Alexander B. Kotlyar

Slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase

NADH-ubiquinone reductase of bovine heart submitochondrial particles as prepared is unable to catalyze either the direct or reverse electron transfer from NADH to ubiquinone. The deactivated state of the enzyme in coupled particles was revealed as: (i) the absence of the rotenone-sensitive, delta mu H(+)-dependent succinate-ferricyanide reductase activity; (ii) a prominent lag in the aerobic succinate-supported, delta mu H(+)-dependent NAD+ reduction; and (iii) a lag in the rotenone-sensitive NADH-ubiquinone reductase or NADH oxidase activities. Being inactive as NADH-ubiquinone reductase (direct or reverse), the enzyme is fully active as rotenone-insensitive NADH-ferricyanide reductase. The enzyme can be activated by preincubation with substrates (NADH or NADPH) only under the conditions where the turnover of the NADH-ubiquinone reductase reaction (but not in the NADH-ferricyanide reductase) occurs. Partial activation of the enzyme was observed when the particles were preincubated with rotenone. Neither NADH under the conditions when the ubiquinone pool was reduced nor succinate plus delta mu H+ or dithionite were able to activate the enzyme. Once activated, the enzyme remains in the active state for quite a long time (more than 5 h at 0 degree C). The deactivation rate is extremely temperature-dependent, being insensitive to NAD+, ferricyanide or succinate. A comparison of the enzyme activation/deactivation kinetics showed that the same mechanism is involved in the slow activation of the direct and reverse electron transfer from NADH to ubiquinone. Activated particles catalyze the aerobic delta mu H(+)-dependent succinate-supported reverse electron transfer in the absence of ATP at a rate comparable with that of NADH-ubiquinone reductase.

Long-range charge transport in single G-quadruplex DNA molecules

DNA and DNA-based polymers are of interest in molecular electronics because of their versatile and programmable structures. However, transport measurements have produced a range of seemingly contradictory results due to differences in the measured molecules and experimental set-ups, and transporting significant current through individual DNA-based molecules remains a considerable challenge. Here, we report reproducible charge transport in guanine-quadruplex (G4) DNA molecules adsorbed on a mica substrate. Currents ranging from tens of picoamperes to more than 100 pA were measured in the G4-DNA over distances ranging from tens of nanometres to more than 100 nm. Our experimental results, combined with theoretical modelling, suggest that transport occurs via a thermally activated long-range hopping between multi-tetrad segments of DNA. These results could re-ignite interest in DNA-based wires and devices, and in the use of such systems in the development of programmable circuits.

Specific High-Affinity Binding of Thiazole Orange to Triplex and G-Quadruplex DNA

Interaction of Thiazole Orange (TO) with double-, triple-, and quadruple-stranded forms of DNA was studied. We have demonstrated by UV-vis absorption, circular dichroism (CD), and fluorescence spectroscopy that TO binds with much higher affinity to triplex and G-quadruplex DNA structures compared to double-stranded (ds) DNA. Complexes of the dye with DNA triplexes and G-quadruplexes are very stable and do not dissociate during chromatography and gel electrophoresis. TO binding to either triple- or quadruple-stranded DNA structures results in a >1000-fold increase in dye fluorescence. The fluorescence titration data showed that TO to triad and tetrad ratios, in tight complexes with the triplex and the G-quadruplex, are equal to 0.5 and 1, respectively. Preferential binding of TO to triplexes and G-quadruplexes enables selective detection of only these DNA forms in gels in the absence of free TO in electrophoresis running buffer. We have also demonstrated that incubation of U2OS cells with submicromolar concentrations of TO results in preferential staining of certain areas in the nucleus in contrast to DAPI which binds to dsDNA and efficiently stains regions that are unstained with TO. We suggest that TO staining may be useful for the detection of noncanonical structural motifs in genomic DNA.

Coupling site I and the rotenone-sensitive ubisemiquinone in tightly coupled submitochondrial particles

The rotenone‐sensitive g = 2.00 low temperature EPR signal attributed to ubisemiquinone is observed in submitochondrial particles during coupled electron transfer from NADH to oxygen and from succinate to NAD+. The signal is seen only in the presence of oligomycin added to induce the respiratory control (7–9 with NADH and 3–4 with succinate) and it disappears in the presence of uncouplers (CCCP or gramicidin D). No reduction of the iron‐sulfur center N‐2 in the presence of 20 mM succinate and cyanide is observed, thus suggesting that N‐2 is not in equilibrium with the ubiquinone pool. A hypothesis is proposed on 7Delta;smH+ generation coupled with electron transfer between iron‐sulfur center N‐2 and the ubiquinone pool.

