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Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chloroflexus aurantiacus . Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides , nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA , and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the Q A pocket of this heterologously expressed reaction center.

DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in various human diseases, including cancer. A rational approach to preventing tumorigenesis involves the use of pharmacologic inhibitors of DNA methylation; these inhibitors should reactivate tumor suppressor genes (TSGs) in tumor cells and restore tumor suppressor pathways. Antroquinonol D (3-demethoxyl antroquinonol), a new DNMT1 inhibitor, was isolated from Antrodia camphorata and identified using nuclear magnetic resonance. Antroquinonol D inhibited the growth of MCF7, T47D, and MDA-MB-231 breast cancer cells without harming normal MCF10A and IMR-90 cells. The SRB assay showed that the 50% growth inhibition (GI50) in MCF7, T47D, and MDA-MB-231 breast cancer cells following treatment with antroquinonol D was 8.01, 3.57, and 25.08 μM, respectively. d-Antroquinonol also inhibited the migratory ability of MDA-MB-231 breast cancer cells in wound healing and Transwell assays. In addition, antroquinonol D inhibited DNMT1 activity, as assessed by the DNMT1 methyltransferase activity assay. As the cofactor SAM level increased, the inhibitory effects of d-antroquinonol on DNMT1 gradually decreased. An enzyme activity assay and molecular modeling revealed that antroquinonol D is bound to the catalytic domain of DNMT1 and competes for the same binding pocket in the DNMT1 enzyme as the cofactor SAM, but does not compete for the binding pocket in the DNMT3B enzyme. An Illumina Methylation 450 K array-based assay and real-time PCR assay revealed that antroquinonol D decreased the methylation status and reactivated the expression of multiple TSGs in MDA-MB-231 breast cancer cells. In conclusion, we showed that antroquinonol D induces DNA demethylation and the recovery of multiple tumor suppressor genes, while inhibiting breast cancer growth and migration potential.

The effects of different concentrations of ubiquinol-10 (Q10H2) on citral's stability were systematically investigated and compared in citral-loaded oil-in-water (O/W) nanoemulsions. Solid phase microextraction gas chromatography (SPME-GC) was employed to monitor the degradation of citral and the formation of off-flavor compounds throughout storage at 25 and 45 °C. The optimum concentration of Q10H2 in the current formulation was determined to be around 0.10 wt % in the system (Q10H2/citral ratio 1:1), which can effectively protect citral from chemical degradation and oxidation. Results suggested, however, that a low concentration of Q10H2 may induce the majority of the ubisemiquinone (Q10(•-))/ubiquinone (Q10) redox transition, which possibly endowed Q10H2 with pro-oxidant properties. Further increase in Q10H2 concentration beyond a certain value also hindered its effect due to the complex properties of radicals involved and the overall environment encountered. With appropriate concentrations of Q10H2 presented in the system, major citral oxidation off-flavor compounds (p-cresol, α,p-dimethylstyrene, p-methylacetophenone), and some of the lipid degradation products can be inhibited to lower levels. In contrast, ubiquinone-10 (Q10) had a negligible effect on citral's chemical stability and off-flavor generation.

Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure-Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged CoQ analogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2'-deoxyuridine (EdU) and a CoQ analogue, with distinct Raman tags was demonstrated.

The aim of this study was to investigate the most important oxidation products of α-tocotrienol (α-T3) along with other tocochromanols in lipid matrices and tocotrienol-rich foods. For this purpose, an efficient molecular distillation procedure was developed for the extraction of analytes, and α-T3-spiked and thermally oxidized natural lipids (lard and wheat germ oil) and α-T3-rich foods (wholemeal rye bread and oil from dried brewer's spent grain) were investigated through HPLC-DAD-F. The following α-T3 oxidation products were extractable from lipid matrices along with tocochromanols: α-tocotrienolquinone (α-T3Q), α-tocotrienolquinone-4a,5-epoxide (α-T3Q-4a,5-E), α-tocotrienolquinone-7,8-epoxide (α-T3Q-7,8-E), 7-formyl-β-tocotrienol (7-FβT3), and 5-formyl-γ-tocotrienol (5-FγT3). Recovery rates were as high as 88% and enrichment factors up to 124. The proposed method allows the investigation of α-T3Q, α-T3Q-4a,5-E, α-T3Q-7,8-E, 7-FβT3, and 5-FγT3 in small quantities (<0.78 μg/g) in lipid matrices, which is necessary for the investigation and analysis of the formation kinetics of these oxidation products in fat, oils, and tocotrienol-rich foods.

