Mutations at the Qo Site of the Cytochrome bc1 Complex Strongly

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Mutations at the Q-Site of the Cytochrome bc Complex Strongly Affect Oxygen Binding 1

Peter Husen, and Ilia A. Solov'yov J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08226 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

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The Journal of Physical Chemistry

Mutations at the Qo-site of the Cytochrome bc1 Complex Strongly Affect Oxygen Binding Peter Husen∗ and Ilia A. Solov’yov∗ Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark E-mail: [email protected]; [email protected] Phone: +45 65502532

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Abstract The homodimeric bc1 protein complex is embedded in membranes of mitochondria and photosynthetic bacteria, where it transports protons across the membrane to maintain an electrostatic potential used to drive ATP synthesis as part of the respiratory or photosynthetic pathways. The reaction cycle of the bc1 complex is driven by series of redox processes involving substrate molecules from the membrane, but occasional side reactions between an intermediate semiquinone substrate and molecular oxygen are suspected to be a source of toxic superoxide, which is believed to be a factor in aging. The present investigation employs molecular dynamics simulations to study the effect of mutations in the Qo binding sites of the bc1 complex on the ability of oxygen molecules to migrate to and bind at various locations within the complex. It is found that the mutations strongly affect the ability of oxygen to bind at the Qo -sites, and, moreover, different behavior of the two monomers of the bc1 complex is observed. The conformational differences at the Qo -sites of the two monomers are studied in detail and discussed. The anionic form of semiquinone was identified as leading to the greatest opportunity for side reactions with oxygen.

Introduction The cytochrome bc1 protein complex, 1,2 also known as complex III, is a part of both the respiratory pathway in eukaryotes, where it is embedded in the inner mitochondrial membrane, and the photosynthetic pathway of photosynthetically active bacteria, where it is embedded in the plasma membrane. The protein complex serves to pump protons across the bioenergetic membrane to maintain an electrostatic potential, which is used by other protein complexes in the membrane to drive ATP synthesis. 3,4 The reaction cycle of the bc1 complex, the Q-cycle, 5,6 is driven by redox reactions with ubiquinol (QH2 ) and ubiquinone (Q) substrate molecules from the membrane. During the Q-cycle, depicted schematically 2 Environment ACS Paragon Plus

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Fig. 1: The bc1 complex and its reaction cycle. The figure depicts the bc1 complex from Rhodobacter capsulatus and illustrates schematically the Q-cycle in one of the monomers. Red labels indicate subunits of monomer A of the bc1 complex, while green labels indicate the subunits of monomer B. During the Q-cycle, two QH2 molecules are oxidized to Q near the positive side of the membrane at the location known as the Qo -site, while one Q is reduced to QH2 near the negative side at the so-called Qi -site. The redox reaction are accompanied by two protons being taken up from the negative side of the membrane and four protons being released to the positive side. One electron from each QH2 leaves the bc1 complex (carried by a cytochrome c2 protein) on the positive side of the membrane, while the other electron is transferred internally in the bc1 complex to the Qi -site, where it is used to reduce Q. in Fig. 1 for one of the two monomers of the bc1 complex, two QH2 are oxidized to Q at the Qo -site near the positive side of the membrane, while one Q is reduced to QH2 at the Qi -site. 5,7,8 The bc1 complex is a homodimer with each monomer consisting of three subunits in the case of Rhodobactor capsulatus: 9,10 cytochrome b (cyt b), cytochrome c1 (cyt c1 ) and the iron sulphur protein (ISP), as shown in Fig. 1. The Q-cycle occurs separately in each of the two monomers, which are shown in Fig. 2 in red and green colors. Essential to the function of the bc1 complex are several prosthetic iron-containing groups, responsible for the electron transfer processes of the Q-cycle. 1,11,12 Each monomer has three heme groups, 3 Environment ACS Paragon Plus


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Fig. 2: The internal structure of the bc1 complex. The bc1 complex is a dimer consisting of two identical monomers, here depicted with red and green colors. Each monomer contains a total of three heme groups, denoted as heme c, heme bL and heme bH , and an Fe2 S2 -cluster, all taking part in the electron transfer processes of the Q-cycle. During the Q-cycle, the Q•− or QH• intermediate is bound at the Qo -site next to the Fe2 S2 cluster of the ISP subunit and the Y302 amino acid residue of cyt. b. The wild-type bc1 complex and the Y302A, Y302C and Y302S mutants with the tyrosine residue substituted by alanine, cysteine and serine, respectively, are studied here computationally. The WT bc1 complex is studied with both semiquinone variants, while the mutants are only studied with the Q•− form. namely heme bL and heme bH in the cyt. b subunit and heme c in cyt. c1 , as well as an iron-sulfur cluster (Fe2 S2 ) in the ISP. The QH2 substrate binds next to the Fe2 S2 -cluster at the Qo -site of the bc1 complex, while the Q substrate binds at heme bH in the Qi -site. 13,14 The Q-cycle leaves the QH2 and Q substrate molecules as radical semiquinones at the intermediate stages of the Q-cycle. 6,15–18 This leads to the risk of stray electron transfer 19–23 reactions leading to production of radical species such as e.g. superoxide, O•− inside 2 ,


