3 research outputs found
Peroxidase versus Peroxygenase Activity: Substrate Substituent Effects as Modulators of Enzyme Function in the Multifunctional Catalytic Globin Dehaloperoxidase
The
dehaloperoxidase-hemoglobin (DHP) from the terebellid polychaete <i>Amphitrite ornata</i> is a multifunctional hemoprotein that
catalyzes the oxidation of a wide variety of substrates, including
halo/nitrophenols, haloindoles, and pyrroles, via peroxidase and/or
peroxygenase mechanisms. To probe whether substrate substituent effects
can modulate enzyme activity in DHP, we investigated its reactiviy
against a panel of <i>o</i>-guaiacol substrates given their
presence (from native/halogenated and non-native/anthropogenic sources)
in the benthic environment that <i>A. ornata</i> inhabits.
Using biochemical assays supported by spectroscopic, spectrometric,
and structural studies, DHP was found to catalyze the H<sub>2</sub>O<sub>2</sub>-dependent oxidative dehalogenation of 4-haloguaiacols
(F, Cl, and Br) to 2-methoxybenzoquinone (2-MeOBQ). <sup>18</sup>O
labeling studies confirmed that O atom incorporation was derived exclusively
from water, consistent with substrate oxidation via a peroxidase-based
mechanism. The 2-MeOBQ product further reduced DHP to its oxyferrous
state, providing a link between the substrate oxidation and O<sub>2</sub> carrier functions of DHP. Nonnative substrates resulted in
polymerization of the initial substrate with varying degrees of oxidation,
with 2-MeOBQ identified as a minor product. When viewed alongside
the reactivity of previously studied phenolic substrates, the results
presented here show that simple substituent effects can serve as functional
switches between peroxidase and peroxygenase activities in this multifunctional
catalytic globin. More broadly, when recent findings on DHP activity
with nitrophenols and azoles are included, the results presented here
further demonstrate the breadth of heterocyclic compounds of anthropogenic
origin that can potentially disrupt marine hemoglobins or function
as environmental stressors, findings that may be important when assessing
the environmental impact of these pollutants (and their metabolites)
on aquatic systems
Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin from <i>Amphitrite ornata</i>
The
marine hemoglobin dehaloperoxidase (DHP) from <i>Amphitrite
ornata</i> was found to catalyze the H<sub>2</sub>O<sub>2</sub>-dependent oxidation of nitrophenols, an unprecedented nonmicrobial
degradation pathway for nitrophenols by a hemoglobin. Using 4-nitrophenol
(4-NP) as a representative substrate, the major monooxygenated product
was 4-nitrocatechol (4-NC). Isotope labeling studies confirmed that
the O atom incorporated was derived exclusively from H<sub>2</sub>O<sub>2</sub>, indicative of a peroxygenase mechanism for 4-NP oxidation.
Accordingly, X-ray crystal structures of 4-NP (1.87 Ă…) and 4-NC
(1.98 Ă…) bound to DHP revealed a binding site in close proximity
to the heme cofactor. Peroxygenase activity could be initiated from
either the ferric or oxyferrous states with equivalent substrate conversion
and product distribution. The 4-NC product was itself a peroxidase
substrate for DHP, leading to the secondary products 5-nitrobenzene-triol
and hydroxy-5-nitro-1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol
(2,4-DNP) but was unreactive against 2,4,6-trinitrophenol (2,4,6-TNP).
pH dependence studies demonstrated increased reactivity at lower pH
for both 4-NP and 2,4-DNP, suggestive of a pH effect that precludes
the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow
UV–visible spectroscopic studies strongly implicate a role
for Compound I in the mechanism of 4-NP oxidation. The results demonstrate
that there may be a much larger number of nonmicrobial enzymes that
are underrepresented when it comes to understanding the degradation
of persistent organic pollutants such as nitrophenols in the environment
Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature
Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B