Understanding the Changes in the Circular Dichroism of Light Harvesting Complex II upon Varying Its Pigment Composition and Organization

In this work we modeled the circular dichroism (CD) spectrum of LHCII, the main light harvesting antenna of photosystem II of higher plants. Excitonic calculations are performed for a monomeric subunit, taken from the crystal structure of trimeric LHCII from spinach. All of the major features of the CD spectrum above 450 nm are satisfactorily reproduced, and possible orientations of the Chl and carotenoid transition dipole moments are identified. The obtained modeling parameters are used to simulate the CD spectra of two complexes with altered pigment composition: a mutant lacking Chls a 611-612 and a complex lacking the carotenoid neoxanthin. By removing the relevant pigment(s) from the structure, we are able to reproduce their spectra, which implies that the alteration does not disturb the overall structure. The CD spectrum of trimeric LHCII shows a reversed relative intensity of the two negative bands around 470 and 490 nm as compared to monomeric LHCII. The simulations reproduce this reversal, indicating that it is mainly due to interactions between chromophores in different monomeric subunits, and the trimerization does not induce observable changes in the monomeric structure. Our simulated spectrum resembles one of two different trimeric CD spectra reported in literature. We argue that the differences in the experimental trimeric CD spectra are caused by changes in the strength of the monomer-monomer interactions due to the differences in detergents used for the purification of the complexes.

A placebo-controlled proof-of-concept study of alirocumab on postprandial lipids and vascular elasticity in insulin-treated patients with type 2 diabetes mellitus

Aim: Type 2 diabetes mellitus (T2DM) is associated with an increased risk of cardiovascular disease (CVD) linked to atherogenic dyslipidaemia and postprandial hyperlipidaemia. Alirocumab, a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor, improves CVD risk by reducing the concentration of low-density lipoprotein-cholesterol (LDL-C). However, effects of PCK9 inhibitors on other aspects of diabetic dyslipidaemia, particularly in the postprandial situation, are less clear. Material and Methods: Twelve male patients with T2DM on an intensive insulin regimen completed a 6-week randomized, double-blind, placebo-controlled, proof-of-concept study. Participants received three biweekly dosages of subcutaneous alirocumab (150 mg) or placebo. Before and after the intervention, fasting and postprandial triglyceride (TG) plasma levels, apolipoprotein (apo) B48, lipoprotein composition isolated by ultracentrifugation, vascular function and markers of inflammation were evaluated. Results: Alirocumab treatment reduced fasting plasma TG levels (between group median change −24.7%; P = 0.018) and fasting apoB48 serum levels (−35.9%; P = 0.039) compared with placebo. Alirocumab reduced the plasma TG area under the curve (AUC) (−26.4%; P = 0.006) and apoB48 AUC (−55.7%; P = 0.046), as well as plasma TG incremental AUC (−21.4%; P = 0.04) and apoB48 incremental AUC (−26.8%; P = 0.02). In addition, alirocumab reduced fasting and postprandial TG levels in very low-density lipoprotein (VLDL) and LDL. Alirocumab improved fasting pulse wave velocity, but no changes in postprandial markers of inflammation were observed. Conclusions: In addition to the well-known LDL-C-reducing effects, 6 weeks of alirocumab treatment lowered both fasting and postprandial plasma TG levels by reducing the TG levels in VLDL and LDL and the concentration of intestinal remnants

Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant

Active-site structure, binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6

The Occurrence of Non-Gaussian Spectral Line Shapes of Molecules in Electrostatically Disordered Media

We calc. the effects of relatively strong elec. fields, corresponding to typical values for the fields inside glasses and proteins, on the spectroscopic properties of a simple two-level mol. Disorder in the elec. field strength is employed as a phys. source for inhomogeneous broadening. A striking result is that a Gaussian probability distribution for the field over the ensemble does not give rise to a Gaussian spectral line shape. The results are used to describe the absorption spectra of the B800 band of the B800-850 light-harvesting complex of photosynthetic purple bacteria. [on SciFinder (R)

