Charged Amino Acid Substitutions Affect Conformation of Neuroglobin and Cytochrome c Heme Groups.

Neuroglobin (Ngb) is a cytosolic heme protein that plays an important role in protecting cells from apoptosis through interaction with oxidized cytochrome c (Cyt c) released from mitochondria. The interaction of reduced Ngb and oxidized Cyt c is accompanied by electron transfer between them and the reduction in Cyt c. Despite the growing number of studies on Ngb, the mechanism of interaction between Ngb and Cyt c is still unclear. Using Raman spectroscopy, we studied the effect of charged amino acid substitutions in Ngb and Cyt c on the conformation of their hemes. It has been shown that Ngb mutants E60K, K67E, K95E and E60K/E87K demonstrate changed heme conformations with the lower probability of the heme planar conformation compared to wild-type Ngb. Moreover, oxidized Cyt c mutants K25E, K72E and K25E/K72E demonstrate the decrease in the probability of methyl-radicals vibrations, indicating the higher rigidity of the protein microenvironment. It is possible that these changes can affect electron transfer between Ngb and Cyt c.

Exploring the Growth Dynamics of Size-Selected Carbon Atomic Wires with In Situ UV Resonance Raman Spectroscopy.

Short carbon atomic wires, the prototypes of the lacking carbon allotrope carbyne, represent the fundamental 1D system and the first stage in carbon nanostructure growth, which still exhibits many open points regarding their growth and stability. An in situ UV resonance Raman approach is introduced for real-time monitoring of the growth of carbon atomic wires during pulsed laser ablation in liquid without perturbing the synthesis environment. Single-chain species' growth dynamics are tracked, achieving size selectivity by exploiting the peculiar optoelectronic properties of carbon wires and the tunability of synchrotron radiation. Diverse solvents are systematically explored, finding size- and solvent-dependent production rates linked to the solvent's C/H ratio and carbonization tendency. Carbon atomic wires' growth dynamics reveal a complex interplay between formation and degradation, leading to an equilibrium. Water, lacking in carbon atoms and reduced polyynes solubility, yields fewer wires with rapid saturation. Organic solvents exhibit enhanced productivity and near-linear growth, attributed to additional carbon from solvent dissociation and low relative polarity. Exploring the dynamics of the saturation regime provides new insights into advancing carbon atomic wires synthesis via PLAL. Understanding carbon atomic wires' growth dynamics can contribute to optimizing PLAL processes for nanomaterial synthesis.

In Situ and Real-Time Monitoring of Mitochondria-Endoplasmic Reticulum Crosstalk in Apoptosis via Surface-Enhanced Resonance Raman Spectroscopy.

The crosstalk between mitochondria and endoplasmic reticula plays a crucial role in apoptotic pathways in which reactive oxygen species (ROS) produced by microsomal monooxygenase (MMO) are believed to accelerate cytochrome c release. Herein, we successfully demonstrate the potential of surface-enhanced resonance Raman spectroscopy (SERRS) for monitoring MMO-derived ROS formation and ROS-mediated cytochrome c release. Silver nanoparticles coated with nickel shells are used as both Raman signal enhancers and electron donors for cytochrome c. SERRS of cytochrome c is found to be sensitive to ROS, allowing for in situ probing of ROS formation with a cell death inducer. Label-free evaluation of ROS-induced apoptosis is achieved by SERRS-based monitoring of cytochrome c release in living cells. This study verifies the capability of SERRS for label-free, in situ, and real-time monitoring of the mitochondria-endoplasmic reticulum crosstalk in apoptosis and provides a novel strategy for the rational design and screening of ROS-inducing drugs for cancer treatment.

Raman Spectroscopy on Free-Base Meso-tetra(4-pyridyl) Porphyrin under Conditions of Low Temperature and High Hydrostatic Pressure.

We present a Raman spectroscopy study of the vibrational properties of free-base meso-tetra(4-pyridyl) porphyrin polycrystals under various temperature and hydrostatic pressure conditions. The combination of experimental results and Density Functional Theory (DFT) calculations allows us to assign most of the observed Raman bands. The modifications in the Raman spectra when excited with 488 nm and 532 nm laser lights indicate that a resonance effect in the Qy band is taking place. The pressure-dependent results show that the resonance conditions change with increasing pressure, probably due to the shift of the electronic transitions. The temperature-dependent results show that the relative intensities of the Raman modes change at low temperatures, while no frequency shifts are observed. The experimental and theoretical analysis presented here suggest that these molecules are well represented by the C2v point symmetry group.

