Exploring the interdependence of calcium and chloride activation of O2 evolution in photosystem II.

Calcium and chloride are activators of oxygen evolution in photosystem II (PSII), the light-absorbing water oxidase of higher plants, algae, and cyanobacteria. Calcium is an essential part of the catalytic Mn4CaO5 cluster that carries out water oxidation and chloride has two nearby binding sites, one of which is associated with a major water channel. The co-activation of oxygen evolution by the two ions is examined in higher plant PSII lacking the extrinsic PsbP and PsbQ subunits using a bisubstrate enzyme kinetics approach. Analysis of three different preparations at pH 6.3 indicates that the Michaelis constant, KM, for each ion is less than the dissociation constant, KS, and that the affinity of PSII for Ca2+ is about ten-fold greater than for Cl-, in agreement with previous studies. Results are consistent with a sequential binding model in which either ion can bind first and each promotes the activation by the second ion. At pH 5.5, similar results are found, except with a higher affinity for Cl- and lower affinity for Ca2+. Observation of the slow-decaying Tyr Z radical, YZ•, at 77 K and the coupled S2YZ• radical at 10 K, which are both associated with Ca2+ depletion, shows that Cl- is necessary for their observation. Given the order of electron and proton transfer events, this indicates that chloride is required to reach the S3 state preceding Ca2+ loss and possibly for stabilization of YZ• after it forms. Interdependence through hydrogen bonding is considered in the context of the water environment that intervenes between Cl- at the Cl-1 site and the Ca2+/Tyr Z region.

A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids.

Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography mass spectrometry (GC-MS) analysis. A one-step derivatization using 100 µL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations (RSDs) of all targeted compounds showed good intra- and inter-day (within 7 days) precision (< 10%), and good stability (< 20%) within 4 days at room temperature (23-25 °C), or 7 days when stored at -20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistics analysis of the concentrations of these target metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.

Chia seed supplementation and disease risk factors in overweight women: a metabolomics investigation.

OBJECTIVE/SETTING: This study assessed the effectiveness of milled and whole chia seed in altering disease risk factors in overweight, postmenopausal women using a metabolomics approach. DESIGN/INTERVENTION: Subjects were randomized to chia seed (whole or milled) and placebo (poppy seed) groups, and under double-blinded procedures ingested 25 g chia seed or placebo supplements each day for 10 weeks. SUBJECTS: Subjects included 62 overweight (body-mass index 25 kg/m(2) and higher), nondiseased, nonsmoking, postmenopausal women, ages 49-75 years, with analysis based on the 56 subjects who completed all phases of the study. OUTCOME MEASURES: Pre- and poststudy measures included body mass and composition, blood pressure and augmentation index, serum lipid profile, inflammation markers from fasting blood samples, plasma fatty acids, and metabolic profiling using gas chromatography-mass spectrometry with multivariate statistical methods including principal component analysis and partial least-square discriminant analysis (PLS-DA). RESULTS: Plasma α-linolenic acid (N=ALA) increased 58% (interaction effect, p=0.002) and eicosapentaenoic acid (EPA) 39% (p=0.016) in the milled chia seed group (N=14) compared to nonsignificant changes in the whole chia seed (N=16) and placebo (N=26) groups. Pre-to-post measures of body composition, inflammation, blood pressure, augmentation index, and lipoproteins did not differ between chia seed (whole or milled) and placebo groups (all interaction effects, p>0.05). Global metabolic difference scores for each group calculated through PLS-DA models were nonsignificant (Q(2)Y<0.40), and fold-changes for 28 targeted metabolites associated with inflammation and disease risk factors did not differ between groups. CONCLUSIONS: Ingestion of 25 g/day milled chia seed compared to whole chia seed or placebo for 10 weeks by overweight women increased plasma ALA and EPA, but had no influence on inflammation or disease risk factors using both traditional and metabolomics-based measures.

An alternative method for calcium depletion of the oxygen evolving complex of photosystem II as revealed by the dark-stable multiline EPR signal.

The dark-stable multiline EPR signal of photosystem II (PSII) is associated with a slow-decaying S(2) state that is due to Ca(2+) loss from the oxygen evolving complex. Formation of the signal was observed in intact PSII in the presence of 100-250 mM NaCl at pH 5.5. Both moderately high NaCl concentration and decreased pH were required for its appearance in intact PSII. It was estimated that only a portion of oxygen evolving complexes was responsible for the signal (about 20% in 250 mM NaCl), based on the loss of the normal S(2)-state multiline signal. The formation of the dark-stable multiline signal in intact PSII at pH 5.5 could be reversed by addition of 15 mM Ca(2+) in the presence of moderately high NaCl, confirming that it was the absence of Ca(2+) that led to its appearance. Formation of the dark-stable multiline signal in NaCl-washed PSII, which lacks the PsbP (23 kDa) and PsbQ (17 kDa) subunits, was observed in about 80% of the sample in the presence of 150 mM NaCl at pH 5.5, but some signal was also observed under normal buffer conditions. In both intact and NaCl-washed PSII, the S(2)Y(Z). signal, which is also characteristic of Ca(2+) depletion, appeared upon subsequent illumination. Formation of the dark-stable multiline signal took place in the absence of Ca(2+) chelator or polycarboxylic acids, indicating that the signal did not require their direct binding as has been proposed previously. The conditions used here were milder than those used to produce the signal in previous studies and included a preillumination protocol to maximize the dark-stable S(2) state. Based on these conditions, it is suggested that Ca(2+) release occurred through protonation of key residues that coordinate Ca(2+) at low pH, followed by displacement of Ca(2+) with Na(+) by mass action at the moderately high NaCl concentration.