Charge Transfer at the Qo-Site of the ... - ACS Publications

Article pubs.acs.org/JPCB

Charge Transfer at the Qo‑Site of the Cytochrome bc1 Complex Leads to Superoxide Production Adrian Bøgh Salo, Peter Husen, and Ilia A. Solov’yov* Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark ABSTRACT: The cytochrome bc1 complex is the third protein complex in the electron transport chain of mitochondria or photosynthetic bacteria, and it serves to create an electrochemical gradient across a cellular membrane, which is used to drive ATP synthesis. The purpose of this study is to investigate interactions involving an occasionally trapped oxygen molecule (O2) at the so-called Qo site of the bc1 complex, which is one of the central active sites of the protein complex, where redox reactions are expected to occur. The investigation focuses on revealing the possibility of the oxygen molecule to influence the normal operation of the bc1 complex and acquire an extra electron, thus becoming superoxide, a biologically toxic free radical. The process is modeled by applying quantum chemical calculations to previously performed classical molecular dynamics simulations. Investigations reveal several spontaneous charge transfer modes from amino acid residues and cofactors at the Qo-site to the trapped O2 molecule.

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INTRODUCTION The cytochrome bc1 complex,1 or ubiquinolcytochrome c oxidoreductase, is found in the inner mitochondrial membrane of eukaryotes and the plasma membrane of photosynthetically active bacteria.1−3 It is the third protein complex in the electron transport chain, which consists of four protein complexes that together serve to maintain an electrochemical gradient across the membrane.4,5 This gradient is fundamentally important as it constitutes the driving force for various biochemical processes, including the synthesis of adenosine triphosphate (ATP) molecules.4,6,7 The bc1 complex is found as a dimer in mitochondrial membranes of all animals and in photosynthetic membranes. In the case of Rhodobacter capsulatus, a species of purple bacteria, each monomer of the dimer consists of three different protein subunits, namely, cytochrome b, cytochrome c1, and a Rieske iron−sulfur protein (ISP), as depicted in Figure 1. The functioning of the bc1 complex delivers an active transport of protons across the membrane, as schematically depicted in Figure 1. This process is initiated upon binding of the substrate molecule quinol at the so-called Qo-site of the protein complex.8−11 Upon binding, the quinol loses one of its electrons, which is passed on to a cytochrome c2 protein in intermembrane space via the iron−sulfur (Fe2S2) cluster, depicted in Figure 2. This primary electron transfer happening at the Qo-site is the first redox step of the general functioning mechanism of the bc1 complex, also known as the Q-cycle, which is summarized graphically in Figure 1.8,10,12−14 In the Qcycle, a total of two quinol (QH2, also sometimes referred to as coenzyme Q10, CoQ10) molecules are bound and oxidized, releasing four protons to the intermembrane space while one © 2016 American Chemical Society

quinone (Q) molecule is reduced by absorbing two protons from the negative side of the membrane.3,10 It is believed that the electron transport chain, and in particular the bc1 complex, is involved in the creation of superoxide,9,15−17 and previous investigations suggest that, during the Q-cycle, oxygen molecules are accidentally trapped near the Qo-site,18,19 and might occasionally be converted to radicals, in particular into superoxide (O2•−), with a further release into the intermembrane space.16 Superoxide is a highly reactive radical anion, and is toxic to the cells even in nanomolar concentrations.20−22 Several studies suggest a relation between the oxidative damage caused by the superoxide radicals and the cellular aging process,15,23−27 which generally became known as the mitochondrial free radical theory of aging.25,28 The present investigation aims to uncover microscopic details about the superoxide anion production at the Qo-site of the bc1 complex, building upon previous studies which have suggested that it forms as a byproduct during intermediate stages of the Q-cycle.9,16,18 Particularly, we use the results of classical molecular dynamics simulations18 of the membrane embedded bc1 complex for defining realistic configurations of the Qo-site, and employ further quantum chemical calculations to describe electron transfer to oxygen. A quantum mechanical description of the process allows establishing the energetics of oxygen binding at the Qo-site and analyzing the spin states of the key fragments involved in the electron transfer processes. Received: October 14, 2016 Revised: November 29, 2016 Published: November 30, 2016 1771

DOI: 10.1021/acs.jpcb.6b10403 J. Phys. Chem. B 2017, 121, 1771−1782

The Journal of Physical Chemistry B

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METHODS

The present study relies on a recent molecular dynamics (MD) study18 of the bc1 complex from Rhodobacter capsulatus (PDB ID: 1ZRT29), and investigates the interaction involving a trapped O2 molecule at the Qo-site through quantum chemical calculations. Setting the Stage. The chosen redox configuration of the bc1 complex is consistent with the recent MD study.18 In particular, the system was considered in a state where the quinol was oxidized, i.e., present in its semiquinone anion (Q•−) form. This state is achieved by dissociating two protons from the bound QH2 molecule and transferring an electron to the iron−sulfur cluster. The semiquinone state of the substrate is short-lived,18,30,31 and the probability of finding the bc1 complex with the substrate in the Q•− form is argued to be on the order of ≃4 × 10−8.18,31 It is, however, this state that is of interest in the context of the present investigation, as it is widely believed that this unstable semiquinone might act as a direct electron donor to the oxygen molecule.3,9,30,32 The two protons from the QH2 were assumed to be transferred to the H156 residue of the ISP subunit, and indirectly to the E295 residue of cyt b, located near the quinol on the edge of the Qo-site as shown in Figure 3, opposite the Fe2S2-cluster. Molecular Dynamics Simulation. The simulated system, as taken from a previous investigation,18 consists of a dimeric bc1 protein complex embedded into a membrane patch, which has been suspended in a water box of 197 Å × 177 Å × 142 Å size using the TIP3P water model33 and a NaCl salt concentration of 0.05 mol/L, also used to neutralize the system. O2 molecules were added into the water phase with a concentration of ∼100 mmol/L, specifically 165 O2 molecules in total. After equilibration of the system, where O2 has diffused into the membrane, the concentration of O2 molecules in the water phase becomes ∼21 mmol/L. This concentration is

