Active site midpoint potentials in different cytochrome c oxidase families

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Active site midpoint potentials in different cytochrome c oxidase families - a computational comparison Margareta R.A. Blomberg Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00093 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Active site midpoint potentials in different cytochrome c oxidase families - a computational comparison Margareta R.A. Blomberg∗ Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91, Stockholm, Sweden.

Contact information for corresponding author: E-mail: [email protected]. Phone: +46-8-16 26 16

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Abstract Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain, reducing molecular oxygen to water. The binuclear active site in CcO comprise a high-spin heme associated with a CuB complex and a redox-active tyrosine. The electron transport in the respiratory chain is driven by increasing midpoint potentials of the involved cofactors, resulting in release of free energy, which is stored by coupling the electron transfer to proton translocation across a membrane, building up an electrochemical gradient. In this context, the midpoint potentials of the active site cofactors in the CcOs are of special interest, since they determine the driving forces for the individual oxygen reduction steps, and thereby affect the efficiency of the proton pumping. It has turned out to be difficult to obtain useful information on some of these midpoint potentials from experiment. However, since each of the reduction steps in the catalytic cycle of oxygen reduction to water corresponds to the formation of an O-H bond, they can be calculated with reasonably high accuracy using quantum chemical methods. From the calculated O-H bond strengths the proton coupled midpoint potentials of the active site cofactors can be estimated. Using models representing the different families of CcO’s (A, B and C), the calculations give midpoint potentials that should be relevant during catalytic turnover. The calculations also suggest possible explanations for why some experimentally measured potentials deviate significantly from the calculated ones, i.e. for CuB in all oxidase families, and for heme b3 in the C-family.


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1. Introduction Cytochrome c oxidases (CcOs) are membrane bound enzymes that catalyse the reduction of molecular oxygen to water as the last step in the respiratory chain in aerobic organisms, see equation (1): O2 + 4H+ + 4e− → 2H2 O (1) The active site of these enzymes, where the chemistry of equation (1) occurs, consists of a high-spin heme group, a copper complex, labelled CuB , and a redox active tyrosine crosslinked to one of the histidine ligands of CuB , all in close vicinity of each other, and referred to as the binuclear center (BNC). The electrons are delivered one by one from a reduced soluble cytochrome c, located on the positive side of the membrane (the periplasm), to the BNC via a set of cofactors, which are low-spin heme groups and/or Cu-complexes. The protons, often referred to as the chemical protons, are transferred from bulk water on the negative side of the membrane (the cytoplasm) to the BNC via one or two proton channels. The CcOs belong to the heme-Cu oxidase (HCuO) superfamily of enzymes, which contains both O2 reducing enzymes and NO reducing enzymes. The oxygen reducing HCuOs are classified into three main families, A, B and C, depending on structural details, mainly the number and type of proton channels, but also with respect to the electron transfer cofactors [1, 2, 3]. The A family contains both mitochrondrial and bacterial CcOs, and also quinol reducing oxidases (e.g. bo3 ), and the most investigated member of the B-family is the ba3 CcO from Thermus thermophilus. The A- and B-family CcOs have a heme a3 in the BNC, and, as elucidated from the different crystal structures, the overall structure of the BNC active site is very similar in these oxidases. The C-family (cbb3 oxidases), on the other hand, has a heme b3 in the BNC, and differs also in that the proximal histidine is hydrogen bonding to a negatively charged glutamate. Molecular oxygen binds to the reduced form of the BNC, the R state with high-spin heme-Fe(II), CuB (I) and TyrOH. It was early shown that the O-O bond is cleaved in the first reaction step, yielding a four electron oxidized form of the BNC, labelled PM , with heme-Fe(IV)=O, CuB (II)OH and a neutral TyrO-radical [4, 5]. The rest of the catalytic cycle consists of four reduction steps, each taking up one electron and one proton to the BNC, leading back to the reduced state R with two new water molecules. A number of spectroscopic methods have been applied to characterize the intermediates involved in this reduction process, which are labelled F, O and E, but there are still uncertainties in the


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exact structure of these intermediates. In particular, it is more difficult to determine the positions of the protons than those of the electrons using spectroscopic methods [6]. The overall exergonicity of the reduction process is determined by the difference in midpoint potential (also termed reduction potential) between the electron donor and the acceptor, molecular oxygen. With cytochrome c as the electron donor, equation (1) becomes exergonic by 50.7 kcal/mol (2.2 eV [7]). A significant part of this free energy is conserved as an electrochemical gradient across the membrane, both via the electrogenic chemistry (taking the electrons and the chemical protons from opposite sides of the membrane) and via so called proton pumping, which means that the chemistry is coupled to proton transfer across the entire membrane. The number of pumped protons varies among the families, between 0.5 and 1 proton pumped per electron. For an overview of the structure and function of respiratory oxidases see e.g. [6, 8, 9, 10, 11, 12]. One of the most interesting aspects of the CcO family of enzymes is the mechanism for the proton pumping. A large amount of knowledge related to the proton pumping has been obtained, but a number of critical questions still remain to be answered [6, 10, 11, 12]. In the context of the proton pumping mechanism, the details of the energetics of the catalytic cycle are of great importance. The driving force in each of the four reduction steps depends on the reduction potential of the active site cofactor that is reduced in that particular step. Early experimental studies on the A-type of CcOs (known to pump four protons per oxygen molecule) indicated that the free energy was not released evenly over the reduction steps, since the midpoint potentials of the BNC cofactors seemed to be quite different [13]. Only the midpoint potentials of the PM and F intermediates were found to be high enough (0.8-1.0 V) [13] to afford proton pumping, while the potentials for the O state (corresponding to CuB (II)) and the E state (corresponding to high-spin hemeFe(III)) seemed to have significantly lower midpoint potentials 0.3-0.4 V, indicating that the corresponding reduction steps would become too endergonic when the electrochemical gradient is present during catalytic turnover. It was then suggested that proton pumping only did occur in two of the four reduction steps, also when a total of four protons per O2 are pumped [14, 15]. However, it was later shown that the proton pumping in the A-type of CcOs actually occurs in all four reduction steps [16, 17], also at a high gradient [18, 19]. To solve the puzzle with the low reduction potentials it was postulated that there exists a “high-energy” metastable state labelled OH , which conserve some of the energy from the previous two reduction steps [16]. The OH state is suggested to be formed directly after the