Virgin argan oil possesses high antioxidant capacity (AC), which may be partially explained by its high content in antioxidant molecules such as polyphenols and tocopherols. However, the content in other antioxidant molecules, for example, coenzyme Q10 (CoQ(10)), coenzyme Q9 (CoQ(9)), and melatonin (Mel), which have been identified in other edible vegetable oils, have not been evaluated in virgin argan oil. Consequently, it was decided to evaluate the contents of CoQ(10), CoQ(9), and Mel in virgin argan oils and compare the results to those obtained in extra virgin olive oils and some varieties of seed oils. By the use of sensitive HPLC-EC/F methods, the results showed that virgin argan oil is a rich source of CoQ(10) and Mel, but no CoQ(9) was detected. Extra virgin olive oil showed higher levels of CoQ(10) and lower levels of Mel than virgin argan oil. Between the seed oil samples, only virgin soybean oil showed higher CoQ(10) and Mel levels than virgin argan oil. The results may be relevant for the contribution of CoQ(10) and Mel to the biological activities of virgin argan oil.

Coenzyme Q (CoQ) is a naturally occurring lipid-soluble quinone that performs multiple functions in all living cells and has become a popular antioxidant supplement, a coadjuvant in the treatment of heart disease, and the object of study for treating neurodegenerative disorders. Although there are many tools for CoQ analysis of microbial and animal samples, there have been relatively few reports of methods for CoQ analysis of green plants. This work describes a method for the routine analysis of coenzyme Q(10) in green leaf tissue of cultivated Nicotiana tabacum (tobacco) using high-performance liquid chromatography (HPLC) with UV detection. The method was applied to the analysis of CoQ(10) in N. tabacum 'KY14' leaves at different stalk positions representing young lanceolate to senescing leaves, and it was found that CoQ(10) increased as leaf position changed down the stalk from 18.69 to 82.68 μg/g fw. The method was also used to observe CoQ(10) in N. tabacum 'NC55' and N. tabacum 'TN90LC' leaves over time, finding that CoQ(10) leaf content remained relatively stable from 3 to 6 weeks but increased in both cultivars at 8 weeks. This method will likely be useful in the analysis of CoQ(10) in the green leaves of other plant species.

A new sensitive and selective method has been developed for the quantification of the total coenzyme Q9 (CoQ9) and coenzyme Q10 (CoQ10) concentration in vegetable oil samples. The coenzyme Q fraction is isolated by solid-phase extraction (SPE) on amino phase eluting with a mixture of heptane:ethyl ether. The organic solvent is evaporated under nitrogen, and the residue is dissolved in a mixture of acetonitrile:tetrahydrofuran and finally is analyzed by reverse-phase high-performance liquid chromatography with a mass detector. The sensitivity of the method is based on the high efficient formation of the radical anions [M (-.)] of CoQ9 and CoQ10 by negative atmospheric pressure ionization. Interferences are minimized by using mass detection of the [M (-.)] ions ( m/ z = 797.5 for CoQ9 and m/ z = 862.5 for CoQ10) in selective reaction monitoring mode ( m/ z = 797.5 --> m/ z = 779.5 and m/ z = 862.5 --> m/ z = 847.5) using a triple-quadrupole mass spectrometer. The method was successfully applied to sunflower, soybean, and rapeseed oils, with a limit of quantification of 0.025 mg/kg for both compounds.

Statin pharmaceuticals, heavily prescribed in the treatment of hypercholesterolemia, are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR). In plants, these compounds also inhibit HMGR, which regulates cytosolic isoprenoid biosynthesis in the mevalonic acid (MVA) pathway. Phytotoxicity was evaluated in the higher aquatic plant Lemna gibba exposed to atorvastatin and lovastatin for 7-days by measuring the concentrations of sterols and ubiquinone; products downstream in the MVA pathway. The efficiency of the parallel and unaffected methylerythritol phosphate pathway (MEP) was also evaluated by measuring the end product, plastoquinone. Statin treatment caused an accumulation of plastoquinone, and unexpectedly, ubiquinone, an artifact likely due to metabolite sharing from the plastidial MEP pathway. Statins were, however, highly phytotoxic to L. gibba and HPLC-UV analysis of plant extracts showed significantly decreased concentrations of both stigmasterol and beta-sitosterol, which are critical components of plant membranes and regulate morphogenesis and development. EC10 values for atorvastatin and lovastatin were as small as 26.1 and 32.8 microg/L, respectively. However, hazard quotients indicated that statins present little risk to the model higher aquatic plant Lemna gibba at environmentally relevant concentrations, even though pathway-specific endpoints were 2-3 times more sensitive than traditional gross morphological endpoints typically used in risk assessment.

The presence of the oxidized and reduced forms of ubiquinones Q(9) and Q(10) was determined in commercial extra virgin olive and seed oils, where the amounts of alpha- and gamma-tocopherols and beta-carotene were also quantitated. Very high concentrations of ubiquinones were found in soybean and corn oils. Furthermore, the total antioxidant capability of each oil was evaluated by measuring total radical-trapping antioxidant parameters (TRAP) in tert-butyl alcohol and using egg lecithin as the oxidizable substrate. These values decreased in the order sunflower > corn > peanut > olive; the highest TRAP, which was found in sunflower oil, was related to the very high amount of alpha-tocopherol. Olive oil, because of the low content of alpha-tocopherol, exhibited a TRAP value approximately one-third that of sunflower oil. TRAP values of corn and soybean oils, in which low amounts of alpha-tocopherol but very high contents of gamma-tocopherol and reduced ubiquinones were present, were intermediate. gamma-Tocopherol exhibited a poor ability of trapping peroxyl radicals in tert-butyl alcohol. This behavior was probably due to the effects of the solvent on the rate of hydrogen abstraction from this phenol.

