Despite being highly toxic, carbon monoxide (CO) is also an essential intracellular signalling molecule. The mechanisms of CO-dependent cell signalling are poorly defined, but are likely to involve ...interactions with heme proteins. One such role for CO is in ion channel regulation. Here, we examine the interaction of CO with K
channels. We find that CO activates K
channels and that heme binding to a CXXHX
H motif on the SUR2A receptor is required for the CO-dependent increase in channel activity. Spectroscopic and kinetic data were used to quantify the interaction of CO with the ferrous heme-SUR2A complex. The results are significant because they directly connect CO-dependent regulation to a heme-binding event on the channel. We use this information to present molecular-level insight into the dynamic processes that control the interactions of CO with a heme-regulated channel protein, and we present a structural framework for understanding the complex interplay between heme and CO in ion channel regulation.
Oxidative stress mediated by reactive oxygen or nitrogen species (ROS/RNS) seems to be implicated in several diseases including neurodegenerative ones. In one of them, namely Alzheimer's disease, ...there is a large body of evidence that the aggregation of the peptide amyloid-beta (Abeta) is implicated in the generation of the oxidative stress. Redox active metal ions play a key role in oxidative stress, either in the production of ROS/RNS by enzymes or loosely bound metals or in the protection against ROS, mostly as catalytic centers in enzymes. In Alzheimer's disease, it is thought that metals (mostly Cu, Fe and heme) can bind to amyloid-beta and that such systems are involved in the generation of oxidative stress. In the present article, we review the role of ROS/RNS produced by redox active Cu ions and heme compounds in the context of the amyloid cascade. We focus on (i) the coordination chemistry of Cu and heme to Abeta; (ii) the role of the corresponding Abeta adducts in the (catalytic) production of ROS/RNS; (iii) the subsequent degradation of Abeta by these reactive species and (iv) the use of antioxidants, in particular metal sequestering compounds and direct antioxidants like polyphenols as a therapeutic strategies.
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► Cu(II) binding to Aβ is dynamic and involves several equilibria. ► The first two amino-acid residues (in blue) of human Aβ have a predominant role in Cu(II) binding. ► Peptide ...sequence alterations (in green and red) are detected; some of them (in red) impact Cu(II) coordination spheres. ► Advanced EPR methods are highly useful to disentangle Cu(II) coordination to Aβ peptides.
Copper ions have been proposed to play a central role in the amyloid cascade process linked to the development of Alzheimer disease (AD). Involvement in both the amyloid-β (Aβ) aggregation process and reactive oxygen species (ROS) production has been considered. In the last 15 years, many studies regarding copper(II) coordination to Aβ have been reported with divergent conclusions and a consensual binding scheme is not reached yet. They include (i) spectroscopic and thermodynamic investigations of copper(II) coordination to chemically modified peptides (mutants, truncated peptides, etc.) and subsequent analysis of the differences obtained with the native Aβ peptide; (ii) spectroscopic characterization of copper(II) coordination to Aβvia direct methods, such as advanced EPR techniques and FTIR spectroscopy combined with the use of 13C, 15N specifically labeled peptides and NMR. More recently, copper(II) coordination to naturally occurring modified peptides of biological relevance such as murine Aβ, H6R and A2V mutants, and truncated forms at position 3, have also been studied.