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reduction of the F state, and it is assumed to relax after a certain time to the O state for which the low CuB (II) and Fe(III) potentials have been observed [17, 20]. The OH state was assumed to differ in structure from the O state, which would result in an elevated midpoint potential of CuB (II), and possibly also of high-spin heme-Fe(III). Data in one experimental study on an A-type of CcO was suggested to show an elevated CuB midpoint potential in the OH state [21], however, several experimental investigations on different A-type of CcOs, designed to prepare the OH state and to determine its properties, could not find an increase in the CuB (II) midpoint potential as compared to the resting O state [22, 23, 24]. The experimentally measured values of the CuB potential fall in the range 0.2-0.4 V, including also the C-type of CcOs [25], and a puzzle thus remains regarding the CuB midpoint potential during catalytic turnover. Another puzzle involving the experimental midpoint potentials of the BNC cofactors concerns the unusual redox properties found for oxidases from the C-family, in particular for the high-spin heme. For the A- and the B-families, which both have a heme a3 in the BNC, the experimentally obtained heme a3 potentials vary between 0.2 and 0.4 V [22, 24, 26, 27, 28, 29, 30, 31], which is reasonably close to that of the cytochrome c donor (0.25 V). For the cbb3 oxidase (family C), on the other hand, most of the experimental values reported for the high-spin heme-Fe(III) potential are below zero, in the range -0.04 to -0.12 V [25, 32]. Clearly this difference may be caused by the change from heme a3 to heme b3 in the BNC, and parallels have been made to cytochrome c dependent nitric oxide reductase (cNOR), which belongs to the same heme-copper oxidase superfamily, and which also has a heme b3 in the active site, with a rather low midpoint potential (0.06 V) [25, 32, 33]. It has also been suggested that the low heme b3 midpoint potential is due to a negative charge in the vicinity of the BNC, which is present only in the cbb3 oxidase [25, 32, 33]. However, such unfavourable redox properties for these CcO enzymes clearly raises questions about how the cbb3 oxidases works [32]. With the above puzzles at hand, it has been questioned whether the experimentally obtained potentials necessarily reflect the redox properties of the active site during turnover [31, 32]. Many of the experimental measurements are made under conditions differing from the working enzymes during oxygen reduction, and they may therefore not catch the relevant midpoint potentials of the cofactors serving as the final electron and proton acceptors in the different reduction steps. Typical for the experimental procedures used to determine reduction potentials is that the ligands of the metals in the BNC active site are not known


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for the different intermediates involved, and neither is the protonation state of different parts of the active site. In contrast to the experimental investigations, the computational approach offers a possibility to follow the stages of the oxygen reduction chemistry. This means that the calculated potential for each cofactor is obtained for what is most likely the relevant intermediate structure, in terms of metal ligands and protonation states, during catalytic turnover. Another advantage with the computational approach is that the calculations can be performed on models of the isolated BNC active site, including only the BNC cofactors themselves and the closest amino acids, thereby providing estimates of what can be termed inherent midpoint potentials. Inherent here refers to what pertains to the BNC active site itself, isolated from the other cofactors involved in the electron transport. The inherent midpoint potentials of the active site cofactors determine the driving forces (or free energies) of the individual reduction steps during catalytic turnover. They are therefore of great interest, although they are evidently not the only factors determining the full mechanisms for the chemistry and the proton pumping. In particular, other factors are more important for the kinetics of the different reaction steps. In spite of the supposition that the basic mechanism for oxygen reduction is the same in the different oxidase families [10], it is now clear that there are significant differencies between the families [34, 35, 36]. To determine the funtional mechanisms of the different oxidases, the present type of computationally obtained properties have to be combined with the large amount of experimental data available concerning the properties of the proton channels to the active site and the properties of the immediate electron donors (the low-spin hemes) [27]. These other aspects of the reduction steps, such as the number and positions of the proton uptake channels leading to the BNC, and the properties of the low-spin hemes (such as their reduction potentials and the couplings between the low-spin and high-spin hemes), are essential for discussions about the reaction mechanisms, but they will not be adressed here. Importantly, the proton coupled reduction potentials that determine the driving forces of the different reduction steps in the CcO catalytic cycle can be described in terms of the chemistry taking place in the BNC. Each reduction step, uptake of one electron and one proton, corresponds to the formation of an O-H bond in the active site. Therefore, the energetics of the reduction steps, and also the individual reduction potentials, can be estimated from the strengths of the different O-H bonds. As discussed in the computational section below, the quantum chemical cluster approach can be used to calculate this kind of relative energies that correspond to the steps in a catalytic cycle. In particular, since the


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charge is not changed, the strength of an O-H bond is not sensitive to a distant surrounding, which means that sufficiently accurate results can be obtained using reasonably sized models (150-200 atoms). The purpose of the present study is to use quantum chemical calculations on models of the CcO BNC-active sites to estimate what may be referred to as inherent reduction potentials for the four cofactors, i.e. the CuB , the tyrosine and the high-spin heme (ferryl and ferric, respectively). Since this part of the active sites (the BNCs) is essentially identical for the A- and the B-families, these properties will be identical and only one model is needed for the two families. Thus, only two different active site models are needed to describe the properties of all three families, one representing the A- and B-families, and one representing the C-family. The former model has a CuB -heme a3 active site. Its structure is taken from a bacterial A-type of CcO, but it serves as a model also for mitochondiral A-types and for B-types of CcOs. The other model has a CuB -heme b3 active site, and the structure is taken from a cbb3 oxidase, which belongs to the C-family. Using these two models, the O-H bond strengths representing each of the four reduction steps in the catalytic cycle can be estimated. Furthermore, the corresponding midpoint potentials for the active site cofactors, which should be relevant during catalytic turnover, can be compared between the families, and also to experimentally obtained potentials. The computational results also suggest possible explanations of the most significant differences between the calculated and the experimental reduction potentials.

