Dibutyltin-3-hydroxyflavone titrates a dissociable component (cofactor) of mitochondrial ATP synthase: An energy-transfer component linked to the ubiquinone pool

Dibutyltin-3-hydroxyflavone, Bu₂Sn(of), is a new fluorescence probe inhibitor of F₁F₀-ATPase and oxidative phosphorylation which inhibits by titration of an unidentified component of F₀. Its site of action is closely related to that of the trialkyltins and of venturicidin. This F₀ component is part of a pool of this component which is present in the heart mitochondrial inner membrane at levels of 5–7 nmol (mg protein)^?1 [18 ± 3 Bu₂Sn(of) sites per mol F₁F₀-ATPase]. However, ATPase activity in submitochondrial particles is near maximally inhibited by titration of approx. three Bu₂Sn(of) sites per mol F₁F₀-ATPase. Over 60% (60–80%) of the Bu₂Sn(of) interaction sites can be lost during the purification of F₁F₀-ATPase from submitochondrial particles. The number of Bu₂Sn(of) interaction sites in various F₁F₀-ATPase preparations is variable. The high numbers of Bu₂Sn(of) sites per mol F₁F₀-ATPase for heart mitochondria (18–21) and submitochondrial particles (15–19.5) decline in ATP synthase (11–15) to the low values obtained in Complex V (7–10.5) and the minimal values observed in highly purified F₁F₀?ATPase (3.5–5.6), thus indicating a variable dissociable component or cofactor of ATP synthase. The Bu₂Sn(of) interaction site, a component of ATP synthase, is responsive to the redox status of the respiratory chain and the interaction with Bu₂Sn(of) is with the reduced form of this component. Fluorescence titration studies show that this component is in redox equilibrium with the ubiquinone pool of the respiratory chain. It is proposed that this redox component serves as an inhibitor titratable cofactor pool which cycles through an F₀ interaction site (or sites) via a system which serves as an energy-transfer link between the respiratory chain and ATP synthase.     

ENDOR spectroscopic and molecular orbital study of the dynamical properties of the side chain in radical anions of ubiquinones Q-1, Q-2, Q-6, and Q-10

The dynamics of the side chain of the radical anions of ubiquinones Q-1 (2,3-dimethoxy-5-methyl-6-[3-methyl-2-butenyl]-1,4-benzoquinone), Q-2, Q-6, and Q-10 have been investigated using electron nuclear double-resonance (ENDOR) spectroscopy. When radicals are produced in the liquid phase, secondary radicals are also formed. The EPR spectra of these additional radicals overlap with the radical of interest. ENDOR spectroscopy was found to be capable for studying the dynamical properties of such conditions. The temperature dependence of the isotropic hyperfine coupling constants of the beta- and gamma-protons of the side chain was measured. The activation energy of the rotation and other dynamical properties of the side chain were calculated assuming that rotation can be modeled by the classical two-jump model. The rotation energy barrier for Q-1 was also determined by the hybrid Hartree-Fock/density functional method UB3LYP with the 6-31G(d) basis set. Calculated results were in good agreement with the experimental results. Despite the numerous parameters affecting the ENDOR linewidth ENDOR spectroscopy was shown to be a potential method for studying the dynamical properties of the mixtures of the radicals. Prominent forbidden transitions appear in the ENDOR spectra when alkali ions are present in the sample. From these transitions measured ENDOR-induced EPR spectra showed an additional doublet and phase transition in electron Zeeman frequency.     

Formation of Temporary Negative Ions and Their Subsequent Fragmentation upon Electron Attachment to CoQ0 and CoQ0H2

Ubiquinone molecules have a high biological relevance due to their action as electron carriers in the mitochondrial electron transport chain. Here, we studied the dissociative interaction of free electrons with CoQ₀, the smallest ubiquinone derivative with no isoprenyl units, and its fully reduced form, 2,3-dimethoxy-5-methylhydroquinone (CoQ₀H₂), an ubiquinol derivative. The anionic products produced upon dissociative electron attachment (DEA) were detected by quadrupole mass spectrometry and studied theoretically through quantum chemical and electron scattering calculations. Despite the structural similarity of the two studied molecules, remarkably only a few DEA reactions are present for both compounds, such as abstraction of a neutral hydrogen atom or the release of a negatively charged methyl group. While the loss of a neutral methyl group represents the most abundant reaction observed in DEA to CoQ₀, this pathway is not observed for CoQ₀H₂. Instead, the loss of a neutral OH radical from the CoQ₀H₂ temporary negative ion is observed as the most abundant reaction channel. Overall, this study gives insights into electron attachment properties of simple derivatives of more complex molecules found in biochemical pathways.     

Voltammetric Probing of Molecular Assemblies of Ubiquinone-10 at the Air–Water Interfaces

The redox properties of ubiquinone 10 (UQ10) placed at the air–water interface were studied using the horizontal touching method with a thin mercury film (TMFE) working electrode and cyclic voltammetry. Changes of pH of the subphase affected the formal potential of the ubiquinone/ubiquinol system exhibiting the participation of protons in the overall reduction of UQ10. The protonation of the semiquinone transition product was found to be the rate determining step. This explains the dependence of the rate constant value on pH. The highest values of rate constants were found at pH over 13. Under these conditions the mechanism of the process is different. The concentration of protons is small, and the availability of the counter ions (i.e., K⁺) becomes crucial for the kinetics of reduction. Their role is to neutralize the negative charge of the redox group following its reduction. The logarithm of rate constants was found to decrease linearly with the increase of surface concentration of ubiquinone. This reflects the influence of intermolecular interactions in the monolayer on the kinetics of the electrode process.     

