Marina Verkhovskaya | University of Helsinki (original) (raw)

Papers by Marina Verkhovskaya

$Research paper thumbnail of The isolation of plasma membranes from a cell wall fraction and some characteristics of the ATPase activity of the membranes$

Fiziologiya Rastenii, Oct 11, 1980

Proceedings of the National Academy of Sciences of the United States of America, Oct 10, 2011

Biochemistry, Dec 15, 2006

The conserved arginine 274 and histidine 224 and 228 residues in subunit NuoCD of complex I from ... more The conserved arginine 274 and histidine 224 and 228 residues in subunit NuoCD of complex I from Escherichia coli were substituted for alanine. The wild-type and mutated NuoCD subunit was expressed on a plasmid in an E. coli strain bearing a nuoCD deletion. Complex I was fully expressed in the H224A and H228A mutants, whereas the R274A mutation yielded approximately 50% expression. Ubiquinone reductase activity of complex I was studied in membranes and with purified enzyme and was 50% and 30% of the wild-type activity in the H224A and H228A mutants, respectively. The activity of R274A was less than 5% of the wild type in membranes but 20% in purified complex I. Rolliniastatin inhibited quinone reductase activity in the mutants with similar affinity as in the wild type, indicating that the quinone-binding site was not significantly altered by the mutations. Ubiquinone-dependent superoxide production by complex I was similar to the wild type in the R274A mutant but slightly higher in the H224A and H228A mutants. The EPR spectra of purified complex I from the H224A and H228A mutants did not differ from the wild type. In contrast, the signals of the N2 cluster and another fastrelaxing [4Fe-4S] cluster, tentatively assigned as N6b, were drastically decreased in the NADH-reduced R274A mutant enzyme but reappeared on further reduction with dithionite. These findings show that the redox potential of the N2 and N6b centers is shifted to more negative values by the R274A mutation. Purified complex I was reconstituted into liposomes, and electric potential was generated across the membrane upon NADH addition in all three mutant enzymes, suggesting that none of the mutations directly affect the proton-pumping machinery.

Biochemistry, May 11, 1999

The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper ... more The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper oxidases which catalyze the reduction of O2 to water linked to translocation of protons across the bacterial or mitochondrial membrane. We have studied the structure of the CuB site in the binuclear heme-copper center of O2 reduction by EXAFS spectroscopy in the fully reduced state of this enzyme, as well as in the reduced CO-liganded states where CO is bound either to the heme iron or to CuB. We find that, in the reduced enzyme, CuB is coordinated by one weakly bound and two strongly bound histidine imidazoles at Cu-N distances of 2.10 and 1.92 A, respectively, and that an additional feature at 2.54 A is due to a highly ordered water molecule that might be weakly associated with the copper. Unexpectedly, the binding of CO to heme iron is found to result in a major conformational change at CuB, which now binds only two equidistant histidine imidazoles at 1.95 A and a chloride ion at 2. 25 A, with elimination of the water molecule and one of the histidines. Attempts to remove the chloride from the enzyme by extensive dialysis did not change this finding, nor did substitution of chloride with bromide. Photolysis of CO bound to the heme iron is known to cause the CO to bind to CuB in a very fast reaction and to remain bound to CuB at low temperatures. In this state, we indeed find the CO to be bound to CuB at a Cu-C distance of 1.85 A, with chloride still bound at 2.25 A and the two histidine imidazoles at a Cu-N distance of 2.01 A. These results suggest that reduction of the binuclear site weakens the bond between CuB and one of its three histidine imidazole ligands, and that binding of CO to the reduced binuclear site causes a major structural change in CuB in which one histidine ligand is lost and replaced by a chloride ion. Whether chloride is a cofactor in this enzyme is discussed.

Biochemistry, Nov 10, 1999

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical ... more The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.

FEBS Letters, Apr 17, 1995

Downhill sodium efllux from right-side-out E. coli membrane vesicles was found to be stimulated b... more Downhill sodium efllux from right-side-out E. coli membrane vesicles was found to be stimulated by negative electric potential, as has been reported earlier [Bassilana et al., Biochemistry 23 (1984) 1015-10221, and in agreement with the concept of electrogenic Na+/nH+ antiporters with n > 1. However, sodium efllux was much more accelerated by positive electric potential, indicating the operation of another sodium transport system. ApH (alkaline inside), created by a pH shift from 8.5 to 6.8 in the medium was found to drive sodium efllux against its concentration gradient, but only when the vesicles had been loaded with boil Na+ and K+. Efllux of K+ against the concentration gradient was also observed under these conditions. When the vesicles were loaded separately with sodium tricine or potassium tricine, no K' efllux and insignificant Na+ efflux were observed. We propose that there are at least two different mechanisms responsible for Na' efilux in E. coli vesicles. One is the Na+/nH+ antiporter previously described, and the other is a novel Na+,K+/mH+ antiporter.