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

Simultaneous Sensing of H2 O, D2 O and HOD through Peroxo Vibrations.

Detection of HOD simultaneously in the presence of a mixture of H2 O and D2 O is still an experimental challenge. Till date, there is no literature report of simultaneous detection of H2 O, D2 O and HOD based on vibrational spectra. Herein we report simultaneous quantitative detection of H2 O, D2 O and HOD in the same reaction mixture with the help of bridged polynuclear peroxo complex in absence and presence of Au nanoparticles on the basis of a peroxide vibrational mode in resonance Raman and surface enhanced resonance Raman spectrum. We synthesize bridged polynuclear peroxo complex in different solvent mixture of H2 O and D2 O. Due to the formation of different nature of hydrogen bonding between peroxide and solvent molecules (H2 O, D2 O and HOD), vibrational frequency of peroxo bond is significantly affected. Mixtures of different H2 O and D2 O concentrations produce different HOD concentrations and that lead to different intensities of peaks positioned at 897, 823 and 867 cm-1 indicating H2 O, D2 O and HOD, respectively. The lowest detection limits (LODs) were 0.028 mole fraction of D2 O in H2 O and 0.046 mole faction of H2 O in D2 O. In addition, for the first time the results revealed that the cis-peroxide forms two hydrogen bonds with solvent molecules.

Protonation of Asp116 and distortion of the all-trans retinal chromophore in Krokinobacter eikastus rhodopsin 2 causes a redshift in absorption maximum upon dehydration.

Water is usually indispensable for protein function. For ion-pumping rhodopsins, water molecules inside the proteins play an important role in ion transportation. In addition to amino acid residues, water molecules regulate the colors of retinal proteins. It was reported that a sodium-pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2), showed a color change from red to purple upon dehydration under crystalline conditions. Here, we applied comprehensive visible and IR absorption spectroscopy and resonance Raman spectroscopy to KR2 in liposomes under hydration-controlled conditions. A large increase in the hydrogen-out-of-plane (HOOP) vibration at 947 (H-C11=C12-H Au mode) and moderate increases at 893 (C7-H and C10-H) and 808 (C14-H) cm-1 were observed under dehydrated conditions, which were assigned by using systematically deuterated retinal. Moreover, the Asn variant at Asp116, which functions as a counter ion for the protonated retinal Schiff base (PRSB), caused a large redshift in the absorption maximum and constitutive increase in the HOOP modes under hydrated and dehydrated conditions. The protonation of a counter ion at Asp116 clearly causes a redshift in the absorption maximum as the all-trans retinal chromophore twists upon dehydration. Namely, the results strongly suggested that water molecules are important for maintaining the hydrogen-bonding network at the PRSB and deprotonation state of Asp116 in KR2.

Validation of resonance Raman spectroscopy-measured skin carotenoid status as a biomarker for fruit and vegetable intake in Korean adults.

Blood carotenoid concentration measurement is considered the gold standard for fruit and vegetable (F&V) intake estimation; however, this method is invasive and expensive. Recently, skin carotenoid status (SCS) measured by optical sensors has been evaluated as a promising parameter for F&V intake estimation. In this cross-sectional study, we aimed to validate the utility of resonance Raman spectroscopy (RRS)-assessed SCS as a biomarker of F&V intake in Korean adults. We used data from 108 participants aged 20-69 years who completed SCS measurements, blood collection and 3-d dietary recordings. Serum carotenoid concentrations were quantified using HPLC, and dietary carotenoid and F&V intakes were estimated via 3-d dietary records using a carotenoid database for common Korean foods. The correlations of the SCS with serum carotenoid concentrations, dietary carotenoid intake and F&V intake were examined to assess SCS validity. SCS was positively correlated with total serum carotenoid concentration (r = 0·52, 95 % CI = 0·36, 0·64, P < 0·001), serum ß-carotene concentration (r = 0·60, 95 % CI = 0·47, 0·71, P < 0·001), total carotenoid intake (r = 0·20, 95 % CI = 0·01, 0·37, P = 0·04), ß-carotene intake (r = 0·30, 95 % CI = 0·11, 0·46, P = 0·002) and F&V intake (r = 0·40, 95 % CI = 0·23, 0·55, P < 0·001). These results suggest that SCS can be a valid biomarker of F&V intake in Korean adults.

