Redox-linked Protonation State Changes in Cytochrome bc 1 Identified by Poisson-Boltzmann Electrostatics Calculations.

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1 Redox-linked Protonation State Changes in Cytochrome bc 1 Identified by Poisson-Boltzmann Electrostatics Calculations Astrid R. Klingen a, Hildur Palsdottir b, Carola Hunte b and G. Matthias Ullmann a a Structural Biology/Bioinformatics Group, University of Bayreuth, Germany; b Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany November 29, 2006 Submitted to Biochim. Biophys. Acta Supplementary Material

2 1. Calculation of s for cofactors of cytochrome bc 1 In Poisson-Boltzmann calculations, every needs to be assigned a certain. For the protein residues in cytochrome bc 1, standard s from the CHARMM22 parameter set [1] were used. Charges for the detergent undecylmaltopyranoside were derived from the standard CHARMM s for glucose. Charges for all other compounds were obtained from density functional theory (DFT) calculations. The resulting s of non-protein compounds of cytochrome bc 1 in all considered protonation and redox forms are listed in Tables 1.2 to The nomenclature used in these tables corresponds to the names in the PDB-deposited cytochrome bc 1 structures 1P84 [2] and 1KB9 [3]. All charges are given as fractions of the elementary charge e. The calculation of s for the Rieske cluster [4, 5] and coenzyme Q [6] have been reported in previous publications from our group. New DFT calculations were performed with the ADF programme suite [7], using functionals VWN [8] and PW91 [9]. Input coordinates for the new DFT calculations were derived from the crystal structures of cytochrome bc 1 from Saccharomyces cerevisiae, unless stated otherwise in the legends of the respective tables. Partial charges were derived from the DFT calculation results by a CHELPG-based algorithm [10] combined with singular value decomposition [11]. Atom radii used in the fitting procedure are given in Table 1.1. These correspond to the radii published by Bondi [12] except for the radius of hydrogen, where we use 1.0 Å instead of Bondi s 1.2 Å. Table 1.1. Atom radii in Å used in the fitting of ic s to the electrostatic potentials obtained from the DFT calculations. type radius C 1.7 H 1.0 N 1.55 O 1.5 S 1.8 Fe 1.4 P 2.0 2

3 Table 1.2. Partial charges in e for the b-type haem groups in their oxidised and reduced form. Input coordinates for the DFT calculations were derived from the high-resolution structure of a small prokaryotic b-type cytochrome [13]. ox red haem FE CHA CHB CHC CHD NA C1A C2A C3A C4A CMA CAA NB C1B C2B C3B C4B CMB CAB CBB NC C1C C2C C3C C4C CMC CAC CBC ND C1D C2D C3D C4D CMD CAD HMC HMC HMC HMB HMB HMB HAA HAA Table 1.2 continued ox red haem HMA HMA HMA HAD HAD HMD HMD HMD HC HAC HBC HBC HBB HBB HAB HA HD HB st ligand His CB CG ND CD CE NE HB HB HE HD HD nd ligand His CB CG ND CD CE NE HB HB HD HE HD

4 Table 1.3. Partial charges in e for haem c 1 in its oxidised and reduced form. Input coordinates for the DFT calculations were derived from the high-resolution structure of cytochrome c from horse mitochondria [14]. ox red haem FE NA NB NC ND C1A C2A C3A C4A C1B C2B C3B C4B C1C C2C C3C C4C C1D C2D C3D C4D CHA HA CHB HB CHC HC CHD HD CMA HMA HMA HMA CAA HAA HAA CMB HMB HMB HMB CAB HAB CBB HXB HXB HXB Table 1.3 continued ox red haem CMC HMC HMC HMC CAC HAC CBC HYC HYC HYC CMD HMD HMD HMD CAD HAD HAD Cys101 SG CB HB HB Cys104 SG CT HB HB His105 CB HB HB ND HD CG NE CD HD CE HE Met225 CB HB HB CG HG HG SD CE HE HE HE

5 Table 1.4a. Partial charges in e for the oxidised Rieske cluster in protonation forms P (both ligand histidines protonated), D1 (His161 deprotonated), D2 (His181 deprotonated), DT (both histidines deprotonated). P D1 D2 DT [Fe 2S 2] FE FE S S His161 N CA C O CB CG ND CD CE NE HN HA HB HB HD HE HE His181 N CA C O CB CG ND CD CE NE HN HA HB HB HD HE HE Table 1.4a continued P D1 D2 DT Cys159 CB SG HB HB Cys178 CB SG HB HB Thr160 C O Leu162 N HN CA HA Cys180 N HN CA HA C O Pro179 C O