Figure 1. Schematic representation of the Q-cycle of the cytochrome bc1 complex. The major steps of electron and proton transfers, induced upon quinol (QH2) and quinone (Q) bindings at the Qo- and Qi-sites, are indicated. Effectively, each of the two monomers of the bc1 complex absorbs two protons from the space within the inner membrane and releases four protons to the intermembrane space, thus maintaining an electrochemical gradient. The bc1 complex, as part of the electron transport chain, plays a vital role in the synthesis of ATP. From each QH2 bound at the Qo-site, one electron travels to the Qisite, while the other one is transferred via cytochrome c1 to a cytochrome c2 from the intermembrane space, which then transports the electron to the so-called complex IV, the fourth protein complex of the electron transport chain.

Various reaction pathways leading to the formation of a superoxide molecule at the Qo-site are observed and characterized, suggesting that the process is indeed plausible.

Figure 2. bc1 complex from Rhodobacter capsulatus10 embedded in a lipid membrane. (A) Highlighted are the cytochrome b (cyt b), cytochrome c1 (cyt c1), and Rieske iron−sulfur proteins (ISP), the latter containing an iron−sulfur (Fe2S2) cluster, which is involved in the initial electron transfer of the Q-cycle.4,10 (B) Internal view of the Qo-site, where the initial electron and proton transfers of the Q-cycle take place. An oxygen molecule, O2, is also indicated in balls-and-sticks representation, positioned in one of its possible localization sites.18 1772


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Figure 3. Qo-site and quinol binding. Quinol molecule transfers an electron to the iron−sulfur cluster, and through intermediate processes releases two protons into the intermembrane space. Before being released into the intermembrane space, the two protons detach from the quinol and go to the H156 residue of the ISP subunit and to the E295 residue of the cyt b, respectively. This leaves the radical semiquinone (Q•−) bound at the Qosite and represents the redox state of the substrate molecule, used in the present study.

significantly higher than the physiological value,18,34 allowing more observations within time scales reachable by atomistic MD simulations. The validity of using such exaggerated concentrations in the model has been tested through auxiliary simulations with low O2 concentrations.18 The MD simulations were performed using NAMD 2.1035 with CHARMM36 force field paramters with CMAP corrections for proteins.36 Further details on the MD simulation protocol can be found in the original publications.18,19 The simulated MD trajectories were analyzed using VMD.37 In particular, O2 molecules near the Qo-site of the bc1 complex were dynamically tracked, and the trapping time for each O2 molecule at the Qo-site was established. The detailed description of O2 diffusion and binding at the Qo-site is discussed in our earlier publication.18 The simulated trajectories revealed several events, where an O2 molecule was trapped at the Qo-site for several hundreds of picoseconds to well over 10 nanoseconds. From a wide range of these identified events, 14 snapshot configurations of the bc1 complex with the bound O2 molecule were selected at random for quantum chemistry analysis, such that the O2 molecule was found within a 5 Å distance from the semiquinone headgroup. Such configurations were considered the most promising states where charge transfer to the oxygen could spontaneously occur. Quantum Chemistry Calculations. The 14 snapshot configurations of the bc1 complex taken from MD simulations were prepared for quantum chemical calculations using VMD37 and the plugin called Molefacture. Since it is presently impossible to consider the entire bc1 complex quantum mechanically, we have for each of the chosen MD snapshots extracted a local environment consisting of several amino acid residues at the Qo-site to model the O2 binding event and the subsequent possible electron transfer. Particularly, we have chosen the residues in direct contact with the O2 molecule, all the charged residues at the Qo-site, except the E295 residue, as well as all the polar ones. While the E295 residue is involved in the proton movement at the Qo-site, it was not included in the quantum mechanical model, partly due to its distance from the proposed reaction center of the superoxide generation, and partly because it is not part of the normal electron transfer pathways away from the Qo-site.32 The resulting Qo-site model included the Fe2S2 group from the ISP, with the attached C133, C155, H135, H156 residues,

the semiquinone molecule headgroup, and the side chains of nearby amino acids, in particular of residues L136, C138, and C155 of the ISP, residues Y147, Y302, V161, I162, L165, L305, F298, R306, and M336 of the cyt c1, and four water molecules found within the Qo-site. In the selected amino acid residues we have considered only side chains, replacing the Cα atoms with CH3 groups using the VMD plugin Molefacture. Four water molecules were also included in the computational model, as they turned out to be close to the reaction site, and the O2 molecule in particular. All the 14 snapshot structures had a total of 263 atoms. The quantum chemical calculations were then carried out using the Gaussian 09 software package,38 where each considered Qo-site configuration has been divided into 12 fragments as summarized in Table 1 and Figure 4. The subdivision into fragments was necessary to define specific redox and spin states Table 1. Summary of Fragment Definitions Used in the Quantum Chemistry Calculationsa fragment no.