Validation and application of an HPLC-EC method for analysis of coenzyme Q10 in blood platelets

Previous studies have indicated that analysis of coenzyme Q10 (CoQ10) in platelets may be clinically useful. The study objectives are to describe, validate and provide application of an HPLC-EC method for platelet CoQ10 analysis. This method analyzes oxidized (ubiquinone-10) and reduced (ubiquinol-10) forms of CoQ10 using two separate injections with the electrochemical analytical cell set at neutral and oxidizing potentials. Results showed that chromatograms were free of interfering peaks. Calibration curves were constructed over a concentration range 116-2317 nmol/L (r(2) = 0.99). The extraction recovery was >95%. The within-run precision CV% was < or =4.2%, and the day-to-day precision was < or =9.9%. Platelets were isolated by differential centrifugation, and frozen at -70 degrees C until analysis. The application of the method was used to compare accumulation of CoQ10 in platelets vs plasma in eight adult volunteers during a 28 day supplementation period (5 mg/kg/day of ubiquinol-10). Mean platelet total CoQ10 was 164 pmol/10(9) cells, and ubiquinol-10:total CoQ10 ratio was 0.56. During supplementation platelet CoQ10 levels were more consistent and predictable than plasma CoQ10 levels. The results indicate that this validated method for platelet ubiquinol-10 and ubiquinone-10 analysis is acceptable for use in the clinical laboratory, and that platelet CoQ10 may have important advantages over plasma during CoQ10 supplementation.     

Supramolecular nanostructures based on bacterial reaction center proteins and quantum dots

Design of the nanostructures based on membrane proteins (the key functional elements of biomembranes) and colloid nanoparticles is a fascinating field at the interface of biochemistry and colloids, nanotechnology and biomedicine. The review discusses the main achievements in the field of ultrathin films prepared from bacterial reaction center proteins and light-harvesting complexes, as well as these complexes tagged with quantum dots. The principles of preparation of these thin films and their structure and properties at different interfaces are described; as well as their characteristics estimated using a combination of the modern interfacial techniques (absorption and fluorescence spectroscopy, atomic force and Brewster angle microscopy, etc.) are discussed. Further approaches to develop the nanostructures based on the membrane proteins and quantum dots are suggested. These supramolecular nanostructures are promising prototypes of the materials for photovoltaic, optoelectronic and biosensing applications.     

Ubiquinone Q9 from a marine isolate of an actinobacterium Nocardia sp.

The major component of a nonpolar fraction of an extract of an actinobacterium Nocardia sp. KMM 3749 isolated from an unidentified marine ascidian was shown, using NMR spectroscopy and mass spectrometry, to be 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone (ubiquinone Q₉).     

Comparison of calculated and experimental FTIR spectra of specifically labeled ubiquinones

Density functional theory has been used to calculate harmonic vibrational frequencies and intensities of unlabeled and labeled ubiquinone-1 in both the gas phase and in solution (CCl₄). Calculations were undertaken using the B3LYP functional and the 6-31G+(d) basis set. Calculations using larger basis sets did not significantly alter the calculated spectra. Calculations for ubiquinone-2, -3, -6 or -8 gave similar results.Calculations show that eight ubiquinone-1 conformations, which differ only in the orientation of their methoxy groups, are likely to coexist in solution at room temperature. The calculated infrared spectra for these eight conformations vary considerably. However, by averaging the Boltzmann weighted spectra for the eight conformers, composite spectra are calculated that successfully model both the frequency and intensity information inherent in the experimental FTIR spectra obtained for both unlabeled and labeled ubiquinones in solution.The calculated spectra presented here provide a robust foundation from which to consider and assess future modeling of the vibrational properties of ubiquinones embedded in protein complexes.     

A Comparative Study on the Reduction Modes for Quinone to Determine Ubiquinone by HPLC with Luminol Chemiluminescence Detection Based on the Redox Reaction

Ubiquinone (UQ) is considered one of the important biologically active molecules in the human body. Ubiquinone determination in human plasma is important for the investigation of its bioavailability, and also its plasma level is considered an indicator of many illnesses. We have previously developed sensitive and selective chemiluminescence (CL) method for the determination of UQ in human plasma based on its redox cycle with dithiothreitol (DTT) and luminol. However, this method requires an additional pump to deliver DTT as a post-column reagent and has the problems of high DTT consumption and broadening of the UQ peak due to online mixing with DTT. Herein, an HPLC (high-performance liquid chromatography) system equipped with two types of online reduction systems (electrolytic flow cell or platinum catalyst-packed reduction column) that play the role of DTT was constructed to reduce reagent consumption and simplify the system. The newly proposed two methods were carefully optimized and validated, and the analytical performance for UQ determination was compared with that of the conventional DTT method. Among the tested systems, the electrolytic reduction system showed ten times higher sensitivity than the DTT method, with a limit of detection of 3.1 nM. In addition, it showed a better chromatographic performance and the best peak shape with a number of theoretical plates exceeding 6500. Consequently, it was applied to the determination of UQ in healthy human plasma, and it showed good recovery (≥97.9%) and reliable precision (≤6.8%) without any interference from plasma components.