In-line connected electrochemical (EC) and diode array (DAD) detectors were compared in the reversed-phase high-performance liquid chromatographic (RP-HPLC) analysis of coenzymes Q(9) and Q(10) in some food materials (beef steak, beef heart, Baltic herring fillet, and rye flour). Coenzymes Q(9) and Q(10) were extracted from the samples using a 5:1 n-hexane-ethanol mixture. Coefficient of variation (CV%) of quadruplicate or quintuplicate determined samples for coenzymes Q(9) and Q(10) was <10 by both EC detector and DAD. Responses of the detection systems were linear in the range evaluated, 10-200 ng/injection, and had correlation coefficients exceeding 0.999. Recoveries of added coenzymes Q(9) and Q(10) varied 73-105% for DAD and 74-103% for EC detector, respectively. Detection limits for coenzymes Q(9) and Q(10) using the DAD system were 4 and 6 ng/injection, respectively, and 0.2 and 0.3 ng/injection by EC detection. Results derived from the two detection systems were generally similar. However, although EC detector was 20-fold more sensitive, the selectivity was, in some cases, poorer than that of DAD.

The hydroubiquinone-cytochrome c2 oxidoreductase (cyt bc1) from Rhodobacter capsulatus has been solubilized according to the dodecyl maltoside method and isolated, and its minimal functional composition has been characterized. We find the complex to be composed of three protein subunits corresponding to polypeptides of cyt b (44 kDa), cyt c1 (33 kDa), and 2Fe2S cluster (24 kDa). A fourth band sometimes discernable at 22 kDa appears to be an artifact of the polyacrylamide gel electrophoresis procedure. Its appearance is shown to be derived from the 2Fe2S cluster subunit by the similarity of the binding of subunit-specific monoclonal antibodies and the identical N-terminal sequence of the 24- and 22-kDa bands. The cofactors of cyt bc1, namely, cyt bH, cyt bL, cyt c1, and the 2Fe2S center, the Qos and Qow domains of the Qo site, and the Qi site appear intact as indicated by their optical and EPR spectral signatures, redox properties, and inhibitor binding. The electron paramagnetic resonance spectrum of the cyt bH heme is altered by antimycin, consistent with a change in the dihedral angle between the ligating histidine imidazoles, while the spectrum of the cyt bL heme is broadened by stigmatellin. The ubiquinone-10 content is variable, ranging from 0.8 to 3 molecules/cyt bc1. Activity studies define this three-subunit cyt bc1 complex as a minimal structure, equipped as the enzyme in the native state and capable of full catalytic activity.

The photoreaction center from Rhodospirillum rubrum contains about 90% protein, 6% pigment, mere traces of lipids, and no cytochromes. It also contains at least 1 mol of ubiquinone and 1 iron atom per mol. Its three-component polypeptide chains were isolated by preparative electrophoresis, and their molar stoichiometry was established as 1:1:1. The amino acid composition of the photoreaction center from strain S1 and from its subunits is reported. The protein as a whole contains about 65% nonpolar residues, and the degree of hydrophobicity of its subunits is alpha less than beta less than gamma. The minimal molecular weight based on the extinction coefficient and on the amino acid content is 90 000. This corresponds to a half-cystine mole number of 6.

Highly purified preparations of the cholate-solubilized respiratory NADH dehydrogenase, isolated from genetically amplified Escherichia coli strains [Jaworowski, A., Campbell, H. D., Poulis, M. I., & Young, I. G. (1981) Biochemistry 20, 2041-2047], have been characterized. Enzyme preparations were shown to contain 70% (w/w) lipid, predominantly phosphatidylethanolamine. One mol of noncovalently bound FAD and approximately 1 mol of ubiquinone/mol of enzyme subunit were detected. The purified enzyme was shown to contain only low levels of Fe and acid-labile S, indicating the absence of iron-sulfur clusters. No Cu, Mo, W, or covalently bound P was detected, and no evidence for other chromophores was obtained from visible and ultraviolet absorption spectra of the purified enzyme or of the delipidated polypeptide prepared by gel filtration in sodium dodecyl sulfate. Protein chemical studies verified that the enzyme consists of a single polypeptide species of Mr 47 000, and the N- and C-terminal cyanogen bromide peptides were identified. The pure enzyme was shown to reconstitute membrane-bound, cyanide-sensitive NADH oxidase activity in membrane vesicles prepared from ndh mutant strains.