formal charge

formal multiplicity

notes

1 2 3 4 5 6 7 8 9 10 11 12

3 2 −2 −2 −1 −1 0 0 −1 0 0 1

−6 5 1 1 1 1 1 1 2 1 3 1

FeA FeB SA SB C133 C153 H135 H156 Q•− C138, C155 O2 the rest

a

The computational model of the Qo-site was subdivided into fragments to improve the initial guess of the wave function in the quantum chemistry calculations. Fragments 1−4 make up the Fe2S2 cluster; fragments 5−8 are the cysteines and histidines attached. Fragment 9 includes the semiquinone headgroup, and fragment 10 includes C138, C155 of the ISP; fragment 12 constitutes the rest of the Qo-site (L136 of the ISP, and Y147, Y302, V161, I162, L165, L305, F298, R306, M336 of cytochrome c1, as well as four water molecules). Fragment 11 includes the O 2 molecule considered in the computations. Figure 4 visually shows the fragment setup. 1773


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calculated for the O2 and the remaining system separately on the basis of the structures of the optimized configurations of the 14 Qo-site models. The electronic densities of the unpaired electrons were determined by calculating the total spin density of the αelectrons (those that have a positive projection of the spin on the principal quantization axis) minus the total spin density of β-electrons (which have a negative projection value on the quantization axis). The individual spin densities of all α- and βelectrons have been computed after solving the self-consistent field equations, which also allowed us to obtain the partial charges and spin density values for all compounds of the Qosite.

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RESULTS The present study aims to investigate quantum mechanically the possibility of superoxide production at the Qo-site of the bc1 complex. Specifically, we focus on the role played by the Fe2S2 cluster, various nearby amino acid residues at the Qo-site, and the extent to which the geometrical position of the oxygen molecule relative to the residues at the Qo-site impacts the possible electron trapping by the O2 molecule. First, the energetics of the investigated Qo-site configurations are discussed. Then, an examination of the spin densities follows, revealing several different electron transfer scenarios, and finally the spatial configuration of the investigated Qo-site configurations is detailed. Energetics of Oxygen Binding at the Qo-Site. The total energies, as used in eq 1, for the optimized Qo-site configurations at different time instances are summarized in Table 2. Figure 5 shows the computed interaction energies between the O2 molecule and the Qo-site of the bc1 complex. The two plots in Figure 5A,B illustrate that the interaction energy of the bound oxygen molecule is 2 orders of magnitude greater for five distinct Qo-site configurations, namely, configurations 5, 6, 7, 10, and 11. These configurations correspond to the situations where a spontaneous electron transfer to the trapped oxygen molecule occur, as follows from the analysis of spin densities compiled in Tables 3 and 4. Here, the spin density values have been computed for each atom, and summed fragmentwise according to the definition in Table 1 and Figure 4. Upon acquiring an electron, the O2 molecule becomes charged, resulting in much stronger Coulomb interactions in the cases where a charge transfer event has occurred, resulting in a noticeable difference in the interaction energy. The results of the quantum chemical calculations, summarized in Figure 5 and Table 2, suggest that it is energetically favorable for an O2 molecule to acquire an electron upon binding, but it also suggests that there are several factors determining whether spontaneous electron transfer is possible, in particular which residues at the Qo-site are capable of donating the electron, and the specific electron transfer pathways leading to such an event. All configurations of the Qosite have been assigned a two-symbol notation, indicating the selected snapshot number and the direction of the O2 spin, being either up (u) or down (d). The magnitude of the interaction energies depicted in Figure 5A indicates that the interaction is weak and is governed by van der Waals forces. Spin Density Analysis. In the following, the spin densities of the fragments defined in Table 1 and Figure 4 are analyzed in order to identify the possible electron donors to a trapped O2

Figure 4. Fragment definition used to set up the quantum chemistry calculations. The Qo-site model has been subdivided into 12 separate fragments in order to define the molecules and atoms of interest in a particular redox and spin state. Especially, the atoms in the Fe2S2 cluster (fragments 1−4) with their attached amino acids (fragments 5−8), as well as the semiquinone and oxygen (fragments 9 and 11, respectively), were handled carefully, so as to ensure that there is a proper antiferromagnetic coupling between the two iron atoms within the Fe2S2, and that the semiquinone is modeled charged prior to a possible electron transfer to the O2 molecule. Fragment definitions and the corresponding residues are summarized in Table 1.

of the Qo-site components, thereby allowing us to improve the initial guess, but no further constraints were imposed on the spin configurations during calculations. Results have been analyzed utilizing a combination of VMD37 and ChemCraft 1.7.39 For the quantum chemical calculations, the CAM-B3LYP hybrid DFT method was employed.40 The Qo-site models were structurally optimized employing the 6-31G basis set.40−42 During optimization, the coordinates of the Cα atoms were frozen to conserve the conformation of the Qo-site obtained from the MD simulations. Since ground state oxygen is populating a triplet state,43 all 14 systems were prepared for the calculations with the O2 molecule having two different projections of its total spin on the principal quantization axis, either pointing upward or downward, yielding a total of 28 structures to be considered. Detailed quantum chemistry analysis was carried out on the optimized structures, employing the 6-311G(d) basis set44 for expanding the electronic wave functions. This basis set yields the triple-ζ accuracy, and has been shown to correctly model the behavior of iron−sulfur clusters,10,42,45−47 including correctly taking into account the antiferromagnetic coupling of the two iron atoms in the Fe2S2 cluster of the ISP subunit, specifically.42,45,48 The interaction energy between the O2 molecule and the rest of the system was derived from the quantum chemistry calculations as ΔE = Etotal − EQ o − EO2

(1)

where Etotal is the total energy of the system (all 263 atoms), EQo is the energy of the system with the O2 molecule removed, and EO2 is the energy of the oxygen molecule. To compute the interaction energies, ΔE’s, the single point energies were 1774