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•− the bc1 complex. As O•− 2 is toxic to cells, and mitochondrial O2 production is believed to

be a factor in aging and age-related diseases, 24 the study of possible mechanisms leading to such production is of great importance. The exact state of semiquinone present transiently at the Qo -site is still controversial, 25 but earlier quantum chemical calculations 14 suggest that both protons dissociate rapidly, when QH2 is oxidized to semiquinone, and experimental studies 15 have found evidence for the presence of the anionic semiquinone, Q•− , at the Qo -site of the bc1 complex from Rhodobacter capsulatus. Due to the existing controversy, however, both the anionic, Q•− and the neutral, QH• forms of the semiquinone substrate were studied in the present computational investigation to evaluate their relative ability to stimulate reactions leading to superoxide production. Experimental studies 26,27 have uncovered that a mutation of the Y302 residue (in case of Rhodobacter capsulatus) of cyt b, shown in Fig. 2, inside the Qo -binding site lead to an increased rate of superoxide production in the bc1 complex. Building upon earlier computational studies 14,22 that established a molecular model of the bc1 complex from Rhodobacter capsulatus with the bound QH2 and Q substrates and identified pathways and binding sites of molecular oxygen in the protein complex, the present investigation tests computationally the effect of Y302 mutations on O2 migration and localization in the bc1 complex as a first step to uncover the role of the Y302 residue in O2 binding and O•− 2 production. In particular, the mutations Y302A, Y302C and Y302S, where the tyrosine is replaced by alanine, cysteine and serine, respectively, are tested in the bc1 complex with Q•− bound at the Qo -site. Due to the high degree of uncertainty surrounding possible superoxide production mechanisms, simulations were carried out over at least 350 ns to provide sufficient statistics for O2 binding events at the multitude of possible binding sites inside the bc1 complex. Such an approach is seen as a more favorable one in contrast to dedicated free energy calculations of specific O2 binding modes. Molecular dynamics simulations in the present investigation reveal a clear effect of 5 Environment ACS Paragon Plus


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the Y302 mutations on O2 binding at the Qo -site. Furthermore, the O2 binding behavior at the Qo -site differs between the two monomers of the bc1 complex. In monomer B of the wild-type (WT) bc1 complex, O2 migrates into the Qo -site from the membrane and occasionally gets trapped near the Q•− and the Fe2 S2 -cluster. This O2 binding mode at the Qo -site is weakened or eliminated by the Y302S mutations, suggesting that the tyrosine residue is involved in trapping the O2 molecule. In monomer A, however, O2 binding at the Qo -site is only observed in some of the mutants, most notably Y302C. In this case, O2 molecules enter the bc1 complex from the water phase through the head of the ISP subunit. Additional MD simulations of the bc1 complex with the neutral semiquinone, QH• , bound at the Qo -site were performed to compare the tendency of O2 trapping for the two different charge states of the substrate. Proximity of molecular oxygen to potential electron donors is a necessary condition for any superoxide production mechanism within the bc1 complex. While experimental mutational studies 26,27 show that the Y302 residue of cyt b significantly influences superoxide production, the present computational investigation describes the function of this residue in the regulation of migration pathways and binding modes of O2 molecules in the bc1 complex.

Methods The system consisting of the bc1 complex from Rhodobacter capsulatus (PDB ID: 1ZRT 28 ) embedded in a lipid bilayer membrane with the substrate molecule Q bound at the Qi -site and either Q•− or QH• bound at the Qo -site was studied using molecular dynamics (MD) simulations with NAMD 2.10 29 and the CHARMM36 force field 30 with CMAP corrections. 31 VMD 1.9.2 32 was used for system preparation, data analysis and visualizations. The bc1 complex embedded in a membrane patch was suspended in a water box of


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197 ˚ A ×177 ˚ A ×142 ˚ A size using the TIP3P water model 33 with a salt (NaCl) concentration of 0.05 mol/L, which was also used to neutralize the system. Oxygen molecules (O2 ) were initially added to the water phase of the system. The computational model used for the bc1 complex is the same as in a previous investigation 22 except for substituting the Y302 residue with the appropriate amino acid for each studied mutant in both monomers of the bc1 complex. The bc1 complex model is based on the 1ZRT crystallographic structure data 28 and force field parameters and partial charges for the heme groups and the quinones originate from previous studies, 14,34,35 later modified 22 to place the bc1 complex in a state with Q•− bound at the Qo -site. In case of the simulation with QH• bound at the Qo -site, the H156 residue of the ISP is considered protonated, where the proton originates from QH2 upon the first proton transfer, while the E295 residue of cyt b remains deprotonated. The force field parameters for the QH• and its coordinating residues at the Qo -site are taken from a recent study. 17 The lipid membrane patch was constructed from a mixture of 406 phosphatidylcholines (PC 18:2/18:2) lipids, 342 phosphatidylethanolamines (PE 18:2/18:2) and 102 cardiolipins (CL 18:2/18:2/18:2), consistent with earlier studies. 14,17,22,34 Parameters for PC and PE lipids were available from CHARMM36, 30,31 but not for CL. Parameters for the latter were instead taken from a previous study 36 for the head groups, while parameters for the lipid tails were still taken from CHARMM36. 30 A total of 165 oxygen molecules were added to the system by randomly replacing bulk water molecules with oxygens. This leads to an oxygen concentration of 21 mmol/L in the bulk water phase after system equilibration, 22 being an artificially high concentration, 2-3 orders of magnitude above physiological levels, 37 which is, however, necessary to obtain sufficient statistics of O2 migrational patterns and binding events. Molecular oxygen was parameterized using the standard CHARMM36 force field. 30 The validity of the artificial concentration of oxygen was discussed extensively in an earlier study, 22 where it was 7 Environment ACS Paragon Plus


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demonstrated that it can be used to determine O2 localization sites within the bc1 complex reasonably well.

MD simulations Simulations were carried out using Langevin dynamics with an integration timestep of 2 fs and periodic boundary conditions. Electrostatic and van der Waals interactions were handled with a smooth cutoff of 12 ˚ A, and long range electrostatic interactions were treated with particle-mesh Ewald (PME) summation method. 38,39 The initial molecular structure used for the simulations was originally adopted from earlier investigations, 14 where it had been equilibrated for a total of 310 ns followed by a production simulation of 360 ns with QH2 bound at the Qo -site of the bc1 complex. After replacing the QH2 with Q•− 22 or QH• , 17 each system was further equilibrated for 3 ns. After introducing a mutation to the Y302 residue of cyt b, the system was equilibrated once again for 3 ns for each mutant considered with the pressure kept at the atmospheric pressure value (NPT ensemble) using the Nosé-Hoover Langevin piston pressure control 40,41 with a piston oscillation period of 200 fs and a decay time of 50 fs. After equilibration, the production MD simulations were carried out maintaining a fixed volume of the system (NVT ensemble). Note that the simulation box before introducing mutations had a size of 197 ˚ A × 177 ˚ A × 142 ˚ A, while during the NPT equilibration for each of the mutants, the simulation box exhibited small changes in each dimension, as summarized in Table 1. The temperature in the simulations was maintained at 310 K using the Langevin thermostat with a damping coefficient of γ = 5 ps−1 , which was previously 22 found to model the diffusive behavior of molecular oxygen accurately enough. Simulations were carried out for the WT bc1 complex and its mutants Y302A, Y302C and Y302S, where the tyrosine Y302 of both cyt b subunits was replaced by alanine, cysteine and serine, respectively. Two repetitions of a ∼ 360 ns MD simulation were carried out for each of the mutants and are 8 Environment ACS Paragon Plus