2. Computational Details The general computational approach used here has been applied and evaluated for a large number of metallo-enzymes, as described in a recent review [37]. Large cluster models of enzyme active sites (150-300 atoms), and quantum mechanical methods (density functional theory) are used to calculate relative energies of different intermediates involved in catalytic enzyme reactions. In general good agreement with observed reaction rates is obtained, indicating that the cluster calculations in most cases fairly well reproduce the relative free energies of the entire enzyme for different active site structures [37]. In the present study quantum mechanical calculations on O2 reduction in CcO are performed on two different cluster models, one with a heme a3 and one with heme b3 in the BNC. Both models include the heme a3 /b3 group, the CuB ion and the redox active tyrosine residue


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that is cross-linked to one of the histidine ligands on CuB . All heme substituents are kept in the models, except the propionate groups plus the long tail of the farnesyl group on heme a3 . Regarding the omission of the propionates it should be pointed out that charged groups in the surrounding of the active site in general have minor effects on the present kind of proton-coupled reduction potentials, since the charge effects are of similar size but with opposite signs on the electron and the proton uptakes. Futhermore, in a previous study on CcO three different BNC-models were used, one without the propionates, one with the propionates (and their surrounding) present and protonated, and one with the propionates (and their surrounding) present and unprotonated, and for all reduction steps the calculated relative energies differed by less than two kcal/mol between all three models [38]. All first shell amino acid ligands to the metals are included in the models, i.e. three histidine ligands on CuB and the proximal histidine on the high-spin heme groups, plus a conserved valine near the BNC. In the CuB -b3 model the negatively charged glutamate hydrogen bonding to the proximal histidine is included, together with a tryptophan that is also hydrogen bonding to the glutamate, because negative groups on the border of the model may result in artificial effects. To make the two models more similar a glycine hydrogen bonding to the proximal histidine is included in the CuB -a3 model. The two models are shown in Fig. 1, and the total number of atoms is about 170 (depending on the state) in the CuB -a3 model, with total charge +1, and about 190 in the CuB -b3 model, with total charge 0. The charge of the CuB -a3 model is obtained from the positively charged CuB -complex, which is combined with a neutral heme-model and neutral amino acids. For the CuB -b3 model the situation is the same, except that one of the amino acids, the glutamate hydrogen bonding to the proximal histidine, is assumed to be deprotonated, i.e. negatively charged. The latter assumption is based on the crystal structure [39], where the hyrogen bonding pattern fits best with a deprotonated Glu323. The same assumption has been made in several provious studies [33, 40, 41]. As mentioned above, the presence of a charged group is not expected to affect the proton coupled reduction potentials significantly, which also means that the pump-protons do not have to be explicitely treated for the present purpose. In contrast, the presence of charged groups, as well as the pump-protons, may have significant effects on the individual electron and proton affinities, in opposite directions, and may therefore affect the details of the reaction mechanisms. The structure of the CuB -a3 model is based on the X-ray coordinates for the Rhodobacter sphaeroides aa3 CcO [42], and it is noted that the BNC structures are close to identical in different a3 -CuB CcOs. The structure of


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Figure 1: The two models used in the calculations, the CuB -a3 model representing the BNC active site of the A- and B-families of CcO and the CuB -b3 model representing the C-family. For both models the structure of the PM state is shown, together with the most important spin-populations. the CuB -b3 model is based on the X-ray coordinates for the Pseudomonas strutzeri cbb3 CcO [39]. During geometry optimizations of the different intermediates a few atoms near the truncations are fixed to the X-ray coordinates to maintain some structural constraints from the surrounding protein. The fixed atoms are the alpha carbons, the hydrogen atoms replacing the peptide bonds plus one carbon atom on the porphyrin. A difference to previous models introduced here is that no coordinates are fixed on the proximal histidine, because the position of this histidine is fixed by its hydrogen bonding to the glutamate and glycine, respectively. The scheme to lock certain coordinates has been carefully tested in a large number of cases [43]. No water molecules are included in the model, apart from the ones formed during the reduction process (see e.g. [44]). The B3LYP-type of functionals have been found to give the best description of transition metal systems [37, 45], and in particular, its limitations are quite well known and can be corrected for. The dispersion corrected hybrid density functional B3LYP-D3 [46, 47] was used, together with a double zeta basis with polarization functions on all second row atoms, to optimize the geometries for the investigated intermediates. To obtain more ac-


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curate energies for the optimized structures a larger basis set was used, the lacv3p+ basis for the metal ions [48] and the large cc-pvtz(-f) basis set for the rest of the atoms, together with the B3LYP∗ -D3 [49] functional (with 15% exact exchange) in single point calculations. Polarization effects on the relative energies from the omitted protein surrounding were estimated using a self-consistent reaction field approach with a dielectric constant of 4.0 [50]. Reasonably small dielectric effects were obtained for relative energies between intermediates with the same charge, and therefore these relative energies are not very sensitive to the choice of dielectric constant. For those O-H bonds where the calculations indicated the largest dielectric effects, calculations were performed also with a dielectric constant of 10.0, which changed the calculated O-H bond strengths by less than half a kcal/mol. Zero-point corrections using the harmonic approximation were taken from the Hessians, calculated at the same level as the geometry optimizations. The Jaguar 7.9 [48] program was used for the calculations with the larger basis set, while geometry optimizations and the Hessian calculations were performed with the Gaussian 09 package [51]. Due to the fixation of coordinates during geometry optimization, accurate entropy effects cannot be obtained from the Hessians. However, approximate estimates of the entropy difference between different intermediate structures can be obtained by projecting out the frequencies corresponding to the fixed coordinates. When this is done for a number of relative energies reported here, small entropy effects of a couple of kcal/mol are found for relative energies on the order of 70-80 kcal/mol. Therefore that kind of entropy effects are neglected, and the only entropy effects on relative energies that are expected to be significant are those that occur when small molecules enter or leave the enzyme. The entropy lost on binding of the gaseous O2 molecule is approximated by the translational entropy for the free molecule (10.8 kcal/mol at room temperature, which is a large part of the calculated total entropy of 14.6 kcal/mol for the oxygen molecule in gas phase). This approximation is supported by previous calculations where more explicit estimates of the entropy effects could be made [52, 53]. For the binding enthalpy of a water molecule to bulk water an empirical value of 12 kcal/mol is used, which includes explicit zero point effects, and which is based on experimental thermodynamics and previous experience [44, 54]. The energetic results reported below are considered as approximate free energies. To estimate the energetics of the reduction steps of the CcO catalytic cycle it is necessary to know the total energy of the electron transferred from cytochrome c plus the proton transferred from bulk water. This energy can be parametrized using experimental results


