12 research outputs found
C-terminal methylation of truncated neuropeptides: An enzyme- assistedextraction artifact involving methanol
Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptidediscovery commonly involves chemical extraction from a tissue source followed by mass spectrometriccharacterization. Ideally, the extraction procedure accurately preserves the sequence and any inher-ent modifications of the native peptides. Here, we present data showing that this is not always true.Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin fam-ily members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated fromfull-length orcokinin precursors as the result of a highly selective peptide modification (peptide trun-cation with C-terminal methylation) that occurs during extraction. These peptides were observed byMALDI-FTMS and LC-Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent,but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanolexcluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC-Q-TOFMS/MS, and comparison with a peptide standard. Extraction substitutingdeuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group,and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe isnot produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears toresult from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conver-sion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. Thisunusual and highly specific extraction-derived peptide conversion exemplifies the need to consider bothchemical and biochemical processes that may modify the structure of endogenous neuropeptides. © 2013 The Authors. Published by Elsevier Inc. All rights reserved
Marinobacter atlanticus electrode biofilms differentially regulate gene expression depending on electrode potential and lifestyle
Marinobacter spp. are opportunitrophs with a broad metabolic range including interactions with metals and electrodes. Marinobacter atlanticus strain CP1 was previously isolated from a cathode biofilm microbial community enriched from a sediment microbial fuel cell. Like other Marinobacter spp., M. atlanticus generates small amounts of electrical current when grown as a biofilm on an electrode, which is enhanced by the addition of redox mediators. However, the molecular mechanism resulting in extracellular electron transfer is unknown. Here, RNA-sequencing was used to determine changes in gene expression in electrode-attached and planktonic cells of M. atlanticus when grown at electrode potentials that enable current production (310 and 510Â mV vs. SHE) compared to a potential that enables electron uptake (160Â mV). Cells grown at current-producing potentials had increased expression of genes for molybdate transport, regardless of planktonic or attached lifestyle. Electrode-attached cells at current-producing potentials showed increased expression of the major export protein for the type VI secretion system. Growth at 160Â mV resulted in an increase in expression of genes related to stress response and DNA repair including both RecBCD and the LexA/RecA regulatory network, as well as genes for copper homeostasis. Changes in expression of proteins with PEP C-terminal extracellular export motifs suggests that M. atlanticus is remodeling the biofilm matrix in response to electrode potential. These results improve our understanding of the physiological adaptations required for M. atlanticus growth on electrodes, and suggest a role for metal acquisition, either as a requirement for metal cofactors of redox proteins or as a possible electron shuttling mechanism
Spectroscopic Investigations of Catalase Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non-thiolate-Ligated Heme Enzyme
We
report on the protonation state of <i>Helicobacter pylori</i> catalase compound II. UV/visible, Mössbauer, and X-ray absorption
spectroscopies have been used to examine the intermediate from pH
5 to 14. We have determined that HPC-II exists in an ironÂ(IV) hydroxide
state up to pH 11. Above this pH, the ironÂ(IV) hydroxide complex transitions
to a new species (p<i>K</i><sub>a</sub> = 13.1) with Mössbauer
parameters that are indicative of an ironÂ(IV)-oxo intermediate. Recently,
we discussed a role for an elevated compound II p<i>K</i><sub>a</sub> in diminishing the compound I reduction potential. This
has the effect of shifting the thermodynamic landscape toward the
two-electron chemistry that is critical for catalase function. In
catalase, a diminished potential would increase the selectivity for
peroxide disproportionation over off-pathway one-electron chemistry,
reducing the buildup of the inactive compound II state and reducing
the need for energetically expensive electron donor molecules
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Reactivity of an FeIV-Oxo Complex with Protons and Oxidants
High-valent Fe-OH species are often invoked as key intermediates but have only been observed in Compound II of cytochrome P450s. To further address the properties of non-heme FeIV-OH complexes, we demonstrate the reversible protonation of a synthetic FeIV-oxo species containing a tris-urea tripodal ligand. The same protonated FeIV-oxo species can be prepared via oxidation, suggesting that a putative FeV-oxo species was initially generated. Computational, Mössbauer, XAS, and NRVS studies indicate that protonation of the FeIV-oxo complex most likely occurs on the tripodal ligand, which undergoes a structural change that results in the formation of a new intramolecular H-bond with the oxido ligand that aids in stabilizing the protonated adduct. We suggest that similar protonated high-valent Fe-oxo species may occur in the active sites of proteins. This finding further argues for caution when assigning unverified high-valent Fe-OH species to mechanisms
Setting an Upper Limit on the Myoglobin Iron(IV)Hydroxide p<i>K</i><sub>a</sub>: Insight into Axial Ligand Tuning in Heme Protein Catalysis
To provide insight into the ironÂ(IV)Âhydroxide
p<i>K</i><sub>a</sub> of histidine ligated heme proteins,
we have probed the
active site of myoglobin compound II over the pH range of 3.9–9.5,
using EXAFS, Mössbauer, and resonance Raman spectroscopies.
We find no indication of ferryl protonation over this pH range, allowing
us to set an upper limit of 2.7 on the ironÂ(IV)Âhydroxide p<i>K</i><sub>a</sub> in myoglobin. Together with the recent determination
of an ironÂ(IV)Âhydroxide p<i>K</i><sub>a</sub> ∼ 12
in the thiolate-ligated heme enzyme cytochrome P450, this result provides
insight into Nature’s ability to tune catalytic function through
its choice of axial ligand
Reactivity of an FeIV-Oxo Complex with Protons and Oxidants
High valent Fe–OH species are often invoked as key intermediates but have only been observed in Compound II of cytochrome P450s. To further address the properties of non-heme Fe(IV)–OH complexes we demonstrate the reversible protonation of a synthetic Fe(IV)–oxo species containing a tris-urea tripodal ligand. The same protonated Fe(IV)–oxo species can be prepared via oxidation, suggesting a putative Fe(V)–oxo species was initially generated. Computational, Mössbauer, XAS, and NRVS studies indicate that protonation of the Fe(IV)–oxo complex most likely occur on the tripodal ligand, which undergoes a structural change that results in the formation of a new intramolecular hydrogen bond with the oxido ligand that aids in stabilizing the protonated adduct. We suggest that similar species for protonated high valent Fe–oxo species may occur in the active sites of proteins. This finding further argues for caution when assigning unverified high valent Fe–OH species to mechanisms
Reactivity of an Fe<sup>IV</sup>-Oxo Complex with Protons and Oxidants
High-valent Fe-OH
species are often invoked as key intermediates
but have only been observed in Compound II of cytochrome P450s. To
further address the properties of non-heme Fe<sup>IV</sup>-OH complexes,
we demonstrate the reversible protonation of a synthetic Fe<sup>IV</sup>-oxo species containing a tris-urea tripodal ligand. The same protonated
Fe<sup>IV</sup>-oxo species can be prepared via oxidation, suggesting
that a putative Fe<sup>V</sup>-oxo species was initially generated.
Computational, Mössbauer, XAS, and NRVS studies indicate that
protonation of the Fe<sup>IV</sup>-oxo complex most likely occurs
on the tripodal ligand, which undergoes a structural change that results
in the formation of a new intramolecular H-bond with the oxido ligand
that aids in stabilizing the protonated adduct. We suggest that similar
protonated high-valent Fe-oxo species may occur in the active sites
of proteins. This finding further argues for caution when assigning
unverified high-valent Fe-OH species to mechanisms