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presented here along with two repetitions of the corresponding simulation for the WT bc1 complex from a previous investigation 22 as indicated in Table 1. The simulation of the WT bc1 complex with the neutral semiquinone, QH• , bound at the Qo -site was carried out over 350 ns. Table 1: MD simulations of the bc1 complex. Two repetitions of the simulations for each mutant of the bc1 complex were carried out, where simulations of the WT bc1 complex with the Q•− substrate were taken from a previous study. 22 Only one simulation for the WT bc1 complex with the QH• substrate was performed. Each simulation was carried out for a duration of about 350 ns. The system dimensions after equilibration in the NPT ensemble are indicated for each of the simulations. Mutation

Qo -site substrate

WT 22

Q•−

WT

QH•

Y302A

Q•−

Y302C

Q•−

Y302S

Q•−

Simulation 1 2 1 1 2 1 2 1 2

Simulation time (ns) 369 373 350 354 368 356 360 357 375

Box size (˚ A×˚ A×˚ A) 197.1 × 176.7 × 142.0 191.3 × 176.4 × 146.2 192.9 × 177.3 × 144.3 196.8 × 176.6 × 142.1 194.0 × 176.1 × 144.6 194.3 × 176.3 × 144.2 196.6 × 173.5 × 144.7 192.7 × 176.6 × 145.2

Results The dynamics and localization of molecular oxygen was studied for three mutants of the bc1 complex, namely Y302A, Y302C and Y302S, each through two repeated MD simulations, and these results were compared with a previous investigation 22 for the WT bc1 complex. A simulation of the WT bc1 complex with the bound QH• substrate at the Qo -site was also performed. First, the binding sites of O2 at the Qo -sites of the WT and mutant bc1 complexes with Q•− bound are revealed, and the differences in the observed behavior between the two monomers of the bc1 complex are discussed. Next, the localization of O2 9 Environment ACS Paragon Plus


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near the heme groups is presented, and, finally, the influence of the substrate charge state on the O2 trapping is evaluated.

O2 binding at the Qo -site with anionic semiquinone The bc1 complex is a dimeric protein complex, and, therefore, the localization of O2 was studied for both of its monomers separately. As previously reported, 22 O2 binds in a cavity at the Qo -site near the Fe2 S2 -cluster in one monomer of the bc1 complex, here denoted as monomer B, when a semiquinone anion, Q•− is bound there. The details of O2 migration into the Qo -site and the timescales involved in O2 diffusion and binding are discussed in detail in a recent study. 22 Figure 3 shows the localization of O2 at the Qo -site of monomer B in simulation 1 for the WT (Fig. 3a) and the three mutants (Fig. 3b-d) indicated by red isosurfaces of the local O2 concentration averaged over the simulated trajectories of the Q•− model. Molecular oxygen was observed to bind at a distance of 8 ˚ A away from the Fe2 S2 cluster in monomer B of the WT bc1 complex (Fig. 3a), but not for any of the mutants. To quantify the difference in O2 binding behavior between the WT and mutants, Fig. 4 shows the rate of O2 encounters at the Qo -site as a function of the distance from the Fe2 S2 -cluster. Specifically, this rate is defined as the number of times, an O2 molecule is observed at a given distance from the Fe2 S2 -cluster per unit simulation time. The distance to the Fe2 S2 cluster was used here as it was found to define intuitively the bound vs non-bound states of O2 at the Qo -site, since the Fe2 S2 -cluster is located at the edge of the cavity containing the Q•− at the Qo -site; the cavity was observed to be the primary channel of O2 migration into the Qo -site. 22 The binding location of O2 at the Qo -site in monomer B of the WT bc1 complex shown in Fig. 3a is related to the peak arising in Fig. 4 and describes encounters of O2 molecules by the Fe2 S2 -cluster at a distance of ∼ 9 ˚ A. The peak arises in both simulations and is ACS Paragon 10 Plus Environment

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Fig. 3: Localization of molecular oxygen in the Qo -site of monomer B. The localization of O2 molecules at the Qo -site of the bc1 complex is indicated by red surfaces, which are isosurfaces representing a trajectory-averaged local oxygen concentration of 1.5 molecules per nm3 in simulation 1. a: Previous studies 22 show that O2 molecules migrate into and bind inside the Qo -site of the bc1 complex. The red region to the right of the Q•− head group indicates the area where O2 binds for tens of nanoseconds at a time in the WT simulations. b: O2 binding localization for the Y302A mutant. Here, O2 binding near the Fe2 S2 -cluster is not observed. Instead, increased O2 localization is found at a distance of 13 ˚ A from the Fe2 S2 -cluster. c-d: No significant regions of increased O2 localization are observed at the Qo -site in the Y302C and Y302S mutants.