Evaluation of a Commercial Device Based on Reflection Spectroscopy as an Alternative to Resonance Raman Spectroscopy in Measuring Skin Carotenoid Levels: Randomized Controlled Trial.

Resonance Raman spectroscopy (RRS) has been used as a reference method for measuring skin carotenoid levels (SCL), which indicate vegetable and fruit intake. However, RRS is not an easy-to-use method in SCL measurement due to its complicated implementation. In this study, a commercial spectrophotometer based on reflection spectroscopy (RS), which is relatively simple and inexpensive, was evaluated to confirm usability compared with RRS in measuring SCL. To investigate the agreement between RS and RRS, eighty participants were randomly assigned to a high-carotenoid diet group (21 mg/day of total carotenoids) or a control-carotenoid diet group (14 mg/day of total carotenoids) during a 6-week whole-diet intervention period and a 4-week tracking period. Strong correlations between the RS and RRS methods were observed at baseline (r = 0.944) and the entire period (r = 0.930). The rate of SCL increase was similar during the diet intervention; however, the initiation of the SCL decrease in RS was slower than in RRS during the tracking period. To confirm the agreement of RS and RRS from various perspectives, new visualization tools and indices were additionally applied and confirmed the similar response patterns of the two methods. The results indicate that the proposed RS method could be an alternative to RRS in SCL measurements.

An in vivo accelerated developmental myelination model for testing promyelinating therapeutics.

BACKGROUND: Therapeutic agents stimulating the process of myelination could be beneficial for the treatment of demyelinating diseases, such as multiple sclerosis. The efficient translation of compounds promoting myelination in vitro to efficacy in vivo is inherently time-consuming and expensive. Thyroid hormones accelerate the differentiation and maturation of oligodendrocytes, thereby promoting myelination. Systemic administration of the thyroid hormone thyroxine (T4) accelerates brain maturation, including myelination, during early postnatal development. The objective of this study was to validate an animal model for rapid testing of promyelinating therapeutic candidates for their effects on early postnatal development by using T4 as a reference compound. METHODS: Daily subcutaneous injections of T4 were given to Sprague Dawley rat pups from postnatal day (PND) 2 to PND10. Changes in white matter were determined at PND10 using diffusion tensor magnetic resonance imaging (DTI). Temporal changes in myelination from PND3 to PND11 were also assessed by quantifying myelin basic protein (MBP) expression levels in the brain using the resonance Raman spectroscopy/enzyme-linked immunosorbent assay (RRS-ELISA) and quantitative immunohistochemistry. RESULTS: DTI of white matter tracts showed significantly higher fractional anisotropy in the internal capsule of T4-treated rat pups. The distribution of total FA values in the forebrain was significantly shifted towards higher values in the T4-treated group, suggesting increased myelination. In vivo imaging data were supported by in vitro observations, as T4 administration significantly potentiated the developmental increase in MBP levels in brain lysates starting from PND8. MBP levels in the brain of animals that received treatment for 9 days correlated with the FA metric determined in the same pups in vivo a day earlier. Furthermore, accelerated developmental myelination following T4 administration was confirmed by immunohistochemical staining for MBP in coronal brain sections of treated rat pups. CONCLUSIONS: T4-treated rat pups had increased MBP expression levels and higher MRI fractional anisotropy values, both indications of accelerated myelination. This simple developmental myelination model affords a rapid test of promyelinating activity in vivo within several days, which could facilitate in vivo prescreening of candidate therapeutic compounds for developmental hypomyelinating diseases. Further research will be necessary to assess the utility of this platform for screening promyelination compounds in more complex demyelination disease models, such us multiple sclerosis.

Influence of the Linker Chemistry on the Photoinduced Charge-Transfer Dynamics of Hetero-dinuclear Photocatalysts.

To optimize light-driven catalytic processes, light-mediated multi-electron transfer dynamics in molecular dyads need to be studied and correlated with structural changes focusing on the catalytically active metastable intermediates. Here, spectro-electrochemistry has been employed to investigate the structure-dependent photoelectron transfer kinetics in catalytically active intermediates of two Ru-Rh catalysts for light-driven NAD+ reduction. The excited-state reactivity of short-lived intermediates was studied along different photoreaction pathways by resonance Raman and time-resolved transient absorption spectro-electrochemistry with sub-picosecond time resolution under operando conditions. The results demonstrate, for the first time, how the bridging ligand serves as a (multi-)electron storage structure, mediates the strength of the electronic coupling of catalytic and photocenter and impacts the targeted electron transfer as well as parasitic electron-transfer kinetics.

Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2.

Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed.

Spectroscopic Studies of Mononuclear Molybdenum Enzyme Centers.

A concise review is provided of the contributions that various spectroscopic methods have made to our understanding of the physical and electronic structures of mononuclear molybdenum enzymes. Contributions to our understanding of the structure and function of each of the major families of these enzymes is considered, providing a perspective on how spectroscopy has impacted the field.

Cis-Trans Reisomerization Precedes Reprotonation of the Retinal Chromophore in the Photocycle of Schizorhodopsin 4.

Recent discoveries of light-driven inward proton-pumping rhodopsins have opened new avenues to exploring the mechanism of unidirectional transport because these proteins transport protons in the opposite direction to conventional proton-pumping rhodopsins, despite their similar protein structure and membrane topology. Schizorhodopsin (SzR) is a newly discovered rhodopsin family of light-driven inward proton pumps. Here, we report time-resolved resonance Raman spectra showing that cis-trans thermal reisomerization precedes reprotonation at the Schiff base of the retinal chromophore in the photocycle of SzR AM_5_00977. This sequence has not been observed for the photocycles of conventional proton-pumping rhodopsins, in which reisomerization follows reprotonation, and thus provides insights into the mechanism of proton uptake to the chromophore during inward proton pumping. The present findings are expected to contribute to controlling the direction of proton transport in engineered proteins.

Iron-Containing Ureases.

Conventional ureases possess dinuclear nickel active sites that are oxygen-stable and require a set of accessory proteins for metallocenter biosynthesis. By contrast, oxygen-labile ureases have active sites containing dual ferrous ions and lack a requirement for maturation proteins. The structures of the two types of urease are remarkably similar, with an active site architecture that includes two imidazoles and a carboxylate ligand coordinated to one metal, two imidazoles coordinated to the second metal, and a metal-bridging carbamylated lysine ligand. The electronic spectrum of the diferric form of the enzyme resembles that of methemerythrin. Resonance Raman spectroscopic analyses confirm the presence of a µ-oxo ligand and indicate the presence of one or more terminal solvent ligands.

Probing the Charge Transfer in a Frustrated Lewis Pair by Resonance Raman Spectroscopy and DFT Calculations.

A classical Lewis adduct derives from a covalent bond between a Lewis acid and a base. When the adduct formation is precluded by means of steric hindrance the association of the respective acid-base molecular system is defined as a frustrated Lewis pair (FLP). In this work, the archetypal FLP Mes3 P/B(C6 F5 )3 was characterized for the first time by resonance Raman spectroscopy, and the results were supported by density functional theory (DFT) calculations. The charge transfer nature of the lowest energy electronic transition, from phosphine to borane, was confirmed by the selective enhancement of the Raman bands associated to the FLP chromophore at resonance condition. Herein, we demonstrate the use of resonance Raman spectroscopy as a distinguished technique to probe the weak interaction involved in FLP chemistry.

Skin carotenoid status and plasma carotenoids: biomarkers of dietary carotenoids, fruits and vegetables for middle-aged and older Singaporean adults.

Skin carotenoid status (SCS) measured by resonance Raman spectroscopy (RRS) may serve as an emerging alternative measurement for dietary carotenoid, fruit and vegetable (FV) intake although its application had not been assessed in a middle-aged and older population in Asia. This cross-sectional study aims to concurrently examine the use of SCS and plasma carotenoids to measure FV and carotenoid intake in a middle-aged and older population, taking into consideration potential socio-demographic and nutritional confounders. The study recruited 103 middle-aged and older adults (mean age: 58 years) in Singapore. Dietary carotenoids and FV, plasma carotenoid concentration and SCS were measured using 3-d food records, HPLC and a biophotonic scanner which utilised RRS, respectively. Adjusted for statistically defined socio-demographic covariates sex, age, BMI, prescription medication and cigarette smoking, plasma carotenoids and SCS showed positive associations with dietary total carotenoids (ßplasma: 0·020 (95 % CI 0·000, 0·040) µmol/l/mg, P = 0·05; ßskin: 265 (95 % CI 23, 506) arbitrary units/mg, P = 0·03) and FV (ßplasma: 0·076 (95 % CI 0·021, 0·132) µmol/l per FV serving, P = 0·008; ßskin: 1036 (95 % CI 363, 1708) arbitrary units/FV serving, P = 0·003). The associations of SCS with dietary carotenoid and FV intake were null with the inclusion of dietary PUFA, fibre and vitamin C as nutritional covariates (P > 0·05). This suggests a potential influence of these nutritional factors on carotenoid circulation and deposition in the skin. In conclusion, SCS, similar to plasma carotenoids, may serve as a biomarker for both dietary carotenoid and FV intake in a middle-aged and older Singaporean population.

