Biomedical subjects

Vladimir P Shinkarev

Publications and source records attributed to Vladimir P Shinkarev.

9 recordsLinked to original sources

PubMed2006

In situ kinetics of cytochromes c1 and c2.

In Rhodobacter sphaeroides chromatophores, cytochromes (cyt) c(1) and c(2) have closely overlapping spectra, and their spectral deconvolution provides a challenging task. As a result, analyses of the kinetics of different cytochrome components of the bc(1) complex in purple bacteria usually report only the sum cyt c(1) + cyt c(2) kinetics. Here we used newly determined difference spectra of individual components to resolve the kinetics of cyt c(1) and c(2) in situ via a least-squares (LS) deconvolution. We found that the kinetics of cyt c(1) and c(2) are significantly different from those measured using the traditional difference wavelength (DW) approach, based on the difference in the absorbance at two different wavelengths specific for each component. In particular, with the wavelength pairs previously recommended, differences in instrumental calibration led to kinetics of flash-induced cyt c(1) oxidation measured with the DW method which were faster than those determined by the LS method (half-time of approximately 120 micros vs half-time of approximately 235 micros, in the presence of antimycin). In addition, the LS approach revealed a delay of approximately 50 micros in the kinetics of cyt c(1) oxidation, which was masked when the DW approach was used. We attribute this delay to all processes leading to the oxidation of cyt c(1) after light activation of the photosynthetic reaction center, especially the dissociation of cyt c(2) from the reaction center. We also found that kinetics of both cyt c(1) and c(2) measured by the DW approach were significantly distorted at times longer than 1 ms, due to spectral contamination from changes in the b hemes. The successful spectral deconvolution of cyt c(1) and c(2), and inclusion of both cytochromes in the kinetic analysis, significantly increase the data available for mechanistic understanding of bc(1) turnover in situ.

Antimycin A↗

PubMed2006

Spectral and kinetic resolution of the bc1 complex components in situ: a simple and robust alternative to the traditional difference wavelength approach.

The kinetics of the cytochrome (cyt) components of the bc(1) complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The "traditional" set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c(tot) (cyt c(1)+cyt c(2)), cyt b(L), cyt b(H), and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c(1) and c(2) is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c(1), c(2), b(L), and b(H)) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc(1) complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc(1) complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.

Electron Transport Complex III↗

PubMed2005

Spectral analysis of the bc(1) complex components in situ: beyond the traditional difference approach.

The cytochrome (cyt) bc(1) complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc(1) complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc(1) complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc(1) turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b(L). From LS analysis of the chromophoric components (RC, c(tot), b(H) and b(L)), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc(1) complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.

Bacterial Chromatophores↗

PubMed2004

Flash-induced oxygen evolution in photosynthesis: simple solution for the extended S-state model that includes misses, double-hits, inactivation, and backward-transitions.

Flash-induced oxygen evolution in higher plants, algae, and cyanobacteria exhibits damped period-four oscillations. To explain such oscillations, Kok suggested a simple phenomenological S-state model, in which damping is due to empirical misses and double-hits. Here we developed an analytical solution for the extended Kok model that includes misses, double-hits, inactivation, and backward-transitions. The solution of the classic Kok model (with misses and double-hits only) can be obtained as a particular case of this solution. Simple equations describing the flash-number dependence of individual S-states and oxygen evolution in both cases are almost identical and, therefore, the classic Kok model does not have a significant advantage in its simplicity over the extended version considered in this article. Developed equations significantly simplify the fitting of experimental data via standard nonlinear regression analysis and make unnecessary the use of many previously developed methods for finding parameters of the model. The extended Kok model considered here can provide additional insight into the effect of dark relaxation between flashes and inactivation.

Algorithms↗

PubMed2003

The modified Q-cycle explains the apparent mismatch between the kinetics of reduction of cytochromes c1 and bH in the bc1 complex.

Crystallographic structures of the bc1 complex from different sources have provided evidence that a movement of the Rieske iron-sulfur protein (ISP) extrinsic domain is essential for catalysis. This dynamic feature has opened up the question of what limits electron transfer, and several authors have suggested that movement of the ISP head, or gating of such movement, is rate-limiting. Measurements of the kinetics of cytochromes and of the electrochromic shift of carotenoids, following flash activation through the reaction center in chromatophore membranes from Rhodobacter sphaeroides, have allowed us to demonstrate that: (i) ubiquinol oxidation at the Qo-site of the bc1 complex has the same rate in the absence or presence of antimycin bound at the Qi-site, and is the reaction limiting turnover. (ii) Activation energies for transient processes to which movement of the ISP must contribute are much lower than that of the rate-limiting step. (iii) Comparison of experimental data with a simple mathematical model demonstrates that the kinetics of reduction of cytochromes c1 and bH are fully explained by the modified Q-cycle. (iv) All rates for processes associated with movement of the ISP are more rapid by at least an order of magnitude than the rate of ubiquinol oxidation. (v) Movement of the ISP head does not introduce a significant delay in reduction of the high potential chain by quinol, and it is not necessary to invoke such a delay to explain the kinetic disparity between the kinetics of reduction of cytochromes c1 and bH.

Antifungal Agents↗

PubMed2003

Oxygen evolution in photosynthesis: simple analytical solution for the Kok model.

