Copper-Catalyzed Glutathione Oxidation is Accelerated by the Anticancer Thiosemicarbazone Dp44mT and Further Boosted at Lower pH.

Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining redox cellular homeostasis. The thiols of two GSH molecules can be oxidized to the disulfide GSSG. The cytosolic GSH/GSSG ratio is very high (>100), and its reduction can lead to apoptosis or necrosis, which are of interest in cancer research. CuII ions are very efficient oxidants of thiols, but with an excess of GSH, CuIn(GS)m clusters are formed, in which CuI is very slowly reoxidized by O2 at pH 7.4 and even more slowly at lower pH. Here, the aerobic oxidation of GSH by CuII was investigated at different pH values in the presence of the anticancer thiosemicarbazone Dp44mT, which accumulates in lysosomes and induces lysosomal membrane permeabilization in a Cu-dependent manner. The results showed that CuII-Dp44mT catalyzes GSH oxidation faster than CuII alone at pH 7.4 and hence accelerates the production of very reactive hydroxyl radicals. Moreover, GSH oxidation and hydroxyl radical production by CuII-Dp44mT were accelerated at the acidic pH found in lysosomes. To decipher this unusually faster thiol oxidation at lower pH, density functional theory (DFT) calculations, electrochemical and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of CuII-Dp44mT on the hydrazinic nitrogen, which favors the rate-limiting reduction step without subsequent dissociation of the CuI intermediate. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of CuII-Dp44mT.

Rapid Discrimination of Neuromyelitis Optica Spectrum Disorder and Multiple Sclerosis Using Machine Learning on Infrared Spectra of Sera.

Neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) are both autoimmune inflammatory and demyelinating diseases of the central nervous system. NMOSD is a highly disabling disease and rapid introduction of the appropriate treatment at the acute phase is crucial to prevent sequelae. Specific criteria were established in 2015 and provide keys to distinguish NMOSD and MS. One of the most reliable criteria for NMOSD diagnosis is detection in patient's serum of an antibody that attacks the water channel aquaporin-4 (AQP-4). Another target in NMOSD is myelin oligodendrocyte glycoprotein (MOG), delineating a new spectrum of diseases called MOG-associated diseases. Lastly, patients with NMOSD can be negative for both AQP-4 and MOG antibodies. At disease onset, NMOSD symptoms are very similar to MS symptoms from a clinical and radiological perspective. Thus, at first episode, given the urgency of starting the anti-inflammatory treatment, there is an unmet need to differentiate NMOSD subtypes from MS. Here, we used Fourier transform infrared spectroscopy in combination with a machine learning algorithm with the aim of distinguishing the infrared signatures of sera of a first episode of NMOSD from those of a first episode of relapsing-remitting MS, as well as from those of healthy subjects and patients with chronic inflammatory demyelinating polyneuropathy. Our results showed that NMOSD patients were distinguished from MS patients and healthy subjects with a sensitivity of 100% and a specificity of 100%. We also discuss the distinction between the different NMOSD serostatuses. The coupling of infrared spectroscopy of sera to machine learning is a promising cost-effective, rapid and reliable differential diagnosis tool capable of helping to gain valuable time in patients' treatment.

Raman Imaging Reveals Accumulation of Hemoproteins in Plaques from Alzheimer's Diseased Tissues.

Hemes have been suggested to play a central role in Alzheimer's disease since they show high peroxidase reactivity when bound to amyloid ß peptides, leading to the production of reactive oxygen species. Here we used Fourier transform infrared and Raman imaging on Alzheimer's diseased mice and human brain tissue. Our finding suggests the accumulation of hemes in the senile plaques of both murine and human samples. We compared the Raman signature of the plaques to the ones of various hemeoproteins and to the hemin-Aß-42 complex. The detected Raman signature of the plaques does not allow identifying the type of heme accumulating in the plaques.

Lessons from combined experimental and theoretical examination of the FTIR and 2D-IR spectroelectrochemistry of the amide I region of cytochrome c.