Biochimica Et Biophysica Acta - Bioenergetics, Dec 1, 2014

Reduction of Complex I (NADH:ubiquinone oxidoreductase I) from Escherichia coli by NADH was inves... more Reduction of Complex I (NADH:ubiquinone oxidoreductase I) from Escherichia coli by NADH was investigated optically by means of an ultrafast stopped-flow approach. A locally designed microfluidic stopped-flow apparatus with a low volume (0.2 μl) but a long optical path (10 mm) cuvette allowed measurements in the time range from 270 μs to seconds. The data acquisition system collected spectra in the visible range every 50 μs. Analysis of the obtained time-resolved spectral changes upon the reaction of Complex I with NADH revealed three kinetic components with characteristic times of b 270 μs, 0.45-0.9 ms and 3-6 ms, reflecting reduction of different FeS clusters and FMN. The rate of the major (τ = 0.45-0.9 ms) component was slower than predicted by electron transfer theory for the reduction of all FeS clusters in the intraprotein redox chain. This delay of the reaction was explained by retention of NAD + in the catalytic site. The fast optical changes in the time range of 0.27-1.5 ms were not altered significantly in the presence of 10-fold excess of NAD + over NADH. The data obtained on the NuoF E95Q variant of Complex I shows that the single amino acid replacement in the catalytic site caused a strong decrease of NADH binding and/or the hydride transfer from bound NADH to FMN.

Elsevier eBooks, 2009

The investigation of the molecular mechanism of the respiratory chain complexes requires determin... more The investigation of the molecular mechanism of the respiratory chain complexes requires determination of the time-dependent evolution of the catalytic cycle intermediates. The ultra-fast freeze-quench approach makes possible trapping such intermediates with consequent analysis of their chemical structure by means of different physical spectroscopic methods (e.g., EPR, optic, and Mössbauer spectroscopies). This chapter presents the description of a setup that allows stopping the enzymatic reaction in the time range from 100 microsec to tens of msec. The construction and production technology of the mixer head, ultra-fast freezing device, and accessories required for collecting a sample are described. Ways of solving a number of problems emerging on freezing of the reaction mixture and preparing the samples for EPR spectroscopy are proposed. The kinetics of electron transfer reaction in the first enzyme of the respiratory chain, Complex I (NADH: ubiquinone oxidoreductase), is presented as an illustration of the freeze-quench approach. Time-resolved EPR spectra indicating the redox state of FeS clusters of the wild-type and mutant (R274A in subunit NuoCD) Complex I from Escherichia coli are shown.

Biochimica Et Biophysica Acta - Bioenergetics, Mar 1, 1996

Respiration-driven Na + transport from Escherichia coli cells and right-side-out membrane vesicle... more Respiration-driven Na + transport from Escherichia coli cells and right-side-out membrane vesicles is strictly dependent on K +. Cells from an E. coli mutant deficient in three major K + transport systems were incapable of accumulating K + or expelling Na + unless valinomycin was added. Membrane vesicles from an E. coli mutant from which the genes encoding the two known electrogenic Na+/nH + antiporters nhaA and nhaB were deleted transported Na + as well as did vesicles from wild-type cells. Quantitative analysis of A~b and ApH showed a high driving force for electrogenic Na+/nH + antiport whether K + was present or not, although Na + transport occurred only in its presence. These results suggest that an Na+/nH + antiporter is not responsible for the Na + transport. Respiration-driven effiux of Na + from vesicles was found to be accompanied by primary uphill effiux of K +. Also, no respiration-dependent effiux of K + was observed in the absence of Na +. Such coupling between Na + and K + fluxes may be explained by the operation of an Na +, K+/H + antiporter previously described in E. coli membrane vesicles (Verkhovskaya, M.L., Verkhovsky, M.I. and WikstrSm, M. (1995) FEBS Lett. 363, 46-48). Active Na + transport is abolished when A~H~ is eliminated by a protonophore, but at low concentrations the protonophore actually accelerated Na + transport. Such an effect may be expected if the Na +, K+/H + antiporter normally operates in tight conjunction with respiratory chain complexes, thus exhibiting some phenomenological properties of a primary redox-linked sodium pump.

Biochimica Et Biophysica Acta - Bioenergetics, 2009

Replacement of glutamate 95 for glutamine in the NADH-and FMN-binding NuoF subunit of E. coli Com... more Replacement of glutamate 95 for glutamine in the NADH-and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5-4.8 times depending on the used electron acceptor. The apparent K m for NADH was 5.2 and 10.4 μM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD + on activity showed that the E95Q mutation caused a 2.4-fold decrease of K i NAD+ in comparison to the wild type enzyme. ADP-ribose, which differs from NAD + by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K i ADP-ribose relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD +. The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from −350 mV to −310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.

European journal of biochemistry, Aug 1, 1989

An alkalo-and halo-tolerant aerobic microorganism has been isolated which, according to microbiol... more An alkalo-and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5s RNA sequence, is a Bacillus similar, but not identical, to B. lichenijormis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na' concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N,N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a Ay which, in the Na' medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K' medium, CCCP discharged Ay even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na' medium and sensitive to CCCP in the K + medium as if Na'-and H+-coupled oxidative phosphorylations were operative in the Na' and K + media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K' increased the Na' uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H'-motive but also Na+-motive terminal oxidase activity.