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Fig. 4: Rate of O2 encounters at the Qo -site. The number of O2 encounters per nanosecond near the Fe2 S2 cluster, representing the Qo -site, evaluated for both monomers of the bc1 complex in the two simulations performed. Peaks in the graphs correspond to distances at which O2 molecules tend to linger and will thus fluctuate around the threshold distance. A clear effect of the Y302 mutation is observed for both simulations in monomer B at the Fe2 S2 –O2 distance of ∼ 9 ˚ A (indicated by dashed rectangles); here, O2 binding is significantly reduced for the mutants compared to the WT case. In monomer A, however, O2 binding within 12 ˚ A away from the Fe2 S2 -cluster tends to increase in the mutants. For the Y302C mutant in particular, a significant peak arises at a distance of 10 ˚ A, where no binding is observed in the WT bc1 complex. Stars indicate peaks corresponding to the sites that are featured in Fig. 3a and Fig. 3b. indicated by dashed rectangles. For the mutants, the height of the peak is significantly reduced, which is consistent with the O2 absence in the binding pocket shown in Figs. 3b-d. Furthermore, the reduced size of the peak in the mutants is consistent between the two repeated simulations. The significant difference in O2 presence at the Qo -site indicates that


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Fig. 5: O2 distance to the Qo -site. Time evolution of the distance to the nearest O2 molecule as measured from the Fe2 S2 -cluster of the bc1 complex for the WT as well as the Y302A, Y302C and Y302S simulations with the Q•− substrate as a function of the simulation time. In monomer B, a number of O2 binding events within 10 ˚ A of the Fe2 S2 -cluster are clearly observed for the WT bc1 complex, while similar binding events are rare for the mutants. Meanwhile, in monomer A, the Y302C mutant shows the strongest tendency to bind O2 molecules at the Qo -site. the mechanical properties of the Y302 are essential for the O2 binding mechanism at this site. Tyrosine is the most bulky of the four studied amino acids and is likely to play a role in keeping an O2 molecule trapped, once it enters the binding pocket. Figure 5 shows the distance to the nearest O2 molecule as measured from the Fe2 S2 cluster as a function of simulation time for the WT bc1 complex and the three mutants. All these simulations assumed the Qo -site to be occupied with the Q•− substrate. Here, O2 binding at a distance of ∼ 9 ˚ A from the Fe2 S2 -cluster in monomer B can also be clearly seen. As reported previously, 22 O2 enters the Qo -site a number of times during the 369 ns ACS Paragon 13 Plus Environment


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trajectory and remains bound for tens of nanoseconds at the Qo -site. The repeated nature of this event means that there are reasonable statistics to differentiate the effect between the WT bc1 complex and the mutants and supports the conclusion that the peaks describing the O2 binding location in Fig. 4 as well as the region of increased O2 localization in Fig. 3 do indeed reflect locations at the Qo -site with a tendency of trapping O2 molecules.

Difference between the monomers of the bc1 complex Figures 4 and 5 show a difference between the two monomers of the bc1 complex. While mutations to Y302 in cyt b of the bc1 complex eliminates or significantly reduces O2 binding at the Qo -site of monomer B, it appears that in monomer A, the mutations instead lead to new locations, while nearly no O2 binding takes place in the WT with the Q•− semiquinone. In particular for the Y302C mutant, significant O2 binding at the Qo -site of monomer A is observed, as follows from Figure 4, which shows increased rates of O2 encounters for Y302C at distances of 8-12 ˚ A from the Fe2 S2 -cluster occurring in both simulations. Figure 5 supports this observation further, as the number of O2 binding events in the case of the Y302C mutant is clearly increased. Figure 6 takes a closer look at O2 binding locations at the Qo -sites of both monomers in the Y302C mutant compared to the WT bc1 complex for both simulations, indicated by red and yellow surfaces. The increased rate of O2 encounters at the Qo -site in monomer A for the Y302C mutant is explained by multiple O2 binding locations at the Qo -site. In simulation 1, O2 binding is observed very close to Q•− at the distance of about 10 ˚ A from the Fe2 S2 -cluster, in the region indicated in Fig. 6 by a red surface above Q•− . In simulation 2, O2 binding is instead observed in the region indicated by a yellow surface close to both Q•− and C302 at a distance of 9 ˚ A from the Fe2 S2 -cluster. By inspecting trajectories of O2 molecules leading to these sites, it was concluded that O2 molecules reaching the Qo -site of monomer A in the Y302C mutant enter the bc1 complex from the water phase on the positive side of the ACS Paragon 14 Plus Environment

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Fig. 6: Localization of molecular oxygen at the Qo -sites of both monomers in WT and Y302C. Regions of increased O2 localization at the Qo -sites of the WT bc1 complex and the Y302C mutant are indicated by red and yellow regions corresponding to the two simulations performed. In monomer B, the O2 localization is largely consistent between the two simulations – here, O2 molecules bind at a distance of around 8 ˚ A from the Fe2 S2 -cluster in the WT, but not in the Y302C mutant. The localization regions for the WT appear orange due to the regions coinciding for the two simulations. For monomer A, O2 only binds near the Fe2 S2 -cluster in the Y302C mutant, but at different locations in the two performed simulations. O2 molecules binding at the Qo -site in monomer B are observed to migrate into the binding site from the memebrane through the channel housing the Q•− tail 22 as indicated by a red arrow in the figure. In monomer A, however, O2 molecules migrate from the water phase through the head of the ISP subunit.



