6 research outputs found
Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants
The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria, in 2015 by Dailey and coworkers triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely H182A and E263Q, involving two key active site residues. Interestingly, both variants are active only towards the physiological substrate coproporphyrin III but inactive towards protoporphyrin IX. In addition, E263 is impairing the final oxidation from ferrous coproheme to ferric coproheme. The characteristics of the active site in terms of the residues involved and the substrate binding properties are discussed by structural and functional means, providing a further contribution to the deciphering of the enigmatic reaction mechanism
Chemistry and Molecular Dynamics Simulations of Heme b‑HemQ and Coproheme-HemQ
Recently, a novel pathway for heme b 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 fromcoproheme to heme b. 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 Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. 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 b-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 × 104 M−1 s−1). 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 b 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 b 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
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
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