Amide I difference spectroscopy is widely used to investigate protein function and structure changes. In this article, we show that the common approach of assigning features in amide I difference signals to distinct secondary structure elements in many cases may not be justified. Evidence comes from Fourier transform infrared (FTIR) and 2D-IR spectroelectrochemistry of the protein cytochrome c in the amide I range, in combination with computational spectroscopy based on molecular dynamics (MD) simulations. This combination reveals that each secondary structure unit, such as an alpha-helix or a beta-sheet, exhibits broad overlapping contributions, usually spanning a large part of the amide I region, which in the case of difference absorption experiments (such as in FTIR spectroelectrochemistry) may lead to intensity-compensating and even sign-changing contributions. We use cytochrome c as the test case, as this small electron-transferring redox-active protein contains different kinds of secondary structure units. Upon switching its redox-state, the protein exhibits a different charge distribution while largely retaining its structural scaffold. Our theoretical analysis suggests that the change in charge distribution contributes to the spectral changes and that structural changes are small. However, in order to confidently interpret FTIR amide I difference signals in cytochrome c and proteins in general, MD simulations in combination with additional experimental approaches such as isotope labeling, the insertion of infrared labels to selectively probe local structural elements will be required. In case these data are not available, a critical assessment of previous interpretations of protein amide I 1D- and 2D-IR difference spectroscopy data is warranted.

Development of a fully automated low-pressure [11 C]CO carbonylation apparatus.

[11 C]carbon monoxide ([11 C]CO) is a versatile synthon for radiolabeling of drug-like molecules for imaging studies with positron emission tomography (PET). We here report the development of a novel, user-friendly, fully automated, and good manufacturing practice (GMP) compliant low-pressure synthesis module for 11 C-carbonylation reactions using [11 C]CO. In this synthesis module, [11 C]CO was reliably prepared from cyclotron-produced [11 C]carbon dioxide ([11 C]CO2 ) by reduction over heated molybdenum and delivered to the reaction vessel within 7 min after end of bombardment, with an overall radiochemical yield (RCY) of 71%. [11 C]AZ13198083, a histamine type-3 receptor ligand, was used as a model compound to assess the functionality of the radiochemistry module. At full batch production conditions (55 µA, 30 min), our newly developed low-pressure 11 C-carbonylation apparatus enabled us to prepare [11 C]AZ13198083 in an isolated radioactivity of 8540 ± 1400 MBq (n = 3). The radiochemical purity of each of the final formulated batches exceeded 99%, and all other quality control tests results conformed with specifications typically set for carbon-11 labeled radiopharmaceuticals. In conclusion, this novel radiochemistry system offers a convenient GMP compliant production drugs and radioligands for imaging studies in human subjects.

Aggregation and Amyloidogenicity of the Nuclear Coactivator Binding Domain of CREB-Binding Protein.

The nuclear coactivator binding domain (NCBD) of transcriptional co-regulator CREB-binding protein (CBP) is an example of conformationally malleable proteins that can bind to structurally unrelated protein targets and adopt distinct folds in the respective protein complexes. Here, we show that the folding landscape of NCBD contains an alternative pathway that results in protein aggregation and self-assembly into amyloid fibers. The initial steps of such protein misfolding are driven by intermolecular interactions of its N-terminal α-helix bringing multiple NCBD molecules into contact. These oligomers then undergo slow but progressive interconversion into ß-sheet-containing aggregates. To reveal the concealed aggregation potential of NCBD we used a chemically synthesized mirror-image d-NCBD form. The addition of d-NCBD promoted self-assembly into amyloid precipitates presumably due to formation of thermodynamically more stable racemic ß-sheet structures. The unexpected aggregation of NCBD needs to be taken into consideration given the multitude of protein-protein interactions and resulting biological functions mediated by CBP.

Acrolein and Copper as Competitive Effectors of α-Synuclein.

Mounting evidence supports the role of amyloidogenesis, oxidative stress, and metal dyshomeostasis in the development of neurodegenerative disorders. Parkinson's Disease is characterized by α-synuclein (αSyn) accumulation and aggregation in brain regions, also promoted by Cu2+ . αSyn is modified by reactive carbonyl species, including acrolein (ACR). Notwithstanding these findings, the interplay between ACR, copper, and αSyn has never been investigated. Therefore, we explored more thoroughly the effects of ACR on αSyn using an approach based on LC-MS/MS analysis. We also evaluated the influence of Cu2+ on the protein carbonylation and how the ACR modification impacts the Cu2+ binding and the production of Reactive Oxygen Species (ROS). Finally, we investigated the effects of ACR and Cu2+ ions on the αSyn aggregation by dynamic light scattering and fluorescence assays. Cu2+ regioselectively inhibits the modification of His50 by ACR, the carbonylation lowers the affinity of His50 for Cu2+ and ACR inhibits αSyn aggregation both in the presence and in the absence of Cu2+ .

Serum-based differentiation between multiple sclerosis and amyotrophic lateral sclerosis by Random Forest classification of FTIR spectra.