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for the overall reaction, which together with the calculated strength of the O-H bond formed in each reduction step gives the exergonicity of the individual reduction steps. The total energy of an electron (from cytochrome c) plus a proton (from bulk water) are here parametrized to make reaction (1) exergonic by 50.7 kcal/mol (2.2 eV), using experimental values for the gas phase energy of molecular oxygen and water, and adding entropy and solvation effects according to the previous paragraph. This gives a total value of 384.6 kcal/mol for the electron plus the proton, very similar to the the value obtained by summing experimental energies of 384.3 kcal/mol, 104.5 kcal/mol (0.25+4.281 V) for an electron from cytochrome c plus 279.8 at pH=7 for a proton in water. Since the calculated O-H bond strengths already include the energy of a free hydrogen atom (313.8 kcal/mol at the present computational level), an energy of 70.8 (384.6-313.8) kcal/mol should be subtracted from the calculated O-H bond strengths to obtain the exergonicites relative to cytochrome c, and thereby the proton coupled midpoint potentials. For the size of models needed in the present study, the best available quantum chemical methods (B3LYP-type of density functional theory) still have certain built-in errors, why a few corrections obtained from comparisons to experiments and/or higher-level calculations on small models need to be added to the pure computational results. First, compared to experiments the calculated bond energy of O2 is too large by 9.6 kcal/mol, and the total binding energy of a water molecule is too small by 1.0 kcal/mol, yielding an error in the exergonicity of equation (1) of 11.6 kcal/mol. However, this error is more or less counterbalanced by errors in chemical bonds formed or cleaved in the BNC, as follows: The heme Fe(IV)=O → heme Fe(III)OH bond is too small at the present level of calculation, by 6-7 kcal/mol as shown by CCSD(T) calculations on small models [55, 56]. A correction of 6.2 kcal/mol is chosen. The TyrO(rad) → TyrOH bond is too small by 3-5 kcal/mol in the calculations as compared to experimental values on the phenol molecule. A correction of 3.4 kcal/mol is chosen. The Cu(II)OH → Cu(I)OH2 bond is 1 kcal/mol too small compared to CCSD(T) calculations on a small model [44], which can be considered to correspond to the error for the water molecule. Finally, as in previous studies [38, 44, 57] the purely calculated reduction potential of high-spin heme-Fe(III) to Fe(II) is assumed to be too small by 8-9 kcal/mol (due to a well-known problem with the DFT-description of certain properties of heme-groups [55, 58, 59, 60]), resulting in a too low energy of the superoxo product obtained as intermediate A. Therefore the high-spin heme Fe(III)OH → Fe(II)OH2 bond is corrected by 9+1 kcal/mol, including a correction of 1 kcal/mol


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for the water molecule, which is counterbalanced by a correction of 9 kcal/mol decreasing the binding energy of the oxygen molecule. This procedure is slightly different from the previous procedure used in several cases [37, 38, 44, 57], where the exergonicity of equation (1) was based only on calculated energies, rather than employing experimental gas phase values as here. The directly calculated values together with an overview of the present corrections are shown in Fig. S1 in Supporting Information. The final results obtained with the present procedure are quite similar to those obtained with the previous procedure, as can be seen from Fig. S2, and also from comparison to previous results using a different model, e.g. in ref. [44]. The advantage with the present procedure is that a compatible procedure can be used to study NO reduction in heme-copper oxidases. Due to a quite large difference in the error in the calculated exergonicity of the overall reactions between oxygen and NO reduction, the original procedure was not compatible between the two reactions. Considering the corrections that has to be introduced in the computational procedure, it is clear that there is an uncertainty of a few kcal/mol in the absolute O-H bonds calculated, but, the similarities between the families, in particular, should be quite reliable.

3. Results and Discussion The most important results of the present study are summarized in Fig. 2. At the top of the Figure one possible reaction scheme for oxygen reduction in CcO is shown. The notation OH will here be used for the oxidized state as formed during catalytic turnover, and with the structure shown in Fig. 2. Omitted from the scheme are the R to A to PM steps, i.e. the oxygen binding and the O-O bond cleavage, since the focus of the present study is the energetics of the four reduction steps and the corresponding reduction potentials. The reduction steps will be discussed below, with regard to which cofactor is reduced, i.e. which O-H bond that is formed. The calculated O-H bond strengths are presented for both models used, which are referred to as CuB -a3 (representing the A- and B- families of CcOs) and CuB -b3 (representing the C- family of CcOs). As explained in Computational Details the sum of the energy of an electron from cytochrome c and a proton from bulk water is parametrized based on experimental data for the overall reaction (1), giving a value of 70.8 kcal/mol that should be subtracted from each O-H bond strength to obtain the exergonicity relative to the cytochrome c donor of each reduction step. By comparing the exergonicity for each reduction step with the midpoint potential of cytochrome c (0.25 V) the midpoint


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Figure 2: Summary of the results for the four reduction steps in the catalytic cycle of CcO. For both models, CuB -a3 (representing the BNC in the A- and B-families of CcOs) and CuB -b3 (representing the BNC in the C- family of CcOs), the calculated O-H bond strengths are given, together with the exergonicities of the corresponding reduction steps relative to the electron donor, and the estimated proton coupled midpoint potentials. potential most relevant during catalytic turnover can be estimated for each cofactor. CuB (II)OH → CuB (I)OH2 : The calculations indicate that copper is the first cofactor to be reduced, in the PM to F step in all families of CcOs, since the product CuB (I)OH2 has the strongest O-H bond that is formed in the reduction process for both models. The calculated O-H bond strength is 87.2 kcal/mol for the CuB -a3 model and 89.2 kcal/mol for the CuB -b3 model, a difference that is within the uncertainty of the calculations. The calculations also show that if another reduction would occur in the PM to F step, for some kinetic reasons, e.g. the TyrO(rad) → TyrOH, then the CuB -reduction may occur in the next step, the F to OH step, which gives the same calculated values (within tenths of a kcal/mol) for the O-H bond strength in CuB (I)OH2 . As shown in Fig. 2, the calculated OH bond strength for CuB (I)OH2 in the active site of CcO corresponds to an exergonicity of 16-18 kcal/mol in the PM to F reduction step when the electron is delivered by cytochrome c, which in turn corresponds to a midpoint potential close to 1 V for CuB . This result is thus stable, both over the different oxidase families, and with respect to in which reduction step this reduction occurs. It is furthermore very similar to previously obtained computational results, using somewhat different CuB -a3 type of models [44]. And it is in sharp contrast to the low experimental values of 0.2-0.3 V obtained when trying to catch the properties of 13 ACS Paragon Plus Environment