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The Journal of Physical Chemistry B Table 2. Energetics of O2 Binding at the Qo-Sitea configuration

O2 spin

Etot (au)

EQo (au)

EO2 (au)

ΔE (kcal/mol)

1u 1d 2u 2d 3u 3d 4u 4d 5u 5d 6u 6d 7u 7d 8u 8d 9u 9d 10u 10d 11u 11d 12u 12d 13u 13d 14u 14d

↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓

−9704.050037 −9704.042460 −9704.075025 −9704.078681 −9704.047388 −9704.043869 −9704.040092 −9704.040095 −9704.107258 −9704.055644 −9704.076526 −9704.085770 −9704.080974 −9704.080792 −9704.042758 −9704.048386 −9704.029003 −9704.029002 −9704.084452 −9704.054682 −9704.085829 −9704.077012 −9704.048619 −9704.048665 −9704.040079 −9704.040055 −9704.035724 −9704.035724

−9553.728268 −9553.718875 −9553.753522 −9553.753870 −9553.726494 −9553.722992 −9553.719588 −9553.719588 −9553.579702 −9553.548918 −9553.578268 −9553.590945 −9553.582079 −9553.582028 −9553.722054 −9553.727599 −9553.708211 −9553.708210 −9553.593576 −9553.581427 −9553.598663 −9553.575299 −9553.725140 −9553.725139 −9553.718012 −9553.717983 −9553.714122 −9553.714127

−150.3166799 −150.3167707 −150.3165502 −150.3165295 −150.3164112 −150.3164004 −150.3163321 −150.3163316 −150.3065786 −150.3064284 −150.3066640 −150.3066161 −150.3068311 −150.3068292 −150.3163827 −150.3163740 −150.3163830 −150.3163823 −150.3065529 −150.3055095 −150.3054953 −150.3068094 −150.3166118 −150.3166174 −150.3166207 −150.3166158 −150.3165320 −150.3165318

−3.1936 −4.2760 −3.1080 −5.1967 −2.8131 −2.8090 −2.6178 −2.6207 −138.6681 −125.6903 −120.2292 −118.1050 −120.5237 −120.4430 −2.7118 −2.7691 −2.7670 −2.7674 −115.6660 −105.2637 −114.0020 −122.3062 −4.3096 −4.3352 −3.4177 −3.4237 −3.1815 −3.1784

The total energies, Etot’s, of the Qo-site computed for 14 configurations assuming two projections of O2 spin, the partial energies of the Qo-site (EQo) and of the O2 molecule (EO2), as used in eq 1 to compute the O2 interaction energy ΔE. Qo-site configurations 5, 6, 7, 10, and 11 stand out with respect to the interaction energy, ΔE, as the values are around 2 orders of magnitude greater than the values for the remaining configurations. The values for Etot, EQo, and EO2 are given in atomic units (au) as used internally in Gaussian, where 1 au ≈ 627.503 kcal/mol, which, for the sake of clarity, is the unit used here for ΔE. All configurations of the Qo-site were assigned a two-symbol notation indicating selected snapshot and direction of the O2 spin, being up (u) or down (d). a

Figure 5. Interaction energies between the O2 molecule and the Qo-site. The Qo-site configurations are numbered as they appear in Table 2, with each configuration represented with two spin projetions of the O2 molecule (u = up and d = down). (A) The interaction energy of the oxygen molecule with the Qo-site calculated using eq 1 in the systems where no charge transfer occurred. (B) The interaction energy between oxygen and the Qo-site computed for configurations with spontaneous charge transfer. The red lines indicate the average values. The average energy in the case without a charge transfer is −3.77 kcal/mol, while in the case of a spontaneous charge transfer event it is −122.7 kcal/mol.

molecule at the Qo-site. Inspection of the spin density values in Tables 3 and 4 suggests that electron transfer to the O2

molecule could involve a manifold of possibilities, as different parts of the Qo-site turn out to be potential electron donors. 1775


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The Journal of Physical Chemistry B Table 3. Spin Densities Subdivided into Fragments for Qo-Site Configurations without Charge Transfera Qo-site

fragments

conf

O2 spin

1 FeA

2 FeB

3 SA

4 SB

5 C133

6 C153

7 H135

8 H156

9 Q•−

10 O2

1u 1d 2u 2d 3u 3d 4u 4d 8u 8d 9u 9d 12u 12d 13u 13d 14u 14d av av

↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓

−3.92 −3.91 −3.91 −3.92 −3.93 −3.89 −3.91 −3.91 −3.89 −3.89 −3.90 −3.90 −3.91 −3.91 −3.93 −3.93 −3.93 −3.93 −3.92 −3.91

3.62 3.62 3.62 3.65 3.62 3.61 3.62 3.62 3.62 3.61 3.61 3.61 3.62 3.62 3.62 3.62 3.62 3.62 3.62 3.62

−0.17 −0.18 −0.18 −0.17 −0.15 −0.18 −0.16 −0.16 −0.17 −0.17 −0.17 −0.17 −0.17 −0.17 −0.15 −0.15 −0.14 −0.14 −0.17 −0.18

−0.14 −0.14 −0.15 −0.21 −0.14 −0.14 −0.16 −0.16 −0.14 −0.13 −0.13 −0.13 −0.15 −0.15 −0.15 −0.15 −0.16 −0.16 −0.14 −0.14

−0.22 −0.21 −0.24 −0.21 −0.22 −0.23 −0.24 −0.24 −0.23 −0.24 −0.23 −0.23 −0.22 −0.22 −0.22 −0.22 −0.22 −0.22 −0.22 −0.21