The challenging diagnosis and differentiation between multiple sclerosis and amyotrophic lateral sclerosis relies on the clinical assessment of the symptoms along with magnetic resonance imaging and sampling cerebrospinal fluid for the search of biomarkers for either disease. Despite the progress made in imaging techniques and biomarker identification, misdiagnosis still occurs. Here we used 2.5 µL of serum samples to obtain the infrared spectroscopic signatures of sera of multiple sclerosis and amyotrophic lateral sclerosis patients and compared them to those of healthy controls. The spectra are then classified with the help of a two-fold Random Forest cross-validation algorithm. This approach shows that infrared spectroscopy is powerful in discriminating between the two diseases and healthy controls by offering high specificity for multiple sclerosis (100%) and amyotrophic lateral sclerosis (98%). In addition, data after six and twelve months of treatment of the multiple sclerosis patients with biotin are discussed.

Triggering Cu-coordination change in Cu(ii)-Ala-His-His by external ligands.

Copper(ii) forms well-known and stable complexes with peptides having histidine at position 2 (Xxx-His) or 3 (Xxx-Zzz-His). Their properties differ considerably due to the histidine positioning. Here we report that in the hybrid motif Xxx-His-His, the Cu(ii)-complexes can be switched between the Xxx-His and the Xxx-Zzz-His coordination modes by addition of external ligands.

Cu(II) Binding to the Peptide Ala-His-His, a Chimera of the Canonical Cu(II)-Binding Motifs Xxx-His and Xxx-Zzz-His.

Peptides and proteins with the N-terminal motifs NH2-Xxx-His and NH2-Xxx-Zzz-His form well-established Cu(II) complexes. The canonical peptides are Gly-His-Lys and Asp-Ala-His-Lys (from the wound healing factor and human serum albumin, respectively). Cu(II) is bound to NH2-Xxx-His via three nitrogens from the peptide and an external ligand in the equatorial plane (called 3N form here). In contrast, Cu(II) is bound to NH2-Xxx-Zzz-His via four nitrogens from the peptide in the equatorial plane (called 4N form here). These two motifs are not mutually exclusive, as the peptides with the sequence NH2-Xxx-His-His contain both of them. However, this chimera has never been fully explored. In this work, we use a multispectroscopic approach to analyze the Cu(II) binding to the chimeric peptide Ala-His-His (AHH). AHH is capable of forming the 3N- and 4N-type complexes in a pH dependent manner. The 3N form predominates at pH ∼ 4-6.5 and the 4N form at ∼ pH 6.5-10. NMR experiments showed that at pH 8.5, where Cu(II) is almost exclusively bound in the 4N form, the Cu(II)-exchange between AHH or the amidated AHH-NH2 is fast, in comparison to the nonchimeric 4N form (AAH). Together, the results show that the chimeric AHH can access both Cu(II) coordination types, that minor changes in the second (or further) coordination sphere can impact considerably the equilibrium between the forms, and that Cu kinetic exchange is fast even when Cu-AHH is mainly in the 4N form.

Far infrared spectroscopy of hydrogen bonding collective motions in complex molecular systems.

Far infrared spectroscopy is a technique that allows the probing of the low frequency region of vibrational spectra and reveals, among others, vibrational modes of inter- and intramolecular hydrogen bonding. Due to their collective nature, these modes are highly sensitive to the conformational state of the molecules as well as to their interactions. Far infrared spectroscopy is thus an emerging technique for the characterization of the low frequency motions of complex molecules, including polymers, peptides, proteins or ionic liquids. This technique is not limited by the molecule's size and can be applied to solids and liquids. An overview of far infrared studies on complex structures and their interactions is given revealing the potential of the approach.

Similarities and differences of copper and zinc cations binding to biologically relevant peptides studied by vibrational spectroscopies.

GHK and DAHK are biological peptides that bind both copper and zinc cations. Here we used infrared and Raman spectroscopies to study the coordination modes of both copper and zinc ions, at pH 6.8 and 8.9, correlating the data with the crystal structures that are only available for the copper-bound form. We found that Cu(II) binds to deprotonated backbone (amidate), the N-terminus and Nπ of the histidine side chain, in both GHK and DAHK, at pH 6.8 and 8.9. The data for the coordination of zinc at pH 6.8 points to two conformers including both nitrogens of a histidine residue. At pH 8.9, vibrational spectra of the ZnGHK complexes show that equilibria between monomers, oligomers exist, where deprotonated histidine residues as well as deprotonated amide nitrogen are involved in the coordination. A common feature is found: zinc cations coordinate to Nτ and/or Nπ of the His leading to the formation of GHK and DAHK multimers. In contrast, Cu(II) binds His via Nπ regardless of the peptide, in a pH-independent manner.