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the OH state [22, 23, 24]. As mentioned in the introduction, one experimental study has been interpreted to show an elevated midpoint potential of CuB [21]. No explicit value was given, but by adding together a number of assumed modifications of the involved potentials given in the paper [21], a value of 0.46 V can be obtained. Although this is an interesting observation, this value is not included below when discussing how to find an explanation for the low values obtained for the CuB potential in several experimental studies. Fe(IV)=O → Fe(III)OH: It is suggested that the oxoferryl cofactor is reduced in the F to OH step, with calculated O-H bond strengths of 83.9 and 85.1 kcal/mol for the CuB -a3 and the CuB -b3 model, respectively. However, as is shown in Fig. 2, the tyrosine O-H bond is very similar in strength, and the order of these two reductions can not be determined based on the present model results. It has been suggested that the presence of a tyrosyl radical in the intermediates is necessary for the proton pumping [38, 44], which would indicate that the oxoferryl group should be reduced first, at least for the A-family, see further below. The latter is also in line with the experimental description of the oxidized (O) state as a high-spin heme a3 Fe(III) state [22, 61]. In any case, the calculated O-H bond strength in the heme Fe(III)OH product is the same, whichever order these two reductions occur in. This corresponds to an exergonicity of 13-14 kcal/mol relative to cytochrome c, and a midpoint potential of 0.8-0.9 V for the heme oxoferryl cofactor, in line with one of the two large experimental midpoint potentials [13]. TyrO(rad) → TyrOH: Assuming the structure of the OH state given in Fig. 2, reduction of the tyrosyl radical is thermodynamically most favourable in the OH to E step. The calculated TyrOH bond strength in this step for the CuB -a3 and the CuB -b3 model is 83.9 and 83.1 kcal/mol, respectively. It can be noted that it was previously suggested that the tyrosyl radical should be kept also in the E intermediate, to allow proton pumping also in the E to R reduction step [38, 44]. However, this was suggested to occur for kinetic reasons, and in the present study the focus is on the inherent thermodynamics of the reduction of the different cofactors. See further below, though. Relative to the cytochrome c donor this reduction step is exergonic by 12-13 kcal/mol, corresponding to a midpoint potential of about 0.8 V for the tyrosine cofactor, which is also in line with one of the two large experimental midpoint potentials [13]. Fe(III)OH → Fe(II)OH2 : Finally, in the E to R reduction step the high-spin heme iron is reduced and the last water O-H bond is formed. The O-H bond strength in the heme 14 ACS Paragon Plus Environment

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Fe(II)OH2 product obtained from the computational procedure is 71.2 and 72.0 kcal/mol, for the CuB -a3 and the CuB -b3 model, respectively. Thus, this is the weakest O-H bond strength in the BNC active site, and it gives a close to thermoneutral reaction relative to the cytochrome c donor. The corresponding midpoint potentials are about 0.3 V, with no big difference between the CuB -a3 and the CuB -b3 models, in contrast to the experimental results with a much lower value for the CuB -b3 oxidases [32]. It is interesting to find that applying the same procedure as in the present study on a model of cNOR [62], which can be referred to as a FeB -b3 system, gives a heme b3 Fe(III) potential of 0.09 V, quite close to the experimental value of 0.06 V [63]. This result indicates that it is the change from copper to a non-heme iron in the BNC that decreases the high-spin heme Fe(III) potential in cNOR, rather than the change from heme a3 to heme b3 . The important conclusion from the present study is that the CuB -a3 and the CuB -b3 enzymes, i.e. all CcO families, seem to have similar inherent high-spin heme Fe(III) midpoint potentials, close to the experimental values for the CuB -a3 oxidases. R → A → PM steps: To complete the catalytic cycles a few words should be said about the energetics of oxygen binding and cleavage. From experiment it is known that molecular oxygen should be weakly bound to the R state, and a binding energy of 0.9 kcal/mol is obtained for the CuB -a3 model, very close to the value of 1 kcal/mol used in previous studies [38, 44]. With the present procedure molecular oxygen is bound by 0.8 kcal/mol in the CuB -b3 model. The O-O bond cleavage step (A to PM ) is found to be exergonic by 6.8 kcal/mol in the CuB -a3 model and 3.7 kcal/mol in the CuB -b3 model. Importantly, when these exergonicities are summed up together with the exergonicites for all four reduction steps given in Fig. 2, a total exergonicity for the entire catalytic cycle of 50.7 kcal/mol is obtained for both systems. Furthermore, the reaction energy of each individual reduction step is found to be very similar in the two systems, the difference in each of the reduction steps is minor, 2 kcal/mol or less, which is within the uncertainty of the computational procedure. It is noted that exactly the same procedure, including corrections, is used for the two models. The experimental midpoint potentials: As mentioned in the introduction, two of the active site reduction potentials were at an early stage found to be large enough for proton pumping, 1.02 V and 0.85 V [13] (corrected for the fact that only one proton is pumped in each step). These two potentials have been considered to correspond to the reduction of the