6 Table 1.4b. Partial charges in e for the reduced Rieske cluster in protonation forms P (both ligand histidines protonated), D1 (His161 deprotonated), D2 (His181 deprotonated). The reducing electron is formally placed at the histidine-coordinated iron [4]. P D1 D2 [Fe 2 S 2 ] FE FE S S His161 N CA C O CB CG ND CD CE NE HN HA HB HB HD HE HE His181 N CA C O CB CG ND CD CE NE HN HA HB HB HD HE HE Table 1.4b continued P D1 D2 Cys159 CB SG HB HB Cys178 CB SG HB HB Thr160 C O Leu162 N HN CA HA Cys180 N HN CA HA C O Pro179 C O

7 Table 1.5. Partial charges in e for coenzyme Q with a tail of one isopren unit in its oxidised and deprotonated quinone (Q) and reduced and protonated quinol (QH 2 ) form. The s of the additional five isopren units (not listed) of the CoQ molecule in the Q i -site are set to zero. Q QH 2 C O HO C O C4M H4M H4M H4M C O C3M H3M H3M H3M C O HO C C1M H1M H1M H1M C C C C C C HA HB H HA HB HC HA HB HC

8 Table 1.6. Partial charges in e for stigmatellin. Partial charges of the hydrophobic part of the tail (not listed) have been set to zero. O C C C3M C O C4A C O C5M C C O C7M C O C8A C H3M H H3M H3M H5M H5M H5M H7M H7M H7M H H H Table 1.6 continued C C C C O C C C C O C C H H H H H H H H H H H H H H H H H H H

9 Table 1.7. Partial charges in e for undecylstigmatellin in its oxidised and reduced form. ox red O C C C3M C O C4A C O C5M C C O C7M C O C8A C H3M H3M H3M H5M H5M H5M H H H7M H7M H7M H H H H Table 1.7 continued ox red C C C C C C C C C C H H H H H H H H H H H H H H H H H H H H H

10 Table 1.8. Partial charges in e for the headgroup of hydroxydioxobenzothiazole in its protonated and deprotonated form. Partial charges of the hydrophobic tail (not listed) have been set to zero. prot deprot S C7A C4A N C H C O C C C O O HO C HA HB

11 Table 1.9. Partial charges in e for the head group of phosphatidylethanolamine in its three protonation forms (prot: doubly protonated, deprotn: deprotonated at amine group, deprotp: deprotonated at phosphate group). Charges of s in the hydrophobic tails (not listed) are set to zero. prot deprotn deprotp C N HN HN HN HA HB C C O3P O1P O2P O4P P HA HB HA HB C C C C O O HA HB HA HB H C C C C O O HA HB HA HB HA HB

12 Table Partial charges in e for the head group of phosphatic acid in its two protonation forms (prot: singly protonated phosphate group with net charge of 1, deprot: deprotonated phosphate group with net charge of 2). Charges of s in the hydrophobic tails (not listed) are set to zero. prot deprot P O O O O C H H C H O O C C H H C H H

13 Table Partial charges in e for the phosphodiester moieties in the head group of cardiolipin in their two protonation forms (prot: protonated phosphodiester with net charge 0, deprot: deprotonated phospodiester with net charge 1). Charges of s in the hydrophobic tails (not listed) are set to zero. prot deprot C O H HO CA OA PA OA OA OA CA HAA HBA HAA HBA CB OB PB OB OB OB CB HAB HBB HAB HBB CB OB OB C C HA HB HA HB Table 1.11 continued prot deprot CB HAB HBB CA OA OA C C HA HB HA HB CA HAA HBA

14 Table Partial charges in e for the head group of the phosphatidylcholin in its two protonation forms. Charges for the cholin moiety are taken from the CHARMM27 parameter set. Charges of s in the hydrophobic tails (not listed) are set to zero. prot deprot N C C C C HA HB HA HB HC HA HB HC HA HB HC C C O O O O P HA HB HO HA HB C O O C C HA HB HA HB C H Table 1.12 continued prot deprot C O O C C HA HB HA HB C HA HB

15 Table Partial charges in e for the head group of the phosphatidylinositol in its two protonation forms. Charges of s in the hydrophobic tails (not listed) are set to zero. prot deprot P O O O O C C C C C C O O O O O C H H H H H H HO HO HO HO HO H H1A H1B C O O Table 1.13 continued prot deprot C C HA HB HA HB C HA HB C O O C C HA HB HA HB C H