A spectroelectrochemical cell for ultrafast two-dimensional infrared spectroscopy.

A spectroelectrochemical cell has been designed to combine electrochemistry and ultrafast two-dimensional infrared (2D-IR) spectroscopy, which is a powerful tool to extract structure and dynamics information on the femtosecond to picosecond time scale. Our design is based on a gold mirror with the dual role of performing electrochemistry and reflecting IR light. To provide the high optical surface quality required for laser spectroscopy, the gold surface is made by electron beam evaporation on a glass substrate. Electrochemical cycling facilitates in situ collection of ultrafast dynamics of redox-active molecules by means of 2D-IR. The IR beams are operated in reflection mode so that they travel twice through the sample, i.e., the signal size is doubled. This methodology is optimal for small sample volumes and successfully tested with the ferricyanide/ferrocyanide redox system of which the corresponding electrochemically induced 2D-IR difference spectrum is reported.

Ultrafast 2D-IR spectroelectrochemistry of flavin mononucleotide.

We demonstrate the coupling of ultrafast two-dimensional infrared (2D-IR) spectroscopy to electrochemistry in solution and apply it to flavin mononucleotide, an important cofactor of redox proteins. For this purpose, we designed a spectroelectrochemical cell optimized for 2D-IR measurements in reflection and measured the time-dependent 2D-IR spectra of the oxidized and reduced forms of flavin mononucleotide. The data show anharmonic coupling and vibrational energy transfer between different vibrational modes in the two redox species. Such information is inaccessible with redox-controlled steady-state FTIR spectroscopy. The wide range of applications offered by 2D-IR spectroscopy, such as sub-picosecond structure determination, IR band assignment via energy transfer, disentangling reaction mixtures through band connectivity in the 2D spectra, and the measurement of solvation dynamics and chemical exchange can now be explored under controlled redox potential. The development of this technique furthermore opens new horizons for studying the dynamics of redox proteins.

New insights into the coordination of Cu(II) by the amyloid-B 16 peptide from Fourier transform IR spectroscopy and isotopic labeling.

Alzheimer's disease is a neurodegenerative disorder in which the formation of amyloid-ß (Aß) aggregates plays a causative role. There is ample evidence that Cu(II) can bind to Aß and modulate its aggregation. Moreover, Cu(II) bound to Aß might be involved in the production of reactive oxygen species, a process supposed to be involved in the Alzheimer's disease. The native Aß40 contains a high affinity binding site for Cu(II), which is comprised in the N-terminal portion. Thus, Aß16 (amino acid 1-16 of Aß) has often been used as a model for Cu(II)-binding to monomeric Aß. The Cu(II)-binding to Aß is pH dependent and at pH 7.4, two different type of Cu(II) coordinations exist in equilibrium. These two forms are predominant at pH 6.5 and pH 9.0. In either form, a variety of studies show that the N-terminal Asp and the three His play a key role in the coordination, although the exact binding of these amino acids has not been addressed. Therefore, we studied the coordination modes of Cu(II) at pH 6.5 and 9.0 with the help of Fourier transform infrared (FTIR) spectroscopy. Combined with isotopic labeling of the amino acids involved in the coordination sphere, the data points toward the coordination of Cu(II) via the carboxylate of Asp1 at both pH values in a pseudobridging monovalent fashion. At low pH, His6 binds copper via Nτ, while His13 and His14 are bound via Nπ. At high pH, direct evidence is given on the coordination of Cu(II) via the Nτ atom of His6. Additionally, this study clearly shows the effect of Cu(II) binding on the protonation state of the His residues where a proton displacement takes places on the nitrogen atoms of the imidazole ring.

A combined far-infrared spectroscopic and electrochemical approach for the study of iron-sulfur proteins.

Herein, we present the development of a far-infrared spectroscopic approach for studying metalloenzyme active sites in a redox-dependent manner. An electrochemical cell with 5 mm path and based on silicon windows was found to be appropriate for the measurement of aqueous solutions down to 200 cm(-1) . The cell was probed with the infrared redox signature of the metal-ligand vibrations of different iron-sulfur proteins. Each Fe-S cluster type was found to show a specific spectral signature. As a common feature, a downshift of the frequency of the Fe-S vibrations was seen upon reduction, in line with the increase of the Fe-S bond. This downshift was found to be fully reversible. Electrochemically induced FTIR difference spectroscopy in the far infrared is now possible, opening new perspectives on the understanding of metalloproteins in function of the redox state.

Zinc inhibition of bacterial cytochrome bc(1) reveals the role of cytochrome b E295 in proton release at the Q(o) site.