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membrane through the head of the ISP subunit. This is in contrast to O2 molecules binding at the Qo -site in monomer B of the WT bc1 complex, which enter from the membrane through the cavity housing the tail of the Q•− . 22 The two paths are indicated schematically in Fig. 6 by the red arrows. Experimental studies 26,27 find an increased O•− formation in the mutants Y302A, 2 Y302C and Y302S compared to the WT bc1 complex. This could indicate that O2 molecules entering the Qo -site from the water phase through the ISP subunit are more relevant to superoxide production than O2 molecules entering from the membrane – either due to the binding locations being more favorable for an electron transfer process or due to the generated O•− 2 being able to leave the bc1 complex more easily. The bc1 complex is a homodimer, so any observed difference in behavior between the monomers are expected to arise due to conformational differences between the monomers. Figure 7 shows the overlayed Qo -sites of both monomers in the WT bc1 complex. The distance between Q•− and the Y302 residue is indicated, and the graphs in the inset shows that this distance is consistently increased in monomer B compared to monomer A. This difference might well explain the different behavior of O2 molecules at the Qo -site. Tyrosine is often involved in gating mechanisms, 42–44 and this is likely to be the case here, as oxygen fails to bind at the Qo -site of monomer B, if this residue is replaced by a less bulky amino acid. In monomer A, the absence of O2 binding at the Qo -site of the WT bc1 complex could be due to Y302 completely blocking the O2 passage into the binding pocket by exhibiting a slightly different conformation, or alternatively, that the shifted tyrosine fails to keep O2 molecules trapped at the Qo -site once they enter. In case of Y302C, the conformation in monomer A opens up other pathways for O2 to enter the Qo -site and bind. The difference between the two monomers could reflect a natural tendency of the bc1 complex to form an asymmetric dimer, at least on the time scales accessible through MD simualations. The simulated molecular structures in the present investigation were prepared starting ACS Paragon 16 Plus Environment

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Fig. 7: Difference between monomers of the bc1 complex. The Qo -sites of the two monomers of the WT bc1 complex are shown superimposed with the trajectory averaged distance between Q•− and Y302 indicated. The graph in the inset shows that this distance is consistently smaller in monomer A compared to monomer B, indicating conformational difference in the two monomers. The distances are measured between the Cγ atom of the tyrosine and the carbon of the Q•− ring bound to the tail. from a similar structure adopted from an earlier investigation 14 consisting of the bc1 complex embedded in a lipid membrane and suspended in a water box, but with the substrate QH2 bound at the Qo -site rather than Q•− . Figure S4 in the supporting information (SI) shows the QH2 –Y302 distance, cf. Figure 7, during the simulations in that earlier study. 14 After around 160 ns, the QH2 bound at the Qo -site of monomer B moves further away



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from the Y302 residue and stays at an increased distance, compared to monomer A, for the remainder of the simulation. The initial structures used for the simulations in the present investigation were prepared from a configuration taken at the end of the trajectory used to prepare Figure S4, and the Q•− –Y302 distance remains increased in monomer B throughout all present simulations as shown in case of the WT bc1 complex in Figure 7.

O2 binding at the heme groups The heme groups of the bc1 complex take part in the electron transfer processes of the Qcycle 6,45 and should, therefore, also be considered as sites, where stray reactions with O2 molecules could occur. Figure 8 shows O2 encounter rate curves similar to those presented in Fig. 4, but computed for each of the three heme groups in each monomer of the bc1 complex, see Fig. 2. Plots of the O2 distance to the heme groups as a function of simulation time, cf. Figure 5 for the Fe2 S2 -cluster, are presented in the SI as Figs. S1-S3. As previously reported, 22 O2 is able to migrate within 5 ˚ A of the central iron atoms of the hemes bH and bL of cyt. b, which facilitate the electron transport from the Qo -site to the Qi -site of the bc1 complex. As such, the possibility of the heme groups acting as electron donors to O2 should not be dismissed. When considering the Y302 mutations, however, no clear effect on O2 binding at the heme groups is observed consistently between simulations. The height of the peak in Fig. 8 corresponding to an O2 binding site at around 5 ˚ A from the heme bH varies between the WT bc1 complex and its mutants, but this variation is not significant compared to the variation between the two repeated simulations, and the time evolution of the distance between O2 and the heme group in Fig. S1, suggests that the observed increases in O2 binding rates are due to very few events in the simulated trajectories. Similarly, in case of heme c in monomer A, one notes the large peaks that suggest O2 binding at a distance of around 9 ˚ A from heme c for simulation 1. This observation is ACS Paragon 18 Plus Environment

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Fig. 8: Rate of O2 encounters at the heme groups. The time averaged number of encounters of O2 molecules per nanosecond at a certain distance from each of the heme groups of the bc1 complex. In particular, the distances to the central Fe atoms of the heme groups are measured. Stars indicate positions depicted in the illustrations to the left of the graphs. No effect of Y302 mutations was observed consistently between the two simulation repetitions. The snapshots to the left are examplary O2 bindings at locations near the heme groups of the WT bc1 complex corresponding to the peaks indicated by stars in the graphs. The peaks at a distance of ∼ 9 ˚ A from heme c in monomer A, which appear for all mutants in simulation 1 but not for simulation 2, are artifacts due to the mutant simulations all starting out with an O2 molecule already bound in simulation 1. ACS Paragon 19 Plus Environment


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considered to be an artifact arising in case of simulation 1 of the mutants, as all those simulations were constructed from the last snapshot of the WT system, where an O2 molecule was trapped near heme c and stayed initially as can be seen from the time evolution of the O2 –heme c distance in Fig. S3 of the SI. The binding location of the O2 molecule in this case is considered real, but the low repeat activity of the binding events and the very long duration of single observed event suggests that the binding statistics is insufficient to make conclusions about O2 binding at this heme group. In simulation 2, the WT and all mutant simulations start from the same initial snapshot, and in this case, neither WT nor the mutants exhibit any significant O2 binding at heme c of monomer A, which further supports that O2 binding at heme c is a rare event.