16 Table Partial charges in e for the head group of the detergent undecylmaltopyranoside. Charges of s in the hydrophobic tail (not listed) are set to zero. C H O C H O C H O HO C H O HO C H O HO C H H O HO Table 1.14 continued C H C H H O H O C H O H O C H O H O C H O C H O CA HA HA

17 2. Calculation of relative energies of the two Q o -site conformations The crystal structures of cytochrome bc 1 from S. cerevisiae [2, 3, 15, 16] reveal two alternative conformations of the Q o -site. The most obvious difference is the orientation of the sidechain of Glu272 of the cytochrome b subunit. In the structure containing stigmatellin, Glu272 is oriented towards the inhibitor and the Rieske iron-sulphur cluster (conformation Glu-FeS). In the structure containing hydroxydioxobenzothiazole (HDBT), it is oriented away from the inhibitor and towards haem b L (conformation Glu-b). To include the conformational flexibility of the Q o -site into Poisson-Boltzmann (PB)/Monte Carlo titration calculations, the energy difference between the two conformations needs to be calculated. By visual inspection of the two crystal structures a fragment of the cytochrome b subunit could be identified that contains all residues undergoing significant conformational changes, namely residues Thr265 to Trp273 and the sidechain of His253. The energy of this fragment in the two different conformations in the environment of completely reduced or oxidised cytochrome bc 1 has been calculated by a combined molecular mechanics (MM)/PB-approach. The conformational energy has two contributions (Fig. 2): G conf = G MM + G PB. G MM = G MM (Glu-FeS) G MM (Glu-b) is the difference in the MM energy of the fragment in the two conformations. This MM contribution was calculated using CHARMM [1]: the fragment in conformations Glu-FeS or Glu-b was placed into a homogeneous dielectric environment (dielectric constant ɛ = 4, Fig. 2), and the relevant contributions to the MM energy were calculated. These contributions are the energies of the dihedral angles, the van der Waals interaction energies of s connected by three covalent bonds, and the electrostatic interaction energies of s that are separated by three or more covalent bonds, as implemented in the CHARMM energy function. The difference in the dihedral, van der Waals and electrostatic energies of the fragment in conformation Glu-FeS and conformation Glu-b is equivalent to G MM. In addition to the difference in MM energies in a homogeneous environment, the electrostatics of the protein/membrane environment can also exert differential effects on the fragment Figure 2. Calculation of the energy difference G conf of the Glu-FeS and Glu-b conformations of the Q o -site of membrane-embedded cytochrome bc 1. ε=80 ε=80 membrane Glu b membrane G conf ε=4 cyt bc 1 ε=4 cyt bc 1 membrane Glu FeS membrane GPB (Glu b ) G PB (Glu FeS) G MM ε=4 ε=4 Glu b Glu FeS 17

18 Table 2. Results from the calculation of the conformational energy difference between the Glu-b and Glu-FeS conformations of the Q o -site of completely oxidised and reduced cytochrome bc 1 in presence of a model membrane. All energies are in kcal/mol. energy contribution oxidised system reduced system Glu-FeS Glu-b Glu-FeS Glu-b CHARMM van der Waals energies CHARMM electrostatic energies CHARMM dihedral energies sum of CHARMM energies G MM MEAD transfer energies G PB resulting G conf = G MM + G PB resulting G conf = G MM + G PB in the two different conformations. This effect of the protein/membrane environment is quantified by PB-calculations: the energy G PB to transfer the fragment from the homogeneous environment of the MM-calculations into the protein environment (Fig. 2) is calculated with MEAD [17], once for the Glu-b conformation and once for the Glu-FeS conformation. The crystal structure obtained with HDBT has been used as protein environment for the Q o -site fragment in both the Glu-FeS and Glu-b conformations since the HDBT-inhibited structure contains additionally refined lipids. Differences between the HDBT- and stigmatellininhibited structures are limited to the Q o -site fragments treated in the MM calculations. The interface between the protein environment and the Q o -site fragment in the Glu-FeS conformation was very mildly energy minimised before the calculation of conformational energies and the MEAD calculations. The minimisation consisted of 1000 steepest decent (SD) steps, 500 molecular dynamics (MD) steps of 0.2 fs at 100 K, 500 MD steps of 0.5 fs at 200 K, 500 MD steps of 0.1 fs at 300 K, 500 MD steps of 0.1 fs at 100 K, 1000 SD steps and 2000 conjugate gradient steps. Only s directly at the interface and s that are connected to the interface s by one covalent bond were allowed to move. The rmsd between the structures before and after minimisation is as low as 0.11 Å. In the protein environment, the protein s carry charges (the protonation state is set to be the doubly protonated form for the histidines, and the neutral form for all other residues), and the dielectric environment is not homogeneous. In the PB-calculations, the charges of the C α s located directly at the interface of the Q o -site fragments and the protein environment, i.e. the C α s of His253, Val264 and Trp273, were set to zero in order to eliminate unphysical coulombic interaction between s connected by covalent bonds. From the difference in the Poisson-Boltzmann energies ( G PB ) together with the difference in MM energy ( G MM ), the conformational energy difference in the protein environment ( G conf ) can be obtained. Table 2 lists results obtained for the different quantities. 18

