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Tyrosinase-generated quinones induce covalent modification, unfolding, and aggregation of human holo-myoglobin.
No full textThe present study describes the pattern of protein modification undergone by human holo-myoglobin by reactive
fluoroquinones enzymatically produced by oxidation of 3-fluorophenol in mild conditions (pH 7.4, 25 °C). The
fluoroquinones react with a number of histidine residues. Surface residues H24, H36, H48, and H82 and the
heme distal histidine H64 were all found to be modified to a significant extent. In contrast, cysteine C110 is not
appreciably affected, possibly because it is not accessible to the fluoroquinones. The sites of protein modification
were assessed by mass spectrometry analysis of the peptide fragments resulting from controlled proteolysis of the
apoprotein. As a consequence of the reaction with quinones, the globular structure of myoglobin becomes more
prone to denaturation by the partial loss of its secondary structure. As a more intriguing consequence, the
fluoroquinones promote the formation of structured aggregates of moderate size that lack the typical morphology
of fibrillar structures
Reactive nitrogen species generated by heme proteins: Mechanism of formation and targets
No full textNitration of tyrosine residues in proteins represents a pathological event that is associated with several human and animal diseases. Besides
the classical pathways of formation of reactive nitrogen species (RNS) by NO oxidation, several studies show that heme peroxidases also play an
important role in RNS generation. The mechanism of generation of these species has been studied in detail focusing on the nitration of several
tyrosine and tryptophan derivatives. Also the O2-storage and O2-carrier heme proteins, myoglobin and hemoglobin, can induce RNS formation
and promote self-nitration and oxidation. These reactions bear biological relevance and, therefore, the identification of the sites of endogenous
modification of these proteins has been carried out by proteomic analysis
Redox Reactivity of the Heme Fe3+/Fe2+ Couple in Native Myoglobins and Mutants with Peroxidase-like Activity
No full textThe reaction enthalpy and entropy for the oneelectron
reduction of the ferric heme in horse heart and
sperm whale aquometmyoglobins (Mb) have been determined
exploiting a spectroelectrochemical approach. Also
investigated were the T67R, T67K, T67R/S92D and T67R/
S92D Mb-H variants (the latter containing a protoheme-Lhistidine
methyl ester) of sperm whale Mb, which feature
peroxidase-like activity. The reduction potential (E¢) in all
species consists of an enthalpic term which disfavors Fe3+
reduction and a larger entropic contribution which instead
selectively stabilizes the reduced form. This behavior differs
from that of the heme redox enzymes and electron
transport proteins investigated so far. The reduction thermodynamics
in the series of sperm whale Mb variants
show an almost perfect enthalpy–entropy compensation,
indicating that the mutation-induced changes in
DH0
rc and DS0
rc are dominated by reduction-induced solvent
reorganization effects. The modest changes in E¢
originate from the enthalpic effects of the electrostatic
interactions of the heme with the engineered charged
residues. The small influence that the mutations exert on
the reduction potential of myoglobin suggests that the increased
peroxidase activity of the variants is not related to
changes in the redox reactivity of the heme iron, but are
likely related to a more favored substrate orientation within
the distal heme cavity
Catalytic Activity, Stability, Unfolding, and Degradation Pathways of Engineered and Reconstituted Myoglobins
No full textThe structural and functional consequences of
engineering a positively charged Lys residue and
replacing the natural heme with a heme-L-His derivative
in the active site of sperm whale myoglobin (Mb) have
been investigated. The main structural change caused by
the distal T67K mutation appears to be mobilization of
the propionate-7 group. Reconstitution of wild-type and
T67K Mb with heme-L-His relaxes the protein fragment
around the heme because it involves the loss of the
interaction of one of the propionate groups which stabilize
heme binding to the protein. This modification
increases the accessibility of exogenous ligands or substrates
to the active site. The catalytic activity of the
reconstituted proteins in peroxidase-type reactions is
thus significantly increased, particularly with T67K Mb.
The T67K mutation slightly reduces the thermodynamic
stability and the chemical stability of Mb during catalysis,
but somewhat more marked effects are observed by
cofactor reconstitution. Hydrogen peroxide, in fact, induces
pseudo-peroxidase activity but also promotes
oxidative damage of the protein. The mechanism of
protein degradation involves two pathways, which depend
on the evolution of radical species generated on
protein residues by the Mb active species and on the
reactivity of phenoxy radicals produced during turnover.
Both protein oligomers and heme-protein cross-links
have been detected upon inactivation
Mechanistic Insight into the Peroxidase Catalyzed Nitration of Tyrosine Derivatives by Nitrite and Hydrogen Peroxide
No full textPeroxidases perform the nitration of tyrosine and tyrosyl
residues in proteins, in the presence of nitrite and hydrogen
peroxide. The nitrating species is still unknown but it is
usually assumed to be nitrogen dioxide. In the present
investigation, the nitration of phenolic compounds derived
from tyrosine by lactoperoxidase and horseradish peroxidase
was studied, with the aim of elucidating themechanism
of the reaction. The results indicate that nitrogen dioxide
cannot be the only nitrating species and suggest the presence
of two simultaneously operative pathways, one proceeding
through enzyme-generated nitrogen dioxide and another
through a more reactive species, assumed to be complexed
peroxynitrite, which is generated by reaction of hydrogen
peroxide with the enzyme–nitrite complex. The importance
of the two pathways depends on peroxide and nitrite concentrations.
With lactoperoxidase, nitration through the
highly reactive intermediate is preferred except at very low
nitrite concentration, while with horseradish peroxidase, the
nitrogen dioxide driven mechanism is preferred except at
very high nitrite concentration.The preferred mechanism for
the two enzymes is that operative in the physiological nitrite
concentration range
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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