Spectroscopic evidence of the role of an axial ligand histidinate in the mechanism of adrenal cytochrome b561.

Adrenal cytochrome b(561) (AdCytb) is the prototype of a widespread protein family that specializes in delivering electrons donated by ascorbic acid for different processes in eukaryotic cells. AdCytb transports redox equivalents from cytoplasmic ascorbate across the membranes of chromaffin granules to support norepinephrine synthesis within their matrix. The interaction of AdCytb with ascorbate is central to a proposed mechanism of AdCytb's function, and a histidine in the active site of AdCytb was suggested to bind cytoplasmic ascorbate and serve as the acceptor of the proton released during ascorbate oxidation. AdCytb contains high- and low-potential hemes but their orientation relative to the matrix and cytoplasmic interfaces of chromaffin granule membrane is disputed. Using a combination of three spectroscopic methods (UV-vis absorption, near-infrared magnetic circular dichroism, and electron paramagnetic resonance), we find that a histidine residue that serves as an axial ligand to the high-potential heme undergoes deprotonation with a pK of ~8.0 and is thus a good candidate for interaction with cytoplasmic ascorbate. The low-potential heme of AdCytb is found to have a pK of ~10.5, making it an unlikely candidate for accepting a proton at physiological pH. UV-vis spectroscopy reveals an additional proton acceptor group in AdCytb with a pK of ~6.5 that is not observed by the other two techniques; whether it plays a role in the mechanism of AdCytb is unknown. We incorporate these results into an updated mechanism of AdCytb reduction predicated on the high-potential heme's localization on the cytoplasmic interface of the chromaffin granule membrane.

All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes.

Antimycin 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 Q(i) site of the bacterial and bovine bc(1) complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q(i) 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 Q(i) pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc(1) complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc(1) complex and only rarely in the bovine bc(1) complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc(1) complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc(1) complex.

Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: I. Equilibrium and modeling studies.

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c(1) in detergent-solubilized bc(1) complex from Rhodobacter sphaeroides. Imidazole binding to cyt c(1) substantially lowers the midpoint potential of the heme and fully inhibits bc(1) complex activity. Temperature dependences showed that binding of Im (K(d) approximately 330 microM, 25 degrees C, pH 8) is enthalpically driven (DeltaH(0) = -56 kJ/mol, DeltaS(0) = -121 J/mol/K), whereas binding of MeIm is 30 times weaker (K(d) approximately 9.3 mM) and is entropically driven (DeltaH(0) = 47 kJ/mol, DeltaS(0)(o) = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c(1) triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c(2) suggested that Im binding to cyt c(1) is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c(2), and c(1), which showed hydrogen bonds formed between the N(delta)H of Im and the cyt c(1) protein, or with a water molecule sequestered with the ligand in the heme cleft.

Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: II. Kinetics and mechanism of binding.

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c(1) of detergent-solubilized bc(1) complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c(1)-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mM Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mM, the rate leveled off, indicating a rate-limiting conformational step with lifetime approximately 1 s; and (iii) at Im concentrations above 100 mM, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.

His92 and His110 selectively affect different heme centers of adrenal cytochrome b(561).

Adrenal cytochrome b(561) (cyt b(561)), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b(561) (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b(H)) peak were seen with mutation of His92; the largest changes in the low-potential (b(L)) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g=3.1 signal (b(H)) but not the g=3.7 signal (b(L)). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b(H) transition; mutations in His110 produced the largest decreases in DeltaA(561) for the b(L) transition. These results indicate that His92 can be considered part of the b(H) heme center, and His110 part of the b(L) heme center, in adrenal cyt b(561).

Intermonomer electron transfer in the bc1 complex dimer is controlled by the energized state and by impaired electron transfer between low and high potential hemes.

The cytochrome bc(1) complex (commonly called Complex III) is the central enzyme of respiratory and photosynthetic electron transfer chains. X-ray structures have revealed the bc(1) complex to be a dimer, and show that the distance between low potential (b(L)) and high potential (b(H)) hemes, is similar to the distance between low potential hemes in different monomers. This suggests that electron transfer between monomers should occur at the level of the b(L) hemes. Here, we show that although the rate constant for b(L)-->b(L) electron transfer is substantial, it is slow compared to the forward rate from b(L) to b(H), and the intermonomer transfer only occurs after equilibration within the first monomer. The effective rate of intermonomer transfer is about 2-orders of magnitude slower than the direct intermonomer electron transfer.

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.

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.

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.

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.

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.

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.

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 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.

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.