In the present review, the objective is to give a report as exhaustive as possible of the literature structural data on copper(II) binding to the Aβ peptides and to its modified forms and to sort out contradicting results. Such discrepancies are mainly due to the unstructured nature of the copper binding site in Aβ. Concomitantly, copper(II) coordination has been revealed to be highly dynamic with equilibrium between amino-acid residues of identical nature for one binding position. As a direct consequence, the copper(II) coordination spheres proposed represent the most reasonable models obtained with data available at present. At physiological pH, two copper(II) binding sites, noted components I and II, coexist. The transition between I and II is pH-driven and the pH where the two components are found in a 1:1 ratio (pKa(I/II)) is approx. 7.8, with I (resp.II) predominant at lower (resp. higher) pH. In I and II, the equatorial binding sites of copper(II) are {NH2 (Asp1), CO (Asp1–Ala2), Nτim (His6), Nπim (His13 or His14)} and {NH2 (Asp1), N− (Asp1–Ala2), CO (Ala2–Glu3), Nτim (His6) or Nim (His13 or His14)}, respectively. I and II were clearly (and by consensus) identified by their EPR parameters, g//=2.27±0.01, A//=183±5×10−4cm−1 and g//=2.23±0.01, A//=160±5×10−4cm−1, respectively. Given examples of copper(II) binding to other naturally occurring Aβ peptides include binding to the murine Aβ peptide, differing from the human Aβ by three point mutations, and to the H6R mutant. Copper(II) binding to murine and human Aβ peptides diverges by the pKa(I/II) value (approx. 6.2 for the former instead of 7.8) and by the nature of the peptide functional group which undergoes deprotonation between I and II, i.e. the Gly5–His6 bond compared with the Asp1–Ala2 bond in the human case. Copper(II) binding to the H6R mutant is characterized by a pKa(I/II) value of approx. 7.3, a decrease induced by the unfavorable coordination of both His13 and His14 in component I.
Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein ...structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.
H4B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation ...(second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H4B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H4B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.
The surface oxidation site (Trp-171) in lignin peroxidase (LiP) required for the reaction with veratryl alcohol a high-redox-potential (1.4 V) substrate, was engineered into Coprinus cinereus ...peroxidase (CiP) by introducing a Trp residue into a heme peroxidase that has similar protein fold but lacks this activity. To create the catalytic activity toward veratryl alcohol in CiP, it was necessary to reproduce the Trp site and its negatively charged microenvironment by means of a triple mutation. The resulting D179W+R258E+R272D variant was characterized by multifrequency EPR spectroscopy. The spectra unequivocally showed that a new Trp radical g values of gx = 2.0035(5), gy = 2.0027(5), and gz = 2.0022(1) was formed after the Fe(IV)=O Por{bullet}⁺ intermediate, as a result of intramolecular electron transfer between Trp-179 and the porphyrin. Also, the EPR characterization crucially showed that Fe(IV)=O Trp-179{bullet} was the reactive intermediate with veratryl alcohol. Accordingly, our work shows that it is necessary to take into account the physicochemical properties of the radical, fine-tuned by the microenvironment, as well as those of the preceding Fe(IV)=O Por{bullet}⁺ intermediate to engineer a catalytically competent Trp site for a given substrate. Manipulation of the microenvironment of the Trp-171 site in LiP allowed the detection by EPR spectroscopy of the Trp-171{bullet}, for which direct evidence has been missing so far. Our work also highlights the role of Trp residues as tunable redox-active cofactors for enzyme catalysis in the context of peroxidases with a unique reactivity toward recalcitrant substrates that require oxidation potentials not realized at the heme site.
Despite crystallographic structures now available and intensive work in the past decades, little is known about the higher redox states of the catalytic cycle of Photosystem II, the enzyme ...responsible for the presence of O2 on Earth and at the beginning of the process that has produced both the biomass and the fossil fuels. In one of the highest oxidation states, the S3-state, only signals at g-values higher than 4 have been detected so far at the X-band. In this work, we report for the first time the complete X-band EPR spectrum for the S3-state of Photosystem II. Simulations show that, for a spin state S = 1, as was previously suggested for S3, it is not possible to account for all the features observed. A satisfactory simulated spectrum was obtained for a spin state S = 3 with zero-field splitting parameters D = 0.175 cm−1 and E/D = 0.275. The detection of the full EPR signal for S3 opens the door for new investigations and a better understanding of the catalytic cycle of Photosystem II.