The light-induced oxidation of water by Photosystem II (PS II) of higher plants, algae, and cyanobacteria, is the main source of atmospheric oxygen. The discovery of the flash-induced period four oscillations in the oxygen evolution made by Pierre Joliot in 1969 has a lasting impact on current photosynthesis research. Bessel Kok explained such oscillations by introducing the cycle of flash-induced transitions of states (S-states) of an oxygen-evolving complex governed by the values of miss and double hit. Although this Kok model has been successfully used over 30 years for interpretation of experimental data in photosynthesis, until now there has been no simple analytical solution for it. Such an analytical solution for individual S-states and for oxygen evolution is presented here. When only the S(1) state is present before flash series, and when both the miss and double hit are zero, the oxygen evolved by the PSII after the n(th) flash, Y(n), is given by the following equation: 4Y(n)=1 + (-1)(n-1)-2 cos((n-1)pi/2). It is found here that binary oscillations of the secondary acceptor semiquinone at the acceptor side of the reaction center of PS II and release of reducing equivalents from reaction center to b(6)f complex can also be determined in the framework of the Kok model. The simple solutions found here for individual S-states, semiquinone, and oxygen evolution provide a basis for quantitative description of the charge accumulation processes at the donor and acceptor sides of PSII. It also provides a rare example of a significant problem in biology, which can be solved analytically.

Algorithms↗

PubMed2002

Modulation of the midpoint potential of the [2Fe-2S] Rieske iron sulfur center by Qo occupants in the bc1 complex.

Following addition of myxothiazol to antimycin-treated chromatophores from Rhodobacter sphaeroides poised at an ambient redox potential (E(h)) of approximately 300 mV, the amplitude of the flash-induced cytochrome c(1) oxidation in the ms range increased, indicating a decrease in the availability of electrons from the immediate donor to c(1), the Rieske iron-sulfur protein (ISP). Because the effect was seen only over the limited E(h) range, we conclude that it is due to a decrease in the apparent midpoint redox potential (E(m)) of the ISP by about 40 mV on addition of myxothiazol. This is in line with the change in E(m) previously seen in direct redox titrations. Our results show that the reduced ISP binds with quinone at the Q(o) site with a higher affinity than does the oxidized ISP. The displacement of ubiquinone by myxothiazol leads to elimination of this preferential binding of the ISP reduced form and results in a shift in the midpoint potential of ISP to a more negative value. A simple hypothesis to explain this effect is that myxothiazol prevents formation of hydrogen bond of ubiquinone with the reduced ISP. We conclude that all Q(o) site occupants (ubiquinone, UHDBT, stigmatellin) that form hydrogen bonds with the reduced ISP shift the apparent E(m) of the ISP in the same direction to more positive values. Inhibitors that bind in the domain of the Q(o) site proximal to heme b(L) (myxothiazol, MOA-stilbene) and displace ubiquinone from the site cause a decrease in E(m) of ISP. We present a new formalism for treatment of the relation between E(m) change and the binding constants involved, which simplifies analysis. Using this formalism, we estimated that binding free energies for hydrogen bond formation with the Q(o) site occupant, range from the largest value of approximately 23 kJ mol(-1) in the presence of stigmatellin (appropriate for the buried hydrogen bond shown by structures), to a value of approximately 3.5 kJ mol(-1) in the native complex. We discuss this range of values in the context of a model in which the native structure constrains the interaction of ISP with the Q(o) site occupant so as to favor dissociation and the faster kinetics of unbinding necessary for rapid turnover.

Binding Sites↗

PubMed2002

Interactions of quinone with the iron-sulfur protein of the bc(1) complex: is the mechanism spring-loaded?

Since available structures of native bc(1) complexes show a vacant Q(o)-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme-substrate complex at the Q(o)-site. This is formed by binding of both ubiquinol (QH(2)) and the dissociated oxidized iron-sulfur protein (ISP(ox)). A binding constant of approximately 14 can be estimated from the displacement of E(m) or pK for quinone or ISP(ox), respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme-product complex is also formed at the Q(o)-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E(m) of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E(m) reflects a binding constant of approximately 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q(o)-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.

Benzoquinones↗

PubMed2002

Modeling of the P700+ charge recombination kinetics with phylloquinone and plastoquinone-9 in the A1 site of photosystem I.

Light activation of photosystem I (PS I) induces electron transfer from the excited primary electron donor P700 (a special pair of chlorophyll a/a' molecules) to three iron-sulfur clusters, F(X), F(A), and F(B) via acceptors A(0) (a monomeric chlorophyll a) and A(1) (phylloquinone). PS I complexes isolated from menA and menB mutants contain plastoquinone-9 rather than phylloquinone in the A(1) site and show altered rates of forward electron transfer from A to [F(A)/F(B)] and altered rates of back electron transfer from [F(A)/F(B)](-) to P700+ (Semenov, A. Y., et al., J. Biol. Chem. 275:23429-23438, 2000). To identify the modified electron transfer steps, we studied the kinetics of flash-induced P700+ reduction in PS I that contains either an intact set or a subset of iron-sulfur clusters F(X), F(A), and F(B) and with the A(1) binding site occupied by phylloquinone or plastoquinone-9. A modeling of the forward and backward electron transfer kinetics in P700-F(A)/F(B) complexes, P700-F(X) cores, and P700-A(1) cores shows that the replacement of phylloquinone by plastoquinone-9 induces a decrease in the free energy gap between A(1) and F(A)/F(B) from approximately -205 mV in wild-type PS I to approximately -70 mV in menA PS I. The +135 mV increase in the midpoint potential of A(1) explains the acceleration in the rate of P700+ dark reduction in menA PS I, and the resulting uphill electron transfer from A(1) to F(X) in menA PS I explains the absence of a contribution from F to the reduction of P700+. This fully quantitative description of PS I relates electron transfer rates, equilibrium constants, and redox potentials, and can be used to predict changes in these parameters upon substitution of electron transfer cofactors.

Carrier Proteins↗