19 3. Model compound pk-values for Poisson-Boltzmann calculations In the framework of PB electrostatics, the energetics of a system with multiple titratable groups can be described by a set of intrinsic pk-values and pairwise interaction energies. The intrinsic pk-value of a certain group is the pk-value the group would have if all other titratable groups in the system were in a certain reference protonation form. By the programme multiflex from the MEAD programme suite [17] intrinsic pk-values are computed as shifts relative to model compound pk-values. The model pk-value of a certain group corresponds to the pk-value it would have as an isolated group in aqueous solution. Table 3 lists experimentally determined model pk-values [18, 19] that have been used in this work. Model compound pk-values for the Rieske ligand histidines have been determined by a combined DFT/PB approach described below. Table 3. Groups in cytochrome bc 1 considered titratable, and corresponding experimentally determined model compound pk-values [18, 19]. group model pk-value arginine 12.0 lysine 10.4 tyrosine 9.6 cysteine 9.1 histidine NδH 6.6 histidine NɛH 7.0 aspartate 4.0 glutamate 4.4 N-terminus 7.5 C-terminus 3.8 haem propionate 4.4 lipid phosphodiester 1.3 phosphatic acid phosphate 6.3 phosphatidyl ethanolamine

20 4. Calculation of model compound pk-values for the Rieske cluster For the Rieske iron-sulphur cluster with its titratable ligand histidine sidechains, experimentally determined model pk-values are not available. We have therefore estimated these values from a combined DFT/PB approach that makes use of the thermodynamic cycle depicted in Fig. 4. The deprotonation energies of the Rieske cluster in vacuum are known from DFT calculations that have been reported earlier, and could be shown to reproduce experimental results in combined DFT/PB calculations [5,20]. The model compound is defined to contain the same set of s that is included into these DFT calculations. The energies to transfer the protonated and deprotonated species ( G trans (AH) and G trans (A ), respectively) from vacuum (ɛ = 1) into the aqueous environment (ɛ = 80) are obtained from PB electrostatics calculations using solvate from the MEAD package. The ionic strength is set to I = 0.1 M in these calculations, the temperature to T = 300 K. The transfer energy of the proton is calculated from the experimentally derived potential of the standard hydrogen electrode [21]. The model compound pk-values were calculated as with R as universal gas constant and pk model = (ln 10 RT ) 1 G aq deprot G aq deprot = Gvac deprot + G trans (A ) + G trans (H + ) G trans (AH). The procedure was followed for all considered one-proton deprotonation reactions of the Rieske cluster in its different redox states. Resulting model compound pk-values are listed in Table 4. Figure 4. Thermodynamic cycle to obtain the model pk-values of the Rieske cluster as isolated group in aqueous solution. AH represents the protonated, and A the deprotonated form of the Rieske cluster. ε vacuum: =1 G AH vac deprot A + H + (AH) G trans G trans ( A ) G trans ( H + ) aq G deprot AH A + H + ε aqueous solution: =80 20

21 Table 4. Model compound pk-values for the ligand histidines of the Rieske centre. The following protonation forms have been considered: both ligand histidines protonated (P), only H161 deprotonated (D1), only H181 deprotonated (D2), both ligand histidines deprotonated (DT, considered only in the oxidised state). redox state deprotonation reaction model pk-value oxidized P D1 9.1 P D2 8.8 D1 DT 8.6 D2 DT 9.0 reduced P D P D