Comments on “Spectroscopic studies of keto–enol tautomeric equilibrium of azo dyes” by M. A. Rauf, S. Hisaindee and N. Saleh, published in RSC Advances 2015, 5, 18097-18110 as a Review Article

The paper mentioned in the title contains inaccuracies and misleading interpretations that misrepresent basic knowledge about tautomerism in azo dyes. This Letter gives a better understanding of the basic principles of the tautomerisation process.</p

Spectroscopy and kinetics of tyrosinase catalyzed trans-resveratrol oxidation

The spectroscopy and kinetics of the tyrosinase catalyzed trans-resveratrol oxidation were investigated by measuring both UV–vis absorption spectra over the 200–500 nm range and Raman spectra over the 600–1800 cm–1 region. Room temperature UV–vis absorption spectra, as a function of time, showed the presence of two isosbestic points located at λ1 = 270 nm and λ2 = 345.5 nm delimiting two different regions: the reactant region around 300 nm, where the absorption decreased with time, and the product region over the low wavelength (λ 390 nm) wavelength zone in which the absorption increased with time until, in both cases, constant values were achieved. A first-order kinetics was deduced with a rate coefficient of k1 = (0.10 ± 0.001) min–1, which turned out to be independent of substrate concentration over the 50–5 μM range; a feature that was rationalized by invoking the limiting case of the Michaelis–Menten scheme appropriate for substrate concentration much lower than the respective Michaelis constant. The observation of the distinct resonance enhanced Raman lines, specifically those peaking at 830 cm–1, 753 cm–1, and 642 cm–1 together with their time evolution, permitted us to gain insight into some crucial features and steps of the catalytic reaction. Namely, that the formation of the so-called trans-resveratrol and tyrosinase SP complex with its O–O bridge plays a crucial role in the first steps of this enzymatic reaction and that the hydroxylation of the ortho C–H bond of the trans-resveratrol OH group occurs after O–O bond cleavage in the tyrosinase active site. The present study makes clear that a class of potential inhibitors of tyrosinase can be found in compounds able to bind the two Cu (II) ions of the enzyme bidentate form

pH-Dependent Complexation of Histamine H₁ Receptor Antagonists and Human Serum Albumin Studied by UV Resonance Raman Spectroscopy

UV resonance Raman spectroscopy was used to characterize the binding of three first-generation histamine H₁ receptor antagoniststripelennamine (TRP), mepyramine (MEP), and brompheniramine (BPA)to human serum albumin (HSA) at pH 7.2 and pH 9.0. Binding constants differ at these pH values, which can be ascribed to the different extent of protonation of the ethylamino side chain of the ligands. We have recently shown [Tardioli et al. <i>J. Raman Spectrosc.</i> <b>2011</b>, <i>42</i>, 1016–1024] that for the solution conformation of TRP and MEP the side chain plays an important role by allowing an internal hydrogen bond with the aminopyridine nitrogen in TRP and MEP. Results presented in this paper suggest that the existence of such molecular structures has serious biological significance on the binding affinity of those ligands to HSA. At pH 7.2, only the stretched conformers of protonated TRP and MEP bind in HSA binding site I. Using UV absorption data, we derived binding constants for the neutral and protonated forms of TRP to HSA. The neutral species seems to be conjugated to a positive group of the protein, affecting both the tryptophan W214 and some of the tyrosine (Y) vibrations. BPA, for which the structure with an intramolecular hydrogen bonded side chain is not possible, is H bound to the indole ring nitrogen of W214, of which the side chain rotates over a certain angle to accommodate the drug in site I. We propose that the protonated BPA is also bound in site I, where the Y150 residue stabilizes the presence of this compound in the binding pocket. No spectroscopic evidence was found for conformational changes of the protein affecting the spectroscopic properties of W and Y in this pH range