All-Atom Molecular Dynamics Simulations Reveal Significant Differences in Interaction between Antimycin and Conserved Amino Acid Residues in Bovine and Bacterial bc1 Complexes

AbstractAntimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Qi site of the bacterial and bovine bc1 complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Qi pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Qi pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc1 complex and only rarely in the bovine bc1 complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc1 complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc1 complex

The structure of chromatophores from purple photosynthetic bacteria fused with lipid-impregnated collodion films determined by near-field scanning optical microscopy

AbstractLipid-impregnated collodion (nitrocellulose) films have been frequently used as a fusion substrate in the measurement and analysis of electrogenic activity in biological membranes and proteoliposomes. While the method of fusion of biological membranes or proteoliposomes with such films has found a wide application, little is known about the structures formed after the fusion. Yet, knowledge of this structure is important for the interpretation of the measured electric potential. To characterize structures formed after fusion of membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides with lipid-impregnated collodion films, we used near-field scanning optical microscopy. It is shown here that structures formed from chromatophores on the collodion film can be distinguished from the lipid-impregnated background by measuring the fluorescence originating either from endogenous fluorophores of the chromatophores or from fluorescent dyes trapped inside the chromatophores. The structures formed after fusion of chromatophores to the collodion film look like isolated (or sometimes aggregated, depending on the conditions) blisters, with diameters ranging from 0.3 to 10 μm (average ≈1 μm) and heights from 0.01 to 1 μm (average ≈0.03 μm). These large sizes indicate that the blisters are formed by the fusion of many chromatophores. Results with dyes trapped inside chromatophores reveal that chromatophores fused with lipid-impregnated films retain a distinct internal water phase

In Situ Kinetics of Cytochromes

ABSTRACT: 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 ∼120 µs vs half-time of ∼235 µs, in the presence of antimycin). In addition, the LS approach revealed a delay of ∼50 µs 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

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)π/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

Spectral analysis of the bc1 complex components in Situ: beyond the traditional difference approach, Biochim. Biophys. Acta 1757

Abstract The cytochrome (cyt) bc 1 complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electrontransport 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

flash-induced

Kinetics of the oxygen evolution step in plants determined fro

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

Modulation of the midpoint potential of the [2Fe–2S] Rieske iron sulfur center by

ABSTRACT: Following addition of myxothiazol to antimycin-treated chromatophores from Rhodobacter sphaeroides poised at an ambient redox potential (E h ) of ∼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 ∼23 kJ mol -1 in the presence of stigmatellin (appropriate for the buried hydrogen bond shown by structures), to a value of ∼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. The cytochrome bc 1 complex (ubiquinol: cytochrome c oxidoreductase and related complexes) plays a central role in free-energy transduction in all major electron-transfer chains Specific inhibitors have provided valuable tools for the elucidation of structure and function of the bc 1 complexes. Over the years, different inhibitors, specific for each quinoneprocessing site, have been identified and studied (4). Inhibitors of the Q o site fall into two main classes, initially exemplified by UHDBT and myxothiazol (5, 6). They have been subdivided into two or three types, called either class I and class II (7), or Q o -II and Q o -III, and Q o -I (4, 8), based on their different effects on the spectra and redox potentials of heme b L or ISP and their effects on the kinetics of cyt c oxidation. Previous work had shown that class II (Q o -I) inhibitors (myxothiazol and MOA-type inhibitors) occupy a domain of the Q o site near heme b L and induce shifts in the spectrum of this heme. They displace more weakly binding inhibitors of either class and all quinone occupants. In the presence of myxothiazol, interactions of the reduced ISP (ISP red ) with † This work was supported by grants from the NIH (GM 53508, GM35438) and USDA 1 Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; cyt, cytochrome; DAD, 2,3,5,6-tetramethyl-p-phenylenediamine, DMSO, dimethyl sulfoxide; ISP, Rieske iron-sulfur protein; PES, N-ethylphenazonium ethosulfate; PMS, N-methylphenazonium methosulfate; Rb., Rhodobacter; RC, photosynthetic reaction center; UHDBT, 5-undecyl-6-hydroxy-4,7-dioxobenzothiazol; b L and bH, low-and highpotential hemes of cytochrome b, respectively; Eh, ambient redox potential; Em, midpoint redox potential; Q, QH2, oxidized and reduced forms of ubiquinone; Qi site (Qo site), quinone reducing (quinol oxidizing) site of bc1 complex