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Figure 3: The O state with an extra proton for the CuB -a3 model and the E state with an extra proton for the CuB -b3 model. The optimized structures are shown together with the most important spin-populations. tyrosyl radical and the Fe(IV)=O, see e.g. [8]. However, the calculated midpoint potentials presented above actually suggest that it might be CuB and Fe(IV)=O that are reduced in those two steps, with calculated values of 0.96-1.05 V and 0.82-0.87 V, respectively. Furthermore, if the OH state as described in Fig. 2 could be experimentally prepared and possible to analyse, the measured midpoint potentials would correspond to the tyrosyl radical and heme a3 Fe(III), with values about 0.8 V and 0.3 V, respectively, according to the calculations, which is also in contrast to the experimental measurements giving 0.2-0.4 V for both reductions in the reductive part of the cycle [22, 23, 24, 26, 27, 28, 29, 30]. Another discrepancy between experimental measurements and the calculations is found for the heme b3 Fe(III) potential, which is calculated to be very similar to the one for heme a3 Fe(III), while most experimental values are significantly lower, by as much as 0.4 V [32], although there are a couple of values above 0.2 V reported [64, 65]. The question is, how to explain these sifnificant discrepancies between some of the calculated midpoint potentials and the corresponding experimental values? In fact, the computational results may give some hints to where to search for a major part of the explanations. As emphasized above 16 ACS Paragon Plus Environment

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each calculated proton coupled reduction potential corresponds to the formation of one new O-H bond, and they are therefore referred to as “chemical”. This implies that if the active site somehow is modified under the particular experimental conditions, the proton coupled reduction that occurs may not any longer correspond to such a chemical reaction step. In general both the electron transfer and the proton transfer contribute to the midpoint potential, and in particular when a stable chemical bond is formed, both may contribute significantly. Regarding the CuB potential the discrepancy between the experimentally measured values and the calculated ones has been reported previously for the CuB -heme a3 oxidases [38, 44], and the same discrepancy is now obtained also for the CuB -heme b3 enzymes. A possible explanation for the difference between the measured values and the calculated ones, which should apply to both systems, has already been suggested [66]. The O-O bond cleavage creates several sites with high proton affinity, and therefore it is likely that if the OH state, with the structure shown in Fig. 2, is left with no more electrons coming in (as in the resting state), another proton is slowly transferred into the BNC, to the tyrosyl radical, which also moves an electron from CuB (I) leading to TyrOH-CuB (II)-OH-Fe(III). This protonated form of the O state is shown in Fig. 3 for the CuB -a3 model, and a very similar type of structure is obtained for the CuB -b3 model. Importantly, the proton is suggested to move from outside the BNC, but it does not have to be an external proton. The point is that the balance between electrons and protons in the BNC has been perturbed, which means that on reduction of this kind of state it is not sure that there is a proton uptake all the way into the BNC, which would clearly affect the measured midpoint potential. A hint of the effect on the midpoint potential can in this case be obtained by calculating the strength of an O-H bond formed by assuming uptake of both an electron and a proton to the BNC, starting from the TyrOH-CuB (II)-OH-Fe(III) structure, i.e. with an “extra” proton. Reducing CuB and forming a water molecule between the metals, gives an O-H bond strength that is 13-14 kcal/mol smaller than the values given in Fig. 2, corresponding to a decrease by 0.5-0.6 V of the CuB potential, approaching the experimental values. Since the product of such a proton coupled reduction step is missing a negative ligand on the high-spin heme Fe(III) group, it is not very stable, and it may very well happen that there is no proton uptake into the BNC. It was suggested in ref. [66] that the protonated O state corresponds to the resting oxidized state, and the transfer of an “extra” proton into the BNC could thus be described as a deactivation of the OH state that is involved during


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turnover. The difference between the reduction potentials of the unprotonated OH state and the protonated O state should explain the missing energy in the sum of the reduction step energies as compared to the energy of the entire reduction process discussed in ref. [8]. It should also be noted that a proton transfer into the BNC may very well be connected with a rather high barrier, leading to enough life-time for the unprotonated state during catalytic turnover [66]. Finally, it is here suggested that it is possible that such an “extra” proton in the BNC may become present under certain experimental conditions, which may explain the failure to prepare a true OH state in several experimental studies. In fact, in all those experiments there has to be more electrons present in the enzyme when starting to reduce molecular oxygen, as compared to during catalytic turnover, which means that it should not be unlikely that there are more easily accessible protons available in the enzyme that leads to the formation of the protonated O state immediately after reduction of the F state, via an internal proton transfer to the BNC. This would explain the low CuB reduction midpoint potentials also in those experiments [22, 24, 26]. In a previous computational study an alternative explanation to the difference between the OH and O states was suggested [20]. In accordance with the original suggestion [17] that the difference between the two states is a difference in geometrical structure, that computational study suggests that the main difference between the OH and O states is obtained by moving a water molecule from what is found to be a less stable position in the OH state to a more stable position in the O state. The main problem with this suggestion is that it is quite unlikely that the activation energy needed to move a water molecule is high enough to explain the necessary life time of the high-energy OH state. It is also clear from the paper that the authors did not manage to find such a barrier [20]. In contrast, in the present suggestion, the OH state is determined to be in the most stable structure for this number of electrons and protons, and its proton coupled reduction potential is still high. And as mentioned above, it is quite likely that the transfer of another proton into the BNC to form the O state with an extra proton, as suggested here, is associated with a rather high barrier, since it is well known that proton transfer processes often are involved in the rate determining steps in enzyme reactions. Finally, it is not possible to compare the calculated midpoint potentials, since the values given in [20] are without charge-compensating protonation. A similar situation as discussed above for the O state actually occurs for the E state in the cbb3 oxidase, for which the calculations predict a significantly higher midpoint potential