The charge state of the semiquinone The main computational model considered in the present investigation assumes the negatively charged semiquinone, where both protons are transferred to the protein after one electron is transferred to the Fe2 S2 -cluster. Since the precise order of events in the Q-cycle is still highly debated, 25 an additional simulation of the WT bc1 complex with the neutral semiquinone variant QH• bound at the Qo -site was carried out to test the influence of the substrate charge state on the modeled O2 dynamics and binding. Figure 9 shows the O2 encounter rates at the Qo -site and the time evolution of the distance between the Fe2 S2 -cluster and the nearest O2 molecule in the simulation. The tendency of O2 binding at the Qo -site is significantly reduced in the case of a bound neutral semiquinone, QH• , as compared to the Q•− variant. In monomer B, the O2 binding rate in the case of the bound QH• substrate is significantly smaller than in the case of the Q•− substrate. Figure 10 shows the localization of O2 at the Qo -site of monomer B with QH• bound. The O2 binding location near the Fe2 S2 -cluster is the same as observed in monomer B in the Q•− model, but the much lower trajectory-averaged O2 concentration of 0.3 nm−3 was used as ACS Paragon 20 Plus Environment

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Fig. 9: O2 binding at the Qo -site with the bound QH• substrate. O2 encounter rates and distance measured from the Fe2 S2 cluster in the simulation with the neutral semiquinone, QH• , at the Qo -site. In both monomers, O2 binding at the Qo -site is significantly reduced compared to the model with the anionic semiquinone (see Fig. 4). In case of monomer B, the O2 encounter rate near the Fe2 S2 -cluster is greatly reduced. isovalue to produce a similarly sized localization region for the QH• model as obtained with the isovalue of 1.5 nm−3 for the Q•− model, shown in Fig. 3. The large difference in O2 binding between the two models means that if both forms of the bound semiquinone substrate are considered possible, the anionic form by far holds the highest risk of side reactions with O2 at the Qo -site. Furthermore, the high sensitivity of O2 behavior to the choice of charge state means that it is crucial to establish the exact sequence of events in the Q-cycle in order to make quantitative predictions of superoxide production rates.

Migration of the ISP Another debated aspect of the Q-cycle is the suggested conformational change of the ISP subunit during the Q-cycle, where the the Fe2 S2 -cluster moves away from the bound substrate at the Qo -site to heme c in the cyt c1 subunit to pass on the electron received from QH2 . 25 There is ample experimental evidence for such a motion of the ISP, 46–49 but the ACS Paragon 21 Plus Environment


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Fig. 10: O2 localization at the Qo -site of monomer B with the bound QH• substrate. a: The isosurfaces representing regions of increased O2 localization are generated using the same concentration isovalue, 1.5 nm−3 , as used to indicate the O2 localization for the Q•− model in Fig. 3. The O2 binding location near the Fe2 S2 -cluster is not resolved for this threshold value in the QH• model. b: Only with the significantly lower concentration isovalue of 0.3 nm−3 is the region of increased O2 presence revealed. In other words, the O2 affinity at the Qo -site is greatly reduced for the bound QH• substrate compared to the Q•− case. exact sequence of events is unclear. The semiquinone substrate at the Qo -site of the bc1 complex, as modeled in both the neutral and anionic forms in the present study, is present immediately after the first electron from QH2 has been passed to the Fe2 S2 -cluster, which represents the earliest state at which, the migration of the ISP might begin. However, such a conformational change was not observed in any of the performed simulations. This could either mean that the ISP migration is not initiated before the next electron transfer from the semiquinone substrate, or it could indicate that the process is too slow to be observed during the simulation time. In the latter case, the simulations show that oxygen has ample time to bind at the Qo -site, or other sites of the bc1 complex, before any significant conformational change occurs. Since the semiquinone state is believed to be very short-lived, 25 the studied configuration of the bc1 complex is then the most relevant one for possible superoxide production.


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Conclusion Single residue mutation of the Y302 residue of the cyt b subunit seems to strongly affect the tendency of oxygen molecules to bind inside the Qo -site of the bc1 complex. In the wild-type bc1 complex, molecular oxygen from the lipid membrane frequently binds at the Qo -site of monomer B, while binding at this location is eliminated or greatly reduced by mutations of Y302. It appears, the tyrosine residue acts as a gate trapping the oxygen molecule at the Qo -site. Contrarily, in monomer A, O2 binding at the Qo -site is increased by mutations, most notably in case of Y302C. In this case, O2 molecules migrate into the Qo -site of the bc1 complex from the water phase through the head of the ISP subunit. The investigation reveals that the difference between the two monomers is due to a small conformational change between the Qo -sites of the two monomers, which is observed through a slightly different distance between the Q•− substrate and the Y302 residue at the Qo -site. Experimental evidence 26,27 finds increased O•− 2 production in the Y302 mutants compared to WT, so assuming O2 molecule entering the Qo -site are key to superoxide production in the bc1 complex, the present results suggest that the O2 binding locations and migration pathways in monomer A are the most relevant ones. O2 binding is also observed near the heme groups of the bc1 complex as reported earlier, 22 but no mutation effect on these bindings could be concluded. In summary, the Y302 residue of cyt b at the Qo -site of the bc1 complex significantly affects the pathways and binding modes of molecular oxygen at the Qo -site, while a radical semiquinone is bound there, and hence is likely to play an important role in the superoxide production mechanism at the Qo -site of the bc1 complex. The charge state of the bound semiquinone is paramount to O2 binding behavior as, apparently, the anionic form, Q•− , stimulates the tendency of O2 binding and hence holds the greatest risk of superoxide production.



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The present investigation reveals that the pathways and binding modes of O2 at the Qo -site are highly sensitive to the precise positioning of the Q•− substrate, which clouds the picture through a multiude of possible O2 binding mechanisms. Further quantum chemical calculations testing possible electron transfers to O2 in various bound positions could help identify the mechanisms relevant to superoxide formation in greater detail.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website and includes (i) graphs of the time evolution of the distances measured for each of the heme groups of the bc1 complex to the nearest O2 molecule in the system and (ii) the Y302–QH2 distance for each monomer of the bc1 complex evaluated for an earlier simulation, 14 from which the initial configuration of the present simulations were prepared.

Acknowledgements The authors thank Angela M. Barragan for providing the data used to produce Figure S4. The authors also acknowledge the Lundbeck Foundation and the Russian Science Foundation (Grant No. 14-12-00342) for financial support. Computational resources for the simulations were provided by the DeiC National HPC Center, SDU.

References (1) Crofts, A. R.; Berry, E. A. Structure and Function of the Cytochrome bc1 Complex of Mitochondria and Photosynthetic Bacteria. Curr. Opin. Struct. Biol. 1998, 8, 501–509.


