3 research outputs found
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