−0.22 −0.22 −0.19 −0.20 −0.23 −0.23 −0.20 −0.20 −0.23 −0.23 −0.23 −0.23 −0.22 −0.22 −0.21 −0.21 −0.21 −0.21 −0.22 −0.22

0.03 0.03 0.03 0.05 0.03 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03

0.01 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.02

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 1.00 1.00 1.00 1.00 1.00 1.00

2.00 −1.99 2.00 −2.00 2.00 −2.00 2.00 −2.00 2.00 −2.00 2.00 −2.00 2.00 −2.00 2.00 −2.00 2.00 −2.00 2.00 −1.99

a The spin densities of the fragments defined in Figure 4 and Table 1 are compiled for those Qo-site configurations where no spontaneous electron transfer occurred. Two projections (u) and (d) of the O2 total spin are considered. From the average values of each column shown in the bottom row, one observes that only small deviations of the individual spin densities of the fragments are present.

Table 4. Summary of Spin Densities Subdivived into Fragments for Configurations with Spontaneous Charge Transfer to the O2 Moleculea

a Similar to Table 3. The text in blue marks significant changes to the spin density on the respective fragments indicating the origin of the electron. The text in red indicates the change in spin density for the electron acceptor, which is the O2 molecule in all cases.

FeA atom of the Fe2S2 cluster. This is the situation depicted in Figure 7A. Once the O2 spin is oppositely aligned to the unpaired FeB electrons (configurations 6d, 10d, 11d), it is only the sulfur atoms of the Fe2S2 cluster donating the electron. It is worth noting that the spin density isosurfaces generated for the cases where only the SA and SB atoms are involved in the electron transfer to the trapped O2 molecule all look similar, as visualized in Figure 7B through the difference spin density surfaces. Configuration 5d, with the spin of the O2 molecule and FeA atom aligned, stands out, as it is the semiquinone, Q•−, that donates the electron here. Finally, configurations 7u and 7d stand out in their own right, as they show similar behavior with both O2 spin orientations, and not only the SA and SB atoms of the Fe2S2 cluster but also the C133 and C153 amino acid residues are involved in the electron transfer process. These two configurations are depicted in Figure 8. Q o -site

The situation where no charge transfer is happening is visualized in Figure 6. Here, the difference between the total spin of all α-electrons (spin up) and β-electrons (spin down) is shown. Additionally, Table 3 shows that all the computed Qosite configurations where no charge transfer is happening have very similar spin density values with a variation of ±0.07. Four different cases where an electron transfer was observed are visualized in Figures 7 and 8. For all renderings of spin densities in Figures 6−8, the isovalue ±0.02 has been used, and the spin density differences have been color coded such that positive values of the difference spin densities are represented with a blue color, while orange represents negative values. A closer examination of the spin densities in Table 4 reveals that in 3 of the cases where spontaneous charge transfer to an O2 molecule takes place (Qo-site configurations 5u, 10u, 11u), the FeB, SA, and SB atoms coherently act as electron donors, while the O2 spin is antiparallel to the unpaired electrons of the 1776


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Figure 6. Spin density isosurfaces of the Qo-site without charge transfer. The densities shown represent the difference between the spin densities of the α-electrons and the β-electrons. The differences in spin densities are color coded such that blue shows the positive value, while orange represents the negative value. The isovalue of ±0.02 was used in the plot. The triplet diradical state of the oxygen is clearly visible, and a single unpaired electron is delocalized on the semiquinone. The electron densities on the two iron atoms of the Fe2S2 cluster seem similar, but the FeB atom features some irregularities, indicating a lower electronic spin density. Only fragments with nonvanishing difference in spin densities at the Qosite are shown.

Figure 8. Further Qo-site configurations where spontaneous electron transfer is possible. The difference densities of α- and β-electrons are plotted similarly to those in Figures 6 and 7. (A) Electron densities in configurations 7u and 7d. In this case, the electron donated from the Fe2S2 cluster was delocalized also over the sulfur atoms of the coordinating cysteine residues, C133 and C153, by a significant contribution, leading to unpaired electron density at these atoms. Only one of the cases is shown here, but data suggests, see Table 4, that the situation is very similar regardless of the orientation of the O2 electron spin. (B) Depiction of Qo-site configuration 5d in the case of O2 electron spin antiparallel to the FeB atom. Here, the semiquinone, Q•−, acts as the sole electron donor to the O2 molecule.

orientation, indicating that this specific configuration is independent from the spin state of the trapped oxygen molecule. Here, the coordinating cysteine residues take part in the charge transfer from the Fe2S2 cluster. In Qo-site configuration 5d with the O2 electrons having spin antiparallel to the FeB atom, the semiquinone acts as the sole electron donor, see Figure 8B. Put together, the spin analysis indicates that while the semiquinone can act as the electron donor, it is actually possible that the additional electron on the O2 molecule is donated by the Fe2S2-cluster or its coordinating amino acids. Spatial Configurations of the Qo-Site. Analysis of the geometrical arrangement of the investigated configurations of the Qo-site indicates that water molecules play a significant role in providing the electron transfer pathways mediating the creation of superoxide at the Qo-site. Most of the configurations with no charge transfer had the O2 molecule at relatively large distances to the Fe2S2 cluster, typically above 9 Å. Proximity to the semiquinone molecule, however, does not seem to contribute to the creation of superoxide. The distance to the semiquinone, measured from the center of the O2 molecule to the center of the semiquinone ring, is in the range 6−9 Å for the configurations where a spontaneous charge transfer was observed. These values are close to the distance where no charge transfer was observed, as it was found to be in the range 5−9 Å. It is clear that while distance to both the Fe2S2-cluster