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(2) Mulkidjanian, A. Y. Ubiquinol Oxidation in the Cytochrome bc1 Complex: Reaction Mechanism and Prevention of Short-circuiting. Biochim. Biophys. Acta – Bioener. 2005, 1709, 5–34. (3) Senior, A. E. ATP Synthesis by Oxidative Phosphorylation. Physiol. Rev. 1988, 68, 177–231. (4) Murray, R. K.; Granner, D. K.; Mayes, P. A.; Rodwell, V. W. Harper’s Illustrated Biochemistry; McGraw-Hill, 2014. (5) Mitchell, P. The Protonmotive Q Cycle: a General Formulation. FEBS Lett. 1975, 59, 137–139. (6) Crofts, A. R.; Shinkarev, V. P.; Kolling, D. R.; Hong, S. The Modified Q-cycle Explains the Apparent Mismatch Between the Kinetics of Reduction of Cytochromes c1 and bH in the bc1 Complex. J. Biol. Chem. 2003, 278, 36191–36201. (7) Crofts, A. R. In The Enzymes of Biological Membranes; Martonosi, A. N., Ed.; Springer, 1985; Vol. 4; pp 347–382. (8) Brandt, U.; Trumpower, B. The Protonmotive Q cycle in Mitochondria and Bacteria. Crit. Rev. Biochem. Mol. 1994, 29, 165–197. (9) Gabellini, N. Organization and Structure of the Genes for the Cytochrome b/c1 Complex in Purple Photosynthetic Bacteria. A Phylogenetic Study Describing the Homology of the b/c1 Subunits Between Prokaryotes, Mitochondria, and Chloroplasts. J. Bioenerg. Biomembr. 1988, 20, 59–83. (10) Gennis, R. B.; Barquera, B.; Hacker, B.; Van Doren, S. R.; Arnaud, S.; Crofts, A. R.; Davidson, E.; Gray, K. A.; Daldal, F. The bc1 Complexes of Rhodobacter sphaeroides and Rhodobacter capsulatus. J. Bioenerg. Biomembr. 1993, 25, 195–209. ACS Paragon 25 Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(11) Rieske, J. S.; MacLennan, D. H.; Coleman, R. Isolation and Properties of an IronProtein from the (Reduced Coenzyme Q)-Cytochrome C Reductase Complex of the Respiratory Chain. Biochem. Biophys. Res. Commun. 1964, 15, 338–344. (12) Reedy, C. J.; Gibney, B. R. Heme Protein Assemblies. Chem. Rev. 2004, 104, 617– 650. (13) Crofts, A. R.; Barquera, B.; Gennis, R. B.; Kuras, R.; Guergova-Kuras, M.; Berry, E. A. Mechanism of Ubiquinol Oxidation by the bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors. Biochemistry 1999, 38, 15807–15826. (14) Barragan, A. M.; Crofts, A. R.; Schulten, K.; Solov’yov, I. A. Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome bc1 Complex. J. Phys. Chem. B 2014, 119, 433–447. (15) Cape, J. L.; Bowman, M. K.; Kramer, D. M. A Semiquinone Intermediate Generated at the Qo Site of the Cytochrome bc1 Complex: Importance for the Q-cycle and Superoxide Production. Proc. Natl. Acad. Sci. USA 2007, 104, 7887–7892. (16) Kolling, D. R.; Samoilova, R. I.; Holland, J. T.; Berry, E. A.; Dikanov, S. A.; Crofts, A. R. Exploration of Ligands to the Qi Site Semiquinone in the bc1 Complex Using High-resolution EPR. J. Biol. Chem. 2003, 278, 39747–39754. (17) Barragan, A. M.; Schulten, K.; Solov’yov, I. A. Mechanism of the Primary Charge Transfer Reaction in the Cytochrome bc1 Complex. J. Phys. Chem. B 2016, DOI: 10.1021/acs.jpcb.6b07394. (18) Postila, P. A.; Kaszuba, K.; Kuleta, P.; Vattulainen, I.; Sarewicz, M.; Osyczka, A.; Róg, T. Atomistic Determinants of Co-enzyme Q Reduction at the Qi-site of the Cytochrome bc1 Complex. Sci. Rep. 2016, 6 . ACS Paragon 26 Plus Environment

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(19) Dröse, S.; Brandt, U. The Mechanism of Mitochondrial Superoxide Production by the Cytochrome bc1 Complex. J. Phys. Chem. 2008, 283, 21649–21654. (20) Guillaud, F.; Dröse, S.; Kowald, A.; Brandt, U.; Klipp, E. Superoxide Production by Cytochrome bc1 Complex: A Mathematical Model. Biochim. Biophys. Acta – Bioener. 2014, 1837, 1643–1652. (21) Rottenberg, H.; Covian, R.; Trumpower, B. L. Membrane Potential Greatly Enhances Superoxide Generation by the Cytochrome bc1 Complex Reconstituted into Phospholipid Vesicles. J. Biol. Chem. 2009, 284, 19203–19210. (22) Husen, P.; Solov’yov, I. A. Spontaneous Binding of Molecular Oxygen at the Qo-Site of the bc1 Complex Could Stimulate Superoxide Formation. J. Am. Chem. Soc. 2016, 138, 12150–12158. (23) Salo, A.; Husen, P.; Solov’yov, I. A. Charge Transfer at the Qo-site of the Cytochrome bc1 Complex Leads to Superoxide Production. Submitted (24) Brand, M. D. The Sites and Topology of Mitochondrial Superoxide Production. Exp. Gerontol. 2010, 45, 466–472. (25) Crofts, A. R.; Lhee, S.; Crofts, S. B.; Cheng, J.; Rose, S. Proton Pumping in the bc1 Complex: A New Gating Mechanism That Prevents Short Circuits. Biochim. Biophys. Acta – Bioener. 2006, 1757, 1019–1034. (26) Song, Z.; Clain, J.; Iorga, B. I.; Yi, Z.; Fisher, N.; Meunier, B. Saccharomyces cerevisiae-Based Mutational Analysis of the bc1 Complex Qo Site Residue 279 To Study the Trade-Off between Atovaquone Resistance and Function. Antimicrob. Agents Chemother. 2015, 59, 4053–4058.