Davydov Splitting, Resonance Effect and Phonon Dynamics in Chemical Vapor Deposition Grown Layered MoS2.

We present comprehensive temperature dependent Raman measurements for chemical vapor deposition grown horizontally aligned layered MoS2in a temperature range of 4-330 K under a resonance condition. Our analysis of temperature dependent phonon frequency shift and linewidth suggests a finite role of three and four phonon anharmonic effect. We observe Davydov splitting of the out-of-plane (A1g) and in-plane (E2g1) modes for both three layer (3L) and few layer (FL) systems. The number of Davydov splitting components are found more in FL compared to 3L MoS2, which suggests that it increases with an increasing number of layers. Further, Davydov splitting is analyzed as a function of temperature. Temperature evaluation of the Raman spectra shows that the Davydov splitting, especially forA1gmode, is very strong and well resolved at low temperature. We observe thatA1gmode shows the splitting at low temperature, whileE2g1mode is split even at room temperature, which suggests a prominent role ofA1gmode in the interlayer interaction at low temperature. Further, an almost 60-fold increase in the intensity of the phonon modes at low temperature clearly shows the temperature dependent tuning of the resonance effect.

Quantitative SERS sensor based on self-assembled Au@Ag heterogeneous nanocuboids monolayer with high enhancement factor for practical quantitative detection.

Accurate and rapid quantitative detection of pesticide and pollutant levels in the actual sample can aid in protecting food security, environmental security, and human health. A high Raman enhancement factor and good repeatability of the surface-enhanced Raman spectroscopy (SERS) substrates are favorable to quantitative analysis. Herein, a quantitative SERS sensor based on constructed self-assembled plasmonic Au@Ag heterogeneous nanocuboids (Au@Ag NCs) monolayer was developed. The sensor was used to quantitatively detect the trace pesticides extracted from pear surfaces and pollutants in fishpond water. Densely packed Au@Ag NCs fabricated into large-scale monolayer films were chemically functionalized using 4-methyl-thiobenzoic acid (4-MBA) at the organic/aqueous interface, in which plentiful nanogaps contribute to increase hotspots. Their sharp corners and edges make the sensor have high SERS performance through providing abundant "hot spots." The obtained optically SERS-based sensor with uniform liquid-state interfacial nanoparticle arrays appeared to have nice SERS performance and uniformity using crystal violet (CV) as a probe molecule. In particular, the proposed SERS sensor was applied for quantitative detection of thiabendazole (TBZ) extracted from pear surfaces and malachite green (MG) in fishpond water down to levels of 0.0105 nM and 0.87 nM for SERS assay respectively. As a result, our proposed SERS quantitative detection strategy is quite preferred to on-site analysis and supervision of contaminant in food samples.

Strategies and Perspectives for UV Resonance Raman Applicability in Clinical Analyses of Human Sperm RNA.

Developing a deeper knowledge about the impact of DNA and RNA epigenetic mutations on sperm production and fertilization performance is essential for selecting best quality samples in Assisted Reproductive Technologies (ART). Indeed, sperm RNAs adenine and guanine are likely to be methylated in low quality RNA sperm samples and their study requires the employment of techniques able to isolate high quality nucleic acids. UV resonance Raman spectroscopy represents a valuable tool that is able to monitor peculiar molecular modifications occurring predominantly in nucleic acids, being less sensitive to the presence of other biological compounds. In this work, we used an UV Resonance Raman (UVRR) setup coupled to a synchrotron radiation source tuned at 250 nm, in order to enhance sperm RNAs adenine and guanine vibrational signals, reducing also the impact of a fluorescence background typically occurring at lower energies. Despite that our protocol should be further optimized and further analyses are requested, our results support the concept that UVRR can be applied for setting inexpensive tools to be employed for semen quality assessment in ART.