The cytochrome (cyt) bc(1) complex (cyt bc(1)) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc(1) is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn(2+)-induced inhibition of Rhodobacter capsulatus cyt bc(1) using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn(2+) binding mechanism and its inhibitory effect on cyt bc(1) function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn(2+) on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn(2+) binding affinity. The kinetic study showed that wild-type cyt bc(1) and its E295V mutant have similar levels of apparent K(m) values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 µM, respectively), whereas their K(I) values for Zn(2+) are 8.3 and 38.5 µM, respectively. The calorimetry-based K(D) values for the high-affinity site of cyt bc(1) are on the same order of magnitude as the K(I) values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn(2+), whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn(2+). Our overall results indicate that the proton-active E295 residue near the Q(o) site of cyt bc(1) can bind directly to Zn(2+), resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q(o) site of cyt bc(1).

Probing the hydrogen bonding structure in the Rieske protein.

The use of the far-infrared spectral range presents a novel approach for analysis of the hydrogen bonding in proteins. Here it is presented for the analysis of Fe--S vibrations (500-200 cm(-1)) and of the intra- and intermolecular hydrogen bonding signature (300-50 cm(-1)) in the Rieske protein from Thermus thermophilus as a function of temperature and pH. Three pH values were adequately chosen in order to study all the possible protonation states of the coordinating histidines. The Fe--S vibrations showed pH-dependent shifts in the FIR spectra in line with the change of protonation state of the histidines coordinating the [2Fe--2S] cluster. Measurements of the low-frequency signals between 300 and 30 K demonstrated the presence of a distinct overall hydrogen bonding network and a more rigid structure for a pH higher than 10. To further support the analysis, the redox-dependent shifts of the secondary structure were investigated by means of an electrochemically induced FTIR difference spectroscopic approach in the mid infrared. The results confirmed a clear pH dependency and an influence of the immediate environment of the cluster on the secondary structure. The results support the hypothesis that structure-mediated changes in the environment of iron--sulfur centers play a critical role in regulating enzymatic catalysis. The data point towards the role of the overall internal hydrogen bonding organization for the geometry and the electronic properties of the cluster.

Spectroscopic analysis of tyrosine derivatives: on the role of the tyrosine-histidine covalent linkage in cytochrome c oxidase.

2'-(1-Imidazolyl)-4-methylphenol (C-N bonding in the ortho' position at the phenyl group), a model compound for a tyrosine-histidine covalent linkage, was studied with a combined electrochemical and UV-vis/IR spectroscopic approach. Electrochemical analysis of the 2'-(1-imidazolyl)-4-methylphenol model compound by the means of cyclic voltammetry yielded a potential of 0.48 vs ferrocene (1.15 V vs NHE) for the oxidation of the deprotonated form, the reaction being kinetically irreversible. A tentative assignment of the electrochemically induced Fourier transform infrared (FTIR) difference infrared spectra is presented that indicates the deprotonation of the tyrosine before oxidation and importantly the strong influence of the solvent on the spectral properties and on the mechanism of radical formation. Fluorescence lifetimes and pre-exponential factors of the tyrosine-histidine model compounds are presented and discussed in comparison to tyrosine. The tyrosine-histidine fluorescence lifetime is found to be solvent dependent. The influence of the solvent on the reaction mechanism is proposed with regard to the mechanism of electron coupled proton transfer in proteins that include covalently linked amino acid side chains, like the cytochrome c oxidase.

Infrared spectroscopic characterization of copper-polyhistidine from 1,800 to 50 cm(-1): model systems for copper coordination.

Poly(L-histidine) and imidazole in the presence of copper cations have been investigated by means of Fourier transform infrared (IR) spectroscopy in the mid- and far-IR spectral range to establish specific marker bands of the copper-coordination site in metalloproteins as a function of pH as well as the effect of the coordination on the amino acid contributions. Whereas the well-known mid-IR region was specific for the secondary structure of the protein mimics, the far-IR region included contributions from the metal-ligand vibrations. The addition of copper led to secondary structure changes of poly(L-histidine) at neutral and basic pD and to specific shifts of ring vibrations. At pD 9.5 the addition of copper deprotonated the nitrogen atoms of the imidazole ring and the backbone. At neutral pD the copper cations were coordinated by the N3 atom of the imidazole ring. Copper-imidazole vibrations at neutral pD were observed at 154 and 128 cm(-1). Signals observed at 313 and 162 cm(-1) were assigned to metal-ligand vibrations arising from copper-poly(L-histidine) complexes with a negatively charged imidazole ring.