Figure 7. Spontaneous charge transfer from the Fe2S2 cluster to the O2 molecule. The difference spin densities for Qo-site configurations with spontaneous electron transfer are plotted similarly to those in Figure 6. (A) In Qo-site configuration 10u, also representative of configurations 5u and 11u, the O2 molecule acquires an electron from the FeB atom, leaving it with the same number of unpaired electrons as the FeA atom. (B) In Qo-site configuration 10d, also representative of configurations 6u, 6d, 10d, and 11d, the SA and SB atoms of the Fe2S2 cluster act as the electron donors to the O2 molecule, without the direct involvement of the FeB atom. Note that only fragments where a nonvanishing difference of electron spin density for α- and β-electrons are shown.

configurations 7u and 7d, see Figure 8A, display very similar behavior for both orientations of the O2 electron spin 1777


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Figure 9. Position of the O2 molecule at the Qo-site is important for the spontaneous electron transfer. (A) In configuration 5d the oxygen molecule is located between the Y302 residue and the semiquinone. The O2 molecule has formed a hydrogen bond with a water molecule bound to Q•−, Y302 and H156. (B) In configuration 12d no charge transfer was observed, but its geometrical similarity to structure A suggests that hydrogen bonds with water molecules might have significance. It is noteworthy that there is no hydrogen bond between the O2 and the water molecule here. (C) Configuration 11u, which is structurally similar to configuration 10u, features an electron transfer to the O2 molecule from the FeB, SA, and SB atoms, and features a hydrogen bonding network with water molecules between the Y302 residue on one side, and the H135 residue of the Fe2S2 cluster on the other side. Configurations 10d and 11d feature similar structures. (D) Configurations 7u and 7d, where the spin orientation of the O2 electrons has no influence. The oxygen molecule is connected with both histidines of the Fe2S2 cluster through hydrogen bonds with water molecules.

and the semiquinone molecule matters, it does not in itself provide much insight. Figure 9 depicts the spatial layout of the Qo-site in four different configurations.A comparison of configuration 5d and 12d suggests that water molecules at the Qo-site play a significant role in mediating the spontaneous electron transfer to the O2 molecule. In Figure 9A, where the O2 molecule has acquired an electron from the semiquinone, the oxygen is located between the semiquinone and the Y302 residue of cytochrome c1. These three molecules are bound through hydrogen bonds to a water molecule connected to the H156 residue of the Fe2S2 cluster of the ISP. In Figure 9B, where no charge transfer was observed even though the O2 molecule is roughly in the same position between the semiquinone and the Y302 residue, there is no hydrogen bond interaction between the O2 molecule and the water molecule. The water molecule has instead formed a second hydrogen bond to the semiquinone. The Qo-site configuration 11u, depicted in Figure 9C, also serves to visualize the spatial arrangement of configurations 10u, 10d, and 11d due to their high structural similarity. The O2

molecule is bound through a hydrogen bond with two water molecules connected to the Y302 residue of the cytochrome c1, and the H135 residue of the ISP Fe2S2 cluster, respectively. All these cases feature an electron transfer from the Fe2S2 cluster. Both 10u and 11u feature the FeB, SA, and SB atoms as electron donors, whereas in configurations 10d and 11d, only the SA and SB are involved. The high degree of structural similarity between these four configurations supports the notion that the orientation of the O2 spin does not significantly influence whether or not a spontaneous electron transfer occurs. Figure 9D depicts Qo-site configurations 7u and 7d, where the same electron transfer scenario was observed for both O2 spin projections. Here, the O2 molecule is located close to the Y302 residue, and several water molecules form a hydrogen bond network around it. These water molecules in turn form hydrogen bonds with the semiquinone and the H156 residue of the Fe2S2 cluster on one side, and the H135 residue of the Fe2S2 cluster on the other. Altogether the performed analysis indicates that the spin orientation of the electrons of the O2 molecule and the specific geometry of the Qo-site have a large influence on which 1778


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change in spin density is observed for the FeB, SA, and SB atoms. The electron is bound more weakly to the FeB atom, as compared to the other five electrons of the FeA atom according to Hund’s rule, and therefore, it can be transferred if the energetics of O2 binding would permit it. Transferring the electron from the FeB atom would leave it with five unpaired electrons, which, according to Hund’s rule, is an energetically favorable state for iron. In case II, the Qo-site configuration 5d features a direct electron transfer from the semiquinone (Q•−) radical to the O2 molecule, with the semiquinone being the sole electron donor. In this case, with the O2 spin projection antiparallel to that of the FeB of the Fe2S2 cluster, an electron transfer from the FeB is not possible. However, case II is unique among the obtained results in that no atoms of the Fe2S2 cluster, or its coordinating residues, have been involved in the charge transfer reaction. Geometrically, this case is very similar to configuration 12 where no charge transfer event was observed, and the visual analysis, depicted in Figure 9, suggests that hydrogen bonds with water molecules enable the electron transfer to the O2 molecule. In case III, one observes that configurations 6u, 6d, 10d, and 11d feature an electron transfer event from the Fe2S2 cluster of the ISP to the trapped O2 molecule without the involvement of the FeB atom. When the O2 spin projection is oppositely aligned to that of the FeB valence electrons, an electron transfer event involving the unpaired electron on the FeB atom is energetically unfavorable, and in this case, the electron donors are the SA and SB atoms. Even with O2 spin up, electron transfer from SA and SB alone to the O2 molecule is possible, as observed for configuration 6u. While this behavior is schematically depicted differently from case I, there are no prominent features in the spatial configuration of the Qo-site to suggest any noticeable difference. It turns out that an electron transfer from SA/SB is possible, as even though each of these sulfur atoms has a closed shell electronic strucutre, this is different in reality, as all the electrons of the Fe2S2 would appear largely delocalized in the entire cluster. Finally, in case IV, the Qo-site configurations 7u and 7d feature an electron transfer from the C133 and C153 residues, which coordinate the Fe2S2 cluster, in addition to the SA and SB atoms of the Fe2S2 cluster itself. It is noteworthy that this behavior is observed for both orientations of the O2 spin, and is similar to the scenario described in case III. The results of the performed investigations demonstrate that, even in cases where the electrons on the FeB atom and the O2 molecule are not aligned favorably for an electron transfer event, the transfer could still occur spontaneously by involving the sulfur atoms of the Fe2S2 cluster, independent of the spin orientation of the radicals on the O2 molecule.

