, upon addition of cyanide, it acts as a proton
acceptor in the formation of the resulting low-spin complex. The proximal
H-bonding network including K141-E210-H160 keeps the enzyme in its
ferric (<i>E</i>°′ = −113 mV) and mainly
five-coordinated high-spin state and is very susceptible to perturbation
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