22 5. Treatment of the Rieske cluster in MEAD calculations The Rieske iron-sulphur cluster has two histidine ligands that can potentially undergo (de)protonation reactions. The Rieske cluster therefore has four different protonation forms: both histidines protonated (P), only H161 ISP deprotonated (D1), only H181 ISP deprotonated (D2), or both histidines deprotonated (DT). In the following, we focus on the situation of the oxidised cluster, where all four possible protonation forms have been considered. The treatment of the reduced cluster is equivalent but more simple, since the doubly deprotonated form does not need to be considered due to its low probability at physiological ph [4, 5]. The multiflex titration programme from the MEAD programme suite only considers conversions between exactly two different forms of a titratable group. Therefore, four separate multiflex calculations were run for the oxidised state of the system, referred to as P D1, P D2, D1 DT and D2 DT in analogy to the different possible deprotonation reactions of the Rieske cluster. The separate multiflex calculations are however not sufficient to decide upon the protonation form of the Rieske cluster in the protein, since the different protonation reactions influence the energetics of each other. These interactions are not accounted for by multiflex calculations considering only a single one-proton deprotonation reaction at a time. An approach based on Monte Carlo (MC) sampling of state energies was thus used in this study, and is outlined in the following. To fully characterise the protonation behaviour of the Rieske cluster, the multiflex calculations listed above are treated like different conformations of a protein. The four different one-proton titration reactions of the oxidised Rieske cluster are assigned relative conformational energies. In the reference state, for which the conformational energies are computed (named MC reference state in the following), the Rieske cluster is in its protonated form, the histidines are protonated, and all other residues are in their neutral protonation form. The fact that the Rieske cluster is in its protonated form in the MC reference state means that the conformational energies have to be computed for the Rieske cluster being in its P form for the P D1 and P D2-titrations, in its D1 form for the D1 DT titration, and in its D2 form for the D2 DT titration (see Table 5). Thus, the relative conformational energies are in fact differences in protonation state energies. Table 5. The MC reference protonation state is defined to be the protonated form of the Rieske cluster for all four different multiflex calculations. Since different one-proton deprotonation reactions are considered in these multiflex calculations, the protonated form of the Rieske cluster has in fact different meanings in terms of the four possible protonation states of the Rieske cluster. The conformational energy is thus a difference between the energies of the different protonation states that result from the definition of the MC reference state and the one-proton deprotonation reaction of the Rieske cluster considered in the respective multiflex calculation. multiflex calculation: P D1 P D2 D1 DT D2 DT Rieske protonation form in the MC reference state: Rieske protonation form for which conformational energy is computed: conformational energy: protonated protonated protonated protonated P P D1 D2 G P D1 conf G P D2 conf G D1 DT conf G D2 DT conf 22

23 Figure 5. Calculation of G D1 DT conf in a protein with two Rieske clusters, e.g. the dimeric form of the cytochrome bc 1 complex. G D2 DT conf is computed analogously. Rieske1: P Rieske2: P ref: MC G P ref Rieske1: P Rieske2: P ref: mflex G Rie1 mflex G D1 >DT conf Rieske1: D1 Rieske2: P ref: mflex Rieske1: D1 Rieske2: D1 ref: MC G D1 ref Rieske1: D1 Rieske2: D1 ref: mflex G Rie2 mflex The differences in protonation state energies are computed relative to the energy assigned to the P D1 multiflex calculation ( G P D1 conf = 0). Since the MC reference state has identical meanings (Rieske cluster in its P form) for the P D1 and P D2 multiflex calculations, they have identical energies assigned ( G P D1 conf = G P D12 conf = 0). According to Fig. 5, the conformational energy G D1 DT conf can be calculated as ( ) G D1 DT conf = G Rie1 mflex + G Rie2 mflex + G P ref G D1 ref. The four contributions can be obtained from the multiflex output (intrinsic pk-values and interaction energies). G Rie1 mflex can be calculated from the intrinsic pk of the first Rieske cluster in the P D1 multiflex run: G Rie2 mflex G Rie1 mflex = ln 10 RT (pk intr,p D1 (Rie1) ph). can be calculated from the intrinsic pk of the second Rieske cluster plus the interaction energy with the first Rieske cluster. The interaction energy has to be added, since the first Rieske cluster is then no longer in its reference state: G Rie2 mflex = ln 10 RT (pk intr,p D1 (Rie1) ph) + W (Rie1, Rie2). The intrinsic pk-values are calculated for the multiflex reference protonation state (ref:mflex in Fig. 5), which is different from the MC reference state (ref:mc in Fig. 5). In order to derive Gconf D1 DT from G Rie1 mflex and GRie2 mflex, the ph-dependent energy differences GD1 ref and G P ref between the MC and multiflex reference protonation states have to be considered. These energy differences are calculated directly from the protonation state energies G (n). The protonation state energies G (n) are computed from the multiflex intrinsic pk-values and interaction energies [18]. 23

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