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than most of the experiments. The reason is that the presence of the negatively charged glutamate hydrogen bonding to the proximal histidine in the active site, may lead to the uptake of an extra proton already before the reduction of the E state, and similar to the protonated O state, this may explain the low experimental high-spin heme-Fe(III) reduction potential in cbb3 . It should first be noted, that it is not as simple as the presence of a negative charge in itself decreases the midpoint potential, since when the electron uptake is coupled to a proton uptake to a nearby site, there are opposite effects of the negative group on the electron and the proton uptake, mainly cancelling the charge effect. Instead, similar to the CuB case, an “extra” proton in the BNC may explain the low high-spin heme-Fe(III) midpoint potential obtained in the experiments for cbb3 oxidases. The E state, with the structure shown in Fig. 2, turns out to have a higher affinity for a proton transfer into the BNC, forming a water molecule between the metals, in the CuB -heme b3 model as compared to the CuB -heme a3 model. The reason is that when the proton enters the BNC, the negative glutamate in the b3 case, takes a proton from the proximal histidine, leading to a negatively charged proximal histidine that stabilizes the high-spin heme-Fe(III) state, see Fig. 3. The same motion of the histidine proton to the glutamate in the protonated E state was observed in previous calculations on models of the cbb3 active site, but the conclusions were different from the present ones [33]. It is here suggested that in most of the experimental measurements of the heme b3 Fe(III) potential in cbb3 oxidases, there may already be an “extra” proton in the BNC before the Fe(III) reduction, which again would result in a decreased midpoint potential, since it does not correspond to the full formation of a O-H bond. Proton pumping: The proton pumping is not the focus of the present study, but since it is one major motivation for paying attention to the individual midpoint potentials in the BNC of the CcO enzymes, in particular for the A-familiy, a few words should still be said about the relation between the results presented above and the proton pumping in the A-family. First of all, to allow proton pumping in all reduction steps in the A-family, the exergonicity of each step has to be large enough, and as pointed out in the introduction, only two of the experimental active site reduction potentials seem to fulfil this criterion [13]. In accordance with the suggestions made on the basis of experimental studies [16, 17], previous computational studies on CuB -heme-a3 models found a high enough reduction potential also for CuB during catalytic turnover [38, 44], which is here further supported by the new results for the CuB -heme b3 model of the cbb3 oxidase. The calculations thus give 19 ACS Paragon Plus Environment

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three midpoint potentials at about 0.8 V or higher, see Fig. 2, which should be high enough to allow proton pumping. This solves part of the puzzle regarding the relation between proton pumping and the energetics in the individual reduction steps in the A-family, but for one of the reduction steps, the E to R step, the calculations support the experimental result with a too low heme Fe(III) reduction potential. At this point it can be noted that proton pumping schemes corresponding to the pumping of only one proton during the O to R steps, and accordingly three protons pumped during the P to O steps have been suggested. One of those was based on the experimental observation that at least one proton is pumped during the O to R steps [67], which challenged the previous suggestion that all protons should be pumed during the P to O steps [14]. A detailed scheme for proton pumping, based on electrostatic repulsion and the electroneutrality principle, was constructed to comply with the observation that one proton is pumped in the reductive phase [67, 68]. Another carefully constructed proton pumping scheme implying that one proton is pumped during the O to R steps was based on electrostatic calculations [69]. On the other hand, based on quantum chemical calculations it was found that an improved energy partitioning over the reduction steps was obtained if the redox-active tyrosine was left unprotonated in the E state, i.e. if the chemical proton in the O to E step ended up in the center of the BNC [70, 71]. As mentioned above in connection with the discussion about the reduction potential of the tyrosine, this is not the thermodynamically preferred structure of the E state. Another aspect of the proton pumping is the mechanism for coupling the electron transfer to the uptake of more than one proton per electron. Additional quantum chemical studies later resulted in the propsal that to secure proton pumping in all four reduction steps, all intermediates should have a structure with a tyrosyl radical [38]. This type of structure is also referred to as an unprotonated tyrosine, since the essential feature of this structure is that the chemical proton goes to the center of the BNC rather than to the tyrosine, which means that the electronic structure may very well more or less correspond to a tyrosinate in combination with a cupric CuB ion, which in turn may explain why no tyrosyl radical has been observed in EPR experiments. The background to the proposal is that it was found that for all intermediates in the catalytic cycle, that kind of structure of the BNC has a significantly higher electron affinity than the corresponding structure with one of the chemical protons located on the tyrosine [38]. A common suggestion for the proton pumping mechanism is that the transfer of a proton to the pump-loading site


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near the BNC active site increases the electron affinity of the BNC so that the electron can enter the BNC. In line with such a coupling mechanism it was suggested that only for the BNC structure with the higher electron affinity, the rather small increase in electron affinity caused by the uptake of a proton to the pump-loading site results in an affinity larger than that of the immediate electron donor low-spin heme a (the A-family of CcO was considered) [38]. In this way, the uptake of the pump-proton would trigger electron transfer from the low-spin heme into the BNC, followed by uptake of the chemical proton and release of the proton in the pump-loading site [38]. A crucial role for the cross-linked tyrosine in the proton pumping mechanism is in line with its conservation in all kinds of cytochrome oxidases [72]. The calculations reported above indicate that in the case of the OH intermediate, such a structure may well be the thermodynamically most stable one. On the other hand, the corresponding structure with an unprotonated tyrosine for the E intermediate, now labelled EH , and described to have the following structure: TyrO(rad)-CuB (I)-H2 O-Fe(II), is not the lowest in energy and would have to be formed for kinetic reasons, most likely connected with the differences between the two proton channels present in the A-family oxidases [38]. Futhermore, as mentioned above it was early suggested that the tyrosine actually should be kept unprotonated until the intermediate R is formed, since this gives an improved energy partitioning between the reduction steps [70, 71], which means that the structure with an unprotonated tyrosine not only allows mechanistically for proton pumping also in the E (or rather EH ) to R step, but it also seems to solve the problem with the low high-spin heme-Fe(III) midpoint potential. The electronic structure of EH as it is written above indicates that the high-spin heme Fe(III) reduction should occur already in the O to E step. However, the calculations show that the EH state mixes in some tyrosinate-Fe(III) character, which means that when the Fe(III) reduction occurs in the O to EH step, the reduction energy is increased by the fact that the larger energy of the tyrosyl reduction is shared between the two reduction steps (O to EH and EH to R) [38]. In this way also the problem with the low high-spin heme Fe(III) potential may be solved, and a catalytic cycle with one proton pumped in each reduction step can be constructed, in spite of one single low reduction potential. The discussion in the previous paragraphs is concerned with the A-family of CcO’s, which are known to pump four protons per oxgen molecule. The A-family is also characterized by having two proton channels to the BNC (the D- and the K-channels) [3], and an immediate electron donor to the BNC, low-spin heme a, with a midpoint potential close to