residues will act as electron donors. In most cases (all but one), either the Fe2S2 cluster itself, or its coordinating amino acid residues, C133 and C153, are involved, and in just a single case, the electron is donated directly from the Q•−.

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DISCUSSION The chosen configurations of the Qo-site with the bound semiquinone forms the basis for the present study, where interactions between a trapped O2 molecule and the entire Qosite are investigated. The semiquinone state is achieved through one electron and two proton transfers from the quinol (QH2) molecule, as schematically depicted in Figure 10; here an electron from the QH2 molecule is transferred and becomes coupled with one of the unpaired valence electrons of the FeB atom of the Fe2S2 cluster of the ISP.

Figure 10. Schematic representation of the primary electron transfer from the QH2 substrate molecule to the ISP. The ground electronic state of the Fe2S2 cluster of the ISP features antifferomagnetic coupling between the electrons of the iron atoms. The electron from the quinol molecule is transferred to the Fe2S2 cluster of the ISP subunit, where it pairs with one of the valence electrons of the FeB atom, thus leaving the quinol in the radical semiquinone state. The FeB atom is considered to be the primary electron acceptor here, as it is closer to the bound QH2 at the Qo-site. See Figure 3 for visual depiction.

It must be noted that the O2 molecule in its ground state is a triplet diradial,43 and thus has two unpaired valence electrons, occupying two molecular orbitals, with spin projections pointing in the same direction on the principal quantization axis. This in turn means that there are three different configurations of the O2 molecule that can be considered. For the purpose of this study, most interesting are the cases where the two reactive electrons have parallel spin projections, being either both spin up or both spin down. Both situations must be considered when performing quantum chemical calculations, as the spin orientation of the electrons on the O2 might readily influence the characteristics of the electron transfer reactions. To provide a clearer interpretation of the electron transfers summarized in Table 4, the most important possible electron transfer events to the oxygen molecule are summarized schematically in Figure 11, which depicts the four identified cases of electron transfer events. Figure 11A shows the cases where the O2 spin projection is parallel to the spin of the FeB valence electrons (O2 spin up), while in Figure 11B, the O2 spin is oppositely directed (O2 spin down). The four electron transfer cases are discussed below in detail. Note that Figure 11 serves an illustrative purpose, and the electrons are assigned to certain fragments following the formal notations introduced in Table 1. Case I corresponds to an electron transfer event observed at the Qo-site configurations 5u, 10u, and 11u, where the paired electron from the FeB atom is transferred to the O2 molecule. It follows from the numerical values in Table 4 that the electron has been delocalized around the Fe2S2 cluster, and therefore, a

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CONCLUSION The details of the electron transfer modes at the Qo-site of the bc1 complex, the third protein complex in the electron transport chain, which drives the synthesis of ATP, have been investigated in order to gain a deeper understanding of the processes that lead to possible superoxide production. It is theorized that reactive oxygen species are a major cause of cellular aging. A combination of molecular dynamics simulations and quantum chemical calculations has been employed to reveal several possible charge transfer modes from the components of the Qo-site to an O2 molecule trapped there. 1779


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Figure 11. Electronic diagrams of possible charge transfer pathways leading to superoxide production. The four identified cases are described in the text. Case I: An electron is transferred directly from the FeB atom of the Fe2S2 cluster, corresponding to the transfers observed in Qo-site configurations 5u, 10u, and 11u. Case II: An electron is transferred directly from the Q•− molecule. This is the case observed in Qo-site configuration 5d. Case III: A delocalized electron is transferred from the SA and SB atoms of the Fe2S2 cluster. This behavior was observed for Qo-site configurations 6u, 6d, 10d, and 11d. Case IV: In configurations 7u and 7d, i.e., for both spin orientations, the electron donated to the O2 molecule originated from the Fe2S2 cluster, but was also delocalized over the covalently bound sulfur atoms of the C133 and C153 residues of the ISP, as illustrated in Figure 8A.

The present study serves as a proof of concept; the calculations show that there is a solid possibility for a trapped O2 molecule to acquire an electron at the Qo-site, thereby disturbing the regular function of the bc1 complex and releasing a harmful superoxide to the cell interior. The computational results reveal several potential electron donor residues. Specifically, situations were identified where either the Fe2S2 cluster, its coordinating cysteine residues, or the semiquinone anion acted as an electron donor. The involvement of the Fe2S2 cluster through one of its iron atoms is dependent on the relative electron spin orientation compared to the O2 molecule, but does not otherwise seem to be a significant limiting factor.