Proceedings of the National Academy of Sciences of the United States of America, Sep 1, 2015

Proceedings of the National Academy of Sciences of the United States of America, Mar 11, 2008

Fig. 1. Redox centers of complex I. The figure is based on the structure of complex I from Thermu... more Fig. 1. Redox centers of complex I. The figure is based on the structure of complex I from Thermus thermophilus (5) and drawn using the program VMD (27). Edge-to-edge distances between the redox centers are shown in ångströ ms (5), except for the distance between cluster N2 and bound ubiquinone (Q), which is center-to-center (16).

Nature, Jul 1, 1999

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which re... more Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane. The enzyme&amp;amp;amp;#39;s catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.

Scientific Reports, Feb 1, 2016

Discovery of the light-driven sodium-motive pump Na +-rhodopsin (NaR) has initiated studies of th... more Discovery of the light-driven sodium-motive pump Na +-rhodopsin (NaR) has initiated studies of the molecular mechanism of this novel membrane-linked energy transducer. In this paper, we investigated the photocycle of NaR from the marine flavobacterium Dokdonia sp. PRO95 and identified electrogenic and Na +-dependent steps of this cycle. We found that the NaR photocycle is composed of at least four steps: NaR 519 + hv → K 585 → (L 450 ↔M 495) → O 585 → NaR 519. The third step is the only step that depends on the Na + concentration inside right-side-out NaR-containing proteoliposomes, indicating that this step is coupled with Na + binding to NaR. For steps 2, 3, and 4, the values of the rate constants are 4×10 4 s-1 , 4.7 × 10 3 M-1 s-1 , and 150 s-1 , respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Δψ ~ 200 mV) generated by a single turnover of NaR incorporated into liposomes and attached to phospholipid-impregnated collodion film. On the basis of these observations, a mechanism of light-driven Na + pumping by NaR is suggested.

FEBS Letters, May 20, 2016

Respiratory Complex I from Escherichia coli may exist in two states, resting (R) and active (A). ... more Respiratory Complex I from Escherichia coli may exist in two states, resting (R) and active (A). The conversion from the R- to A-forms occurs spontaneously upon turnover. The fast resting-to-active (R/A) transition of membrane-bound and purified Complex I was studied with the stopped-flow technique by following NADH oxidation either by absorption decay at 340 nm or using the fluorescent pH indicator, trisodium 8-hydroxypyrene-1,3,6-trisulfonate (pyranine). The R/A transition of Complex I from E. coli occurs upon its turnover in a time interval of ~ 1.5 s. Comparisons between the bacterial Complex I R/A transition and the active/deactive transition of mitochondrial Complex I are discussed.

Heliyon, 2017

Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active... more Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein: dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.

Molecular Microbiology, Nov 7, 2011

The C-terminus of the NuoL subunit of Complex I includes a long amphipathic a-helix positioned pa... more The C-terminus of the NuoL subunit of Complex I includes a long amphipathic a-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C-and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic a-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic a-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long a-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed.

Biochimica Et Biophysica Acta - Bioenergetics, Aug 1, 1994

The oxygen-reactive haem in the binuclear site is given the subscript '3' irrespective of haem st... more The oxygen-reactive haem in the binuclear site is given the subscript '3' irrespective of haem structure in honour of Dr. David Keilin who discovered and named the oxygen-reactive cytochrome a 3. Copper of the binuclear site has the subscript 'B' in honour of Drs. Helmut Beinert and Bob van Gelder, who discovered its functionality and closeness to haem iron.

Nature, Dec 1, 1999

especially as the fully reduced enzyme is out of electrostatic balance 7. The partial reversibili... more especially as the fully reduced enzyme is out of electrostatic balance 7. The partial reversibility of the catalytic cycle by an imposed electric field led to the postulate that the transitions between the 'peroxy' (P) and 'oxoferryl' (F) intermediates, and between the F and 'oxidized' (O) intermediates, are coupled to the pumping of two protons each 2. O~ is now 4 postulated to be the next intermediate after F and to have the energy to pump two protons, but to decay to the de-energized O. With the existence of O~, a reversal of O to F and then to P with equal energy inputs for pumping two protons each is impossible, as the reversal of O to F would now require the input of sufficient energy to translocate four charges. This is in contrast to the experiment represented in Fig. 3 of ref. 2, which shows that 2.2 charges are transported for the reversal from O to F. Starting from fully reduced cytochrome c oxidase, a catalytic cycle in which only three protons are pumped (two during oxidation and one during re-reduction) 7 fits the data of Fig. 3 in ref. 4 as well as a twoplus-two cycle, fits the data of Fig. 2 in ref. 4 better than a two-plus-two cycle, and avoids any need for the energy-rich state O~.