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that of cytochrome c, 0.25 V. For the B- and the C-families the situation is quite different with regard to proton pumping. These oxidases are most often considered to pump only two protons per oxygen molecule, i.e. on the average 0.5 proton per electron [10]. The most striking structural difference between the CcO families is that the B- and the C-families have only one proton channel (the K-analogue channel) [3], which should transfer both chemical and pumped protons. Since the single proton channel passes more or less through the redox active tyrosine, it should not be possible to form the type of kinetically preferred intermediates with a tyrosyl radical (unprotonated tyrosine) suggested to be involved for the A-family, which most likely leads to decreased proton pumping for the B- and the Cfamilies. Furthermore, the B- and the C-families have a different immediate electron donor to the BNC (low-spin heme b), with a different midpoint potential as compared to the lowspin heme a in the A-family, which may affect the proton pumping, both mechanistically and energetically. Finally, the negative charge in the vicinity of the BNC in the C-family, which does not affect the O-H bond strength significantly, and thus not the proton coupled midpoint potential, may still have a significant effect on the separate electron and proton affinities (in opposite directions) in the BNC. This may very well modify the mechanism and energetics for the proton pumping in these enzymes, which is in line with the experimental observations that, in particular the A- and the B-families have significantly different reaction mechanisms [34, 35, 36].

4. Conclusions Quantum chemical calculations on BNC-active site models are used to estimate the inherent reduction potentials of the active site cofactors of different families of heme-copper oxidases. The strength of the O-H bond formed in each of the four reduction steps in the catalytic cycle of oxygen reduction to water is calculated using density functional theory. By comparing these bond strengths to the energy of the electron transferred from the donor and the proton transferred from bulk water, an estimate of the total exergonicity of each reduction step is obtained. Finally, by comparison to the potential of the electron donor (cytochrome c) the proton coupled reduction potentials can be deduced from the exergonicity of each reduction step. The calculations indicate that all families have similar active site inherent reduction potentials, i.e. for CuB , tyrosine and the high-spin heme (both ferryl and ferric). This result is in contrast to some experimental studies, which report a significantly


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lower high-spin heme-Fe(III) potential for the C-family as compared to both the A- and the B-families. The calculations also indicate that CuB has the largest reduction potential in the BNC for all CcO families, suggesting that it is the first cofactor to be reduced after the O-O bond cleavage, which is also a striking difference to the experimentally reported potentials for CuB . Possible sources for the differences between experiment and theory are indicated. It is suggested that the reduction potentials obtained from the calculations may be more relevant for catalytic turnover than some of the experimental ones, and the connection between the computational results and proton pumping in the A-family is briefly discussed. Finally, the results indicate that the mechanistic differences found between the families most likely should be explained by kinetic factors together with differences in the location of the proton pathways, which are not the focus of the present study.

ASSOCIATED CONTENT Supporting Information Available: Contains Fig S1 and S2 giving an overview of the directly calculated relative energies and the different procedures used for corrections, together with cartesian coordinates for the most important structures.

Funding This work was supported by the Swedish Research Council (grant number 2016-03721). Computer time was provided by the Swedish National Infrastructure for Computing.

References [1] Pereira, M.M., Santana, M., Teixeira, M. (2001) A novel scenario for the evolution of haem-copper oxygen reductases, Biochim. Biophys. Acta 1505, 185-208. [2] Hemp, J., Gennis, R.B. (2008) Diversity of the heme-copper superfamily in archaea: Insights from genomics and structural modeling, Results Probl. Cell Differ. 45, 1-31. ¨ [3] Lee, H.J., Reimann, J., Huang, Y., Adelroth, P. (2012) Functional proton transfer pathways in the heme-copper oxidase superfamily, Biochim. Biophys. Acta 1817, 537544.


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[4] Proshlyakov, D.A., Pressler, M.A., Babcock, G.T. (1998) Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase, Proc. Natl. Acad. Sci. U.S.A. 95, 8020-8025. [5] Fabian, M., Wong, W.W., Gennis, R.B., Palmer, G. (1999) Mass spectrometric determination of dioxygen bond splitting in the “peroxy” intermediate of cytochrome c oxidase, Proc. Natl. Acad. Sci. U.S.A. 96, 13114-13117. [6] Brzezinski, P., Gennis, R.B. (2008) Cytochrome c oxidase: Exciting progress and remaining mysteries, J. Bioenerg. Biomembr. 40, 521-531. [7] Brzezinski, P. (2004) Redox-driven membrane-bound proton pumps, Trends Biochem. Sci. 29, 380-387. [8] Kaila, V.R.I., Verkhovsky, M.I., Wikström, M. (2010) Proton-Coupled Electron Transfer in Cytochrome Oxidase, Chem. Rev. 110, 7062-7081. [9] Popovic, D.M., Leontyev, I.V., Beech, D.G. and Stuchebrukhov, A.A. (2010) Similarity of cytochrome c oxidases in different organisms, Proteins 78, 2691-2698. [10] Han, H., Hemp, J., Pace, L.A., Ouyang, H., Ganesan, K., Hyeob Roh, J., Daldal, F., Blanke, S.R., Gennis, R.B. (2011) Adaption of aerobic respiration to low O2 environments, Proc Natl Acad Sci USA 108, 14109-14114. [11] Rich, P.R. (2017) Mitochondrial cytochrome c oxidase: catalysis, coupling and controversies, Biochemical Society Transactions 45 813-829. [12] Wikström, M., Krab, K., Sharma, V. (2018) Oxygen Activation and Energy Conservation by Cytochrome c Oxidase, Chem. Rev. 118, 2469-2490. [13] Wikström, M., Morgan, J.E. (1992) The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction, J Biol Chem 267, 10266-10273. [14] Wikström, M. (1989) Identification of the electron transfers in cytochrome c oxidase that are coupled to proton-pumping, Nature 338, 776-778. [15] Morgan, J.E., Verkhovsky, M.I., Wikström, M. (1994) The histidine cycle: A new model for proton translocation in the respiratory heme-copper oxidases, J. Bioenerg. Biomemb.26, 599-608. 24 ACS Paragon Plus Environment

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Biochemistry

Graphical abstract


Active site midpoint potentials in different cytochrome c oxidase families

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