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(27) Lee, D.-W.; Selamoglu, N.; Lanciano, P.; Cooley, J. W.; Forquer, I.; Kramer, D. M.; Daldal, F. Loss of a Conserved Tyrosine Residue of Cytochrome b Induces Reactive Oxygen Species Production by Cytochrome bc1. J. Biol. Chem. 2011, 286, 18139– 18148. (28) Berry, E. A.; Huang, L.-S.; Saechao, L. K.; Pon, N. G.; Valkova-Valchanova, M.; Daldal, F. X-ray Structure of Rhodobacter capsulatus Cytochrome bc1: Comparison with its Mitochondrial and Chloroplast Counterparts. Photosyn. Res. 2004, 81, 251– 275. (29) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J. Comp. Chem. 2005, 26, 1781–1802. (30) MacKerell, A. D.; Feig, M.; Brooks, C. L. Extending the Treatment of Backbone Energetics in Protein Force Fields: Limitations of Gas-phase Quantum Mechanics in Reproducing Protein Conformational Distributions in Molecular Dynamics Simulations. J. Comp. Chem. 2004, 25, 1400–1415. (31) Mackerell, A. D. Empirical Force Fields for Biological Macromolecules: Overview and Issues. J. Comp. Chem. 2004, 25, 1584–1604. (32) Humphrey, W.; Dalke, A.; Schulten, K. VMD – Visual Molecular Dynamics. Journal of Molecular Graphics 1996, 14, 33–38. (33) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926–935. (34) Postila, P. A.; Kaszuba, K.; Sarewicz, M.; Osyczka, A.; Vattulainen, I.; Róg, T. Key Role of Water in Proton Transfer at the Qo-site of the Cytochrome bc1 Complex ACS Paragon 28 Plus Environment

Page 28 of 31

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Predicted by Atomistic Molecular Dynamics Simulations. Biochim. Biophys. Acta – Bioener. 2013, 1827, 761–768. (35) Kaszuba, K.; Postila, P. A.; Cramariuc, O.; Sarewicz, M.; Osyczka, A.; Vattulainen, I.; Róg, T. Parameterization of the Prosthetic Redox Centers of the Bacterial Cytochrome bc1 Complex for Atomistic Molecular Dynamics Simulations. Theor. Chem. Acc. 2013, 132, 1–13. (36) Aguayo, D.; González-Nilo, F. D.; Chipot, C. Insight into the Properties of Cardiolipin Containing Bilayers from Molecular Dynamics Simulations, Using a Hybrid All-atom/United-atom Force Field. J. Chem. Theory Comput. 2012, 8, 1765–1773. (37) Gnaiger, E.; Lassnig, B.; Kuznetsov, A.; Rieger, G.; Margreiter, R. Mitochondrial Oxygen Affinity, Respiratory Flux Control and Excess Capacity of Cytochrome c Oxidase. J. Exp. Biol. 1998, 201, 1129–1139. (38) Ewald, P. P. Die Berechnung optischer und elektrostatischer Gitterpotentiale. Ann. Phys. 1921, 369, 253–287. (39) Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An N log (N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089–10092. (40) Martyna, G. J.; Tobias, D. J.; Klein, M. L. Constant Pressure Molecular Dynamics Algorithms. J. Chem. Phys. 1994, 101, 4177–4189. (41) Feller, S. E.; Zhang, Y.; Pastor, R. W.; Brooks, B. R. Constant Pressure Molecular Dynamics Simulation: The Langevin Piston Method. J. Chem. Phys. 1995, 103, 4613–4621. (42) Lee, C.-Y. A Possible Biological Role of the Electron Transfer Between Tyrosine and Tryptophan. FEBS letters 1992, 299, 119–123. ACS Paragon 29 Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(43) Beene, D. L.; Price, K. L.; Lester, H. A.; Dougherty, D. A.; Lummis, S. C. Tyrosine Residues that Control Binding and Gating in the 5-hydroxytryptamine3 Receptor Revealed by Unnatural Amino Acid Mutagenesis. J. Neurosci. 2004, 24, 9097–9104. (44) Wellens, A.; Lahmann, M.; Touaibia, M.; Vaucher, J.; Oscarson, S.; Roy, R.; Remaut, H.; Bouckaert, J. The Tyrosine Gate as a Potential Entropic Lever in the Receptor-binding Site of the Bacterial Adhesin FimH. Biochemistry 2012, 51, 4790– 4799. (45) Sarewicz, M.; Osyczka, A. Electronic Connection Between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling. Physiol. Rev. 2014, 95, 219–243. (46) Zhang, Z.; Huang, L.; Shulmeister, V. M.; Chi, Y.-I.; Kim, K. K.; Hung, L.-W.; Crofts, A. R.; Berry, E. A.; Kim, S.-H. Electron Transfer by Domain Movement in Cytochrome bc1. Nature 1998, 392, 677–684. (47) Darrouzet, E.; Valkova-Valchanova, M.; Moser, C. C.; Dutton, P. L.; Daldal, F. Uncovering the [2Fe2S] Domain Movement in Cytochrome bc1 and its Omplications for Energy Conversion. Proc. Natl. Acad. Sci. USA 2000, 97, 4567–4572. (48) Tian, H.; White, S.; Yu, L.; Yu, C.-A. Evidence for the Head Domain Movement of the Rieske Iron-Sulfur Protein in Electron Transfer Reaction of the Cytochrome bc1 Complex. J. Biol. Chem. 1999, 274, 7146–7152. (49) Darrouzet, E.; Daldal, F. Protein-protein Interactions Between Cytochrome b and the Fe-S Protein Subunits During QH2 Oxidation and Large-scale Domain Movement in the bc1 Complex. Biochemistry 2003, 42, 1499–1507.


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Fig. 11: For Table of Contents Only


Mutations at the Qo Site of the Cytochrome bc1 Complex Strongly

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