The number of different configurations of the Qo-site is sufficient to demonstrate several possible electron transfer reactions at the Qo-site of the bc1 complex in the presence of an O2 molecule and, given the outcome of the quantum chemical calculations, serve well to illustrate that electron transfer from residues involved in quinol binding at the Qo-site to a nearby O2 molecule is possible. In fact, in approximately one-third, i.e., 10 out of 28 of the investigated systems, there was a spontaneous electron transfer event happening. While the systems were selected randomly from the MD simulations,18 more data is needed in order to give a proper estimate of the electron transfer rate constant. 1780


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Cytochrome bc1 and Their Mechanistic Implications. J. R. Soc., Interface 2016, 13, 20160133. (12) Mitchell, P. The Protonmotive Q Cycle: A General Formulation. FEBS Lett. 1975, 59-2, 137−139. (13) Trumpower, B. L. Evidence for a Protonmotive Q Cycle Mechanism of Electron Transfer Through the Cytochrome bc1 Complex. Biochem. Biophys. Res. Commun. 1976, 70, 73−80. (14) Crofts, A.; Hong, S.; Ugulava, N.; Barquera, B.; Gennis, R.; Guergova-Kuras, M.; Berry, E. Pathways for Proton Release During Ubihydroquinone Oxidation by the bc(1) Complex. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 10021−10026. (15) Muller, F. The Nature and Mechanism of Superoxide Production by the Electron Transport Chain: Its Relevance to Aging. J. Am. Aging Assoc. 2000, 23, 227−253. (16) Muller, F. L.; Liu, Y.; Van Remmen, H. Complex III Releases Superoxide to Both Sides of the Inner Mitochondrial Membrane. J. Biol. Chem. 2004, 279, 49064−49073. (17) Dröse, S.; Brandt, U. The Mechanism of Mitochondrial Superoxide Production in the Cytochrome bc1 Complex. J. Biol. Chem. 2008, 283, 21649−21654. (18) 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. (19) Husen, P.; Solov’yov, I. A. Mutations at the Qo-Site of the Cytochrome bc1 Complex Strongly Affect Oxygen Binding. J. Phys. Chem. B 2016, DOI: 10.1021/acs.jpcb.6b08226. (20) Watts, R. J.; Washington, D.; Howsawkeng, J.; Loge, F. J.; Teel, A. L. Comparative Toxicity of Hydrogen Peroxide, Hydroxyl Radicals, and Superoxide Anion to Escherichia Coli. Adv. Environ. Res. 2003, 7, 961−968. (21) Benov, L. How Superoxide Radical Damages the Cell. Protoplasma 2001, 217, 33−36. (22) Solov’yov, I. A.; Schulten, K. Magnetoreception Through Cryptochrome May Involve Superoxide. Biophys. J. 2009, 96, 4804− 4813. (23) Sastre, J.; Pallardó, F. V.; Viña, J. Mitochondrial Oxidative Stress Plays a Key Role in Aging and Apoptosis. IUBMB Life 2000, 49, 427− 435. (24) Lenaz, G. Role of Mitochondria in Oxidative Stress and Ageing. Biochim. Biophys. Acta, Bioenerg. 1998, 1366, 53−67. (25) Miquel, J. An Integrated Theory of Aging as the Result of Mitochondrial-DNA Mutation in Differentiated Cells. Arch. Gerontol. Geriatr. 1991, 12, 99−117. (26) Harman, D. Aging: A Theory Based on Free Radical and Radiation Chemistry. J. Gerontol. 1956, 11, 298−300. (27) Ladiges, W.; Wanagat, J.; Preston, B.; Loeb, L.; Rabinovitch, P. A Mitochondrial View of Aging, Reactive Oxygen Species and Metastatic Cancer. Aging Cell 2010, 9, 462−465. (28) Miquel, J.; Economos, A. C.; Fleming, J.; Johnson, J. E., Jr. Mitochondrial Role in Cell Aging. Exp. Gerontol. 1980, 15, 575−591. (29) Berry, R. A.; Huang, L. S.; Saechao, L. K.; Pon, N. G.; ValkovaValchanova, M.; Daldal, F. X-Ray Structure of Rhodobacter Capsulatus Cytochrome bc(1): Comparison with Its Mitochondrial and Chloroplast Counterparts. Photosynth. Res. 2004, 81, 251−275. (30) Osyczka, A.; Sarewicz, M. Electronic Connection Between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling. Physiol. Rev. 2015, 95, 219−243. (31) 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, Bioenerg. 2006, 1757, 1019−1034. (32) Crofts, A. R.; Holland, J. T.; Victoria, D.; Kolling, D. R. J.; Dikanov, S. A.; Gilbreth, R.; Lhee, S.; Kuras, R.; Kuras, M. G. The Qcycle Reviewed: How Well Does a Monomeric Mechanism of the bc1 Complex Account for the Function of a Dimeric Complex? Biochim. Biophys. Acta, Bioenerg. 2008, 1777, 1001−1019.

Finally, it is also worth noting that the results obtained in this study suggest that hydrogen bond interactions between the O2 molecule and water molecules otherwise involved, directly or indirectly, in the processes at the Qo-site could enable the generation of superoxide at the Qo-site of the bc1 complex. The water molecules form a network of hydrogen bonds with the components of the Qo-site, and the present investigation suggests that it is by forming hydrogen bonds to this network that the O2 molecule is able to acquire an electron. Specifically, a charge transfer event was only observed for the Qo-site configurations where the O2 molecule had formed hydrogen bonds with the water molecules. A logical next step is to investigate the details of the rate of formation of superoxide at the Qo-site. While our current study has provided insight into the electron transfer pathways, more calculations are necessary to establish a complete multiscale description of superoxide (O2•−) formation inside the bc1 complex.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors would like to thank the Lundbeck Foundation and the Russian Science Foundation (Grant 14-12-00342) for financial support. Computational resources for the simulations were provided by the DeiC National HPC Center, SDU.

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