Ubisemiquinone in the NADH-ubiquinone reductase region of the mitochondrial respiratory chain

Coupled bovine heart submitochondrial particles exhibit a rotenone‐sensitive g = 2.00 low‐temperature EPR signal attributable to ubisemiquinone which is observed during steady‐state electron transfer from NADH to oxygen or from succinate to NAD+ in Δµ̃H+‐dependent reverse electron transfer. Quantitation of the signal under optimal conditions gives a value for maximal semiquinone of approx. 0.5 spins per spin of the fully reduced NADH dehydrogenase iron‐sulfur center N‐2. The intensity of the signal is drastically reduced when electron transfer from NADH to oxygen is blocked by cyanide or in the case of the reverse electron transfer from succinate to NAD+ being prevented by anaerobiosis. Only those particles which contain ‘turnover‐preconditioned” NADH‐ubiquinone reductase demonstrate the ubisemiquinone signal together with N‐1, N‐2, N‐3 and N‐4 iron‐sulfur centers. The spin relaxation characteristics of the rotenone‐sensitive ubisemiquinone signal point to its interaction with one of the rapidly relaxing (4Fe‐4S) centers, most probably N‐2.

Active/de-active transition of respiratory complex I in bacteria, fungi, and animals

Mammalian complex I (NADH:ubiquinone oxidoreductase) exists as a mixture of interconvertible active (A) and de-activated (D) forms. The A-form is capable of NADH:quinone-reductase catalysis, but not the D-form. Complex I from the bacterium Paracoccus denitrificans, by contrast, exists only in the A-form. This bacterial complex contains 32 fewer subunits than the mammalian complex. The question arises therefore if the structural complexity of complex I from higher organisms correlates with its ability to undergo the A/D transition. In the present study, it was found that complex I from the bacterium Escherichia coli and from non-vertebrate organisms (earthworm, lobster, and cricket) did not show the A/D transitions. Vertebrate organisms (carp, frog, chicken), however, underwent similar A/D transitions to those of the well-characterized bovine complex I. Further studies showed that complex I from the lower eukaryotes, Neurospora crassa and Yarrowia lipolytica, exhibited very distinct A/D transitions with much lower activation barriers compared to the bovine enzyme. The A/D transitions of complex I as they relate to structure and regulation of enzymatic activity are discussed.

Effect of Ca2+ ions on the slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase.

Slow active/inactive transition of the membrane-bound mitochondrial NADH-ubiquinone reductase (Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158) is sensitive to Ca2+ and other divalent cations. Millimolar concentrations of Ca2+ drastically reduce the rate of the turnover-dependent activation of NADH-ubiquinone reductase. When NADH oxidase, the rotenone-sensitive NADH-ubiquinone reductase or the succinate-supported delta mu H+-dependent NAD+ reduction were initiated by the deactivated enzyme preparations all the three activities were strongly inhibited by Ca2+; no sensitivity of these reactions to Ca2+ was observed when the assays were started by the activated enzyme preparations. The affinity of the deactivated enzyme to polyvalent cations was in the following order: Ni2+ greater than Co2+ greater than La3+ greater than Mn2+ greater than Ca2+ approximately Mg2+ greater than Ba2+. Monovalent metal cations had no effect on the slow turnover-dependent enzyme activation. The apparent affinity of the deactivated enzyme to Ca2+ was strongly pH-dependent. The KCa2+ values of 5.7 mM and 0.6 mM at pH 7.5 and 8.5 were determined from the presteady-state kinetics parameters. The spontaneous temperature-dependent deactivation of the enzyme was insensitive to Ca2+. Ca2+ increases the reactivity of the enzyme sulfhydryl group in the deactivated preparations towards N-ethylmaleimide. This effect was also used to quantitate Ca2+ affinity for the enzyme. The KCa2+ values of 1.2 mM and 0.4 mM at pH 8.0 and 9.0, respectively, were determined. The data obtained suggest that Ca2+ content in the mitochondrial matrix may play an important role in the control of NADH oxidation by the respiratory chain.

Interaction of the membrane-bound succinate dehydrogenase with substrate and competitive inhibitors

The protective effect of dicarboxylates on the active-site-directed inhibition of the membrane-bound succinate dehydrogenase by N-ethylmaleimide, steady-state kinetics methods for Ki and Ks determinations, and equilibrium studies were employed to quantitate the relative affinities of succinate, fumarate, malonate and oxaloacetate to the reduced and oxidized species of the enzyme. A more than 10-fold difference in the relative affinities of the reduced and oxidized succinate dehydrogenase to succinate, fumarate and oxaloacetate is found, whereas the reactivity of the active-site sulphydryl group does not depend on the redox state of the enzyme. The redox-state-dependent changes in the affinity of the membrane-bound succinate dehydrogenase to oxaloacetate can be quantitatively accounted for by a 10-fold increase in the rate of dissociation of the enzyme-inhibitor complex which occurs upon reduction of the enzyme. The data obtained give no support for either the existence of a sulphydryl group other than the active-site one important for the catalysis or for the presence of a separate dicarboxylate-specific regulatory site in the succinate dehydrogenase molecule.

Biomaterial engineered electrodes for bioelectronics

A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode. With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed. This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface. Biasing of the electrode at a negative potential (-0.3 V vs. SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface. Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps. While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1. The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c. The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant. At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed. Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water. Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.

In vitro synthesis of uniform poly(dG)–poly(dC) by Klenow exo− fragment of polymerase I

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo− fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.