The electronic properties of the Mn₄OₓCa cluster in the S₂ state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band ⁵⁵Mn-ENDOR using photosystem II preparations ...isolated from the thermophilic cyanobacterium T. elongatus and higher plants (spinach). The data presented here show that there is very little difference between the two species. Specifically it is shown that: (i) only small changes are seen in the fitted isotropic hyperfine values, suggesting that there is no significant difference in the overall spin distribution (electronic coupling scheme) between the two species; (ii) the inferred fine-structure tensor of the only Mnᴵᴵᴵ ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the Mnᴵᴵᴵ ion has the same coordination sphere in both sample preparations; and (iii) the data from both species are consistent with only one structural model available in the literature, namely the Siegbahn structure Siegbahn, P. E. M. Accounts Chem. Res.2009, 42, 1871–1880, Pantazis, D. A. et al., Phys. Chem. Chem. Phys.2009, 11, 6788–6798. These measurements were made in the presence of methanol because it confers favorable magnetic relaxation properties to the cluster that facilitate pulse-EPR techniques. In the absence of methanol the separation of the ground state and the first excited state of the spin system is smaller. For cyanobacteria this effect is minor but in plant PS II it leads to a break-down of the ST=½ spin model of the S₂ state. This suggests that the methanol–OEC interaction is species dependent. It is proposed that the effect of small organic solvents on the electronic structure of the cluster is to change the coupling between the outer Mn (MnA) and the other three Mn ions that form the trimeric part of the cluster (MnB, MnC, MnD), by perturbing the linking bis-μ-oxo bridge. The flexibility of this bridging unit is discussed with regard to the mechanism of O-O bond formation.
1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a ...facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity.
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•ACC Oxidase, a non-heme iron dependent enzyme, was reconstituted with Cu(II).•Several active site variants were studied.•cw and pulsed EPR spectroscopies were used to characterize the binding of copper and of the substrate.•DFT calculations were used to provide structural models.
Three peroxomanganese(III) complexes MnIII(O2)(mL5 2)+, MnIII(O2)(imL5 2)+, and MnIII(O2)(N4py)+ supported by pentadentate ligands (mL5 2 = N-methyl-N,N′,N′-tris(2-pyridylmethyl)ethane-1,2-diamine, ...imL5 2 = N-methyl-N,N′,N′-tris((1-methyl-4-imidazolyl)methyl)ethane-1,2-diamine, and N4py = N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) were generated by treating Mn(II) precursors with H2O2 or KO2. Electronic absorption, magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD data demonstrate that these complexes have very similar electronic transition energies and ground-state zero-field splitting parameters, indicative of nearly identical coordination geometries. Because of uncertainty in peroxo (side-on η2 versus end-on η1) and ligand (pentadentate versus tetradentate) binding modes, density functional theory (DFT) computations were used to distinguish between three possible structures: pentadentate ligand binding with (i) a side-on peroxo and (ii) an end-on peroxo, and (iii) tetradentate ligand binding with a side-on peroxo. Regardless of the supporting ligand, isomers with a side-on peroxo and the supporting ligand bound in a tetradentate fashion were identified as most stable by >20 kcal/mol. Spectroscopic parameters computed by time-dependent (TD) DFT and multireference SORCI methods provided validation of these isomers on the basis of experimental data. Hexacoordination is thus strongly preferred for peroxomanganese(III) adducts, and dissociation of a pyridine (mL5 2 and N4py) or imidazole (imL5 2) arm is thermodynamically favored. In contrast, DFT computations for models of FeIII(O2)(mL5 2)+ demonstrate that pyridine dissociation is not favorable; instead a seven-coordinate ferric center is preferred. These different results are attributed to the electronic configurations of the metal centers (high spin d5 and d4 for FeIII and MnIII, respectively), which results in population of a metal-peroxo σ-antibonding molecular orbital and, consequently, longer M–Operoxo bonds for peroxoiron(III) species.
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