Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump which converts atmospheric oxygen to water and couples the oxygen reduction reaction to the creation of a membrane proton gradient. The structure of the enzyme has been solved; however, the mechanism of proton pumping is still poorly understood. Recent calculations from this group indicate that one of the histidine ligands of enzyme's CuB center, His291, may play the role of the pumping element. In this paper, we report on the results of calculations that combined first principles DFT and continuum electrostatics to evaluate the energetics of the key energy generating step of the model-the transfer of the chemical proton to the binuclear center of the enzyme, where the hydroxyl group is converted to water, and the concerted expulsion of the proton from delta-nitrogen of His291 ligand of CuB center. We show that the energy generated in this step is sufficient to push a proton against an electrochemical membrane gradient of about 200 mV. We have also re-calculated the pKa of His291 for an extended model in which the whole Fe(a3)-CuB center with their ligands is treated by DFT. Two different DFT functionals (B3LYP and PBE0), and various dielectric models of the protein have been used in an attempt to estimate potential errors of the calculations. Although current methods of calculations do not allow unambiguous predictions of energetics in proteins within few pKa units, as required in this case, the present calculation provides further support for the proposed His291 model of CcO pump and makes a specific prediction that could be targeted in the experimental test.     

Coupled electron and proton transfer reactions during the O → E transition in bovine cytochrome c oxidase

A combined DFT/electrostatic approach is employed to study the coupling of proton and electron transfer reactions in cytochrome c oxidase (CcO) and its proton pumping mechanism. The coupling of the chemical proton to the internal electron transfer within the binuclear center is examined for the O → E transition. The novel features of the His291 pumping model are proposed, which involve timely well-synchronized sequence of the proton-coupled electron transfer reactions. The obtained pK_as and E_ms of the key ionizable and redox-active groups at the different stages of the O → E transition are consistent with available experimental data. The PT step from E242 to H291 is examined in detail for various redox states of the hemes and various conformations of E242 side-chain. Redox potential calculations of the successive steps in the reaction cycle during the O → E transition are able to explain a cascade of equilibria between the different intermediate states and electron redistribution between the metal centers during the course of the catalytic activity. All four electrometric phases are discussed in the light of the obtained results, providing a robust support for the His291 model of proton pumping in CcO. This article is part of a Special Issue entitled: Respiratory oxidases.     

Theoretical and computational analysis of the membrane potential generated by cytochrome c oxidase upon single electron injection into the enzyme

We have developed theory and the computational scheme for the analysis of the kinetics of the membrane potential generated by cytochrome c oxidase upon single electron injection into the enzyme. The theory allows one to connect the charge motions inside the enzyme to the membrane potential observed in the experiments by using data from the “dielectric topography” map of the enzyme that we have created. The developed theory is applied for the analysis of the potentiometric data recently reported by the Wikström group [I. Belevich, D.A. Bloch, N. Belevich, M. Wikström and M.I. Verkhovsky, Exploring the proton pump mechanism of cytochrome c oxidase in real time, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 2685-2690] on the O to E transition in Paracoccus denitrificans oxidase. Our analysis suggests, that the electron transfer to the binuclear center is coupled to a proton transfer (proton loading) to a group just “above” the binuclear center of the enzyme, from which the pumped proton is subsequently expelled by the chemical proton arriving to the binuclear center. The identity of the pump site could not be determined with certainty, but could be localized to the group of residues His326 (His291 in bovine), propionates of heme a₃, Arg 473/474, and Trp164. The analysis also suggests that the dielectric distance from the P-side to Fe a is 0.4 or larger. The difficulties and pitfalls of quantitative interpretation of potentiometric data are discussed.     

Molecular dynamics simulation of water in cytochrome c oxidase reveals two water exit pathways and the mechanism of transport

We have examined the network of connected internal cavities in cytochrome c oxidase along which water produced at the catalytic center is removed from the enzyme. Using combination of structural analysis, molecular dynamics simulations, and free energy calculations we have identified two exit pathways that connect the Mg²⁺ ion cavity to the outside of the enzyme. Each pathway has a well-defined bottleneck, which determines the overall rate of water traffic along the exit pathway, and a specific cooperative mechanism of passing it. One of the pathways is going via Arg438/439 (in bovine numbering) toward the Cu_A center, approaching closely its His204_B ligand and Lys171_B residue; and the other is going toward Asp364 and Thr294. Comparison of the pathways among different aa₃-type enzymes shows that they are well conserved. Possible connections of the finding to redox-coupled proton pumping mechanism are discussed. We propose specific mutations near the bottlenecks of the exit pathways that can test some of our hypotheses.     

Spectroscopic study on the communication between a heme a3 propionate, Asp399 and the binuclear center of cytochrome c oxidase from Paracoccus denitrificans

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a₃, including Asp399 and the Cu_B ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.     

Two conformational states of Glu242 and pKas in bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain of aerobic organisms. It catalyses the reduction of atmospheric oxygen to water, and couples this reaction to proton pumping across the membrane; this process generates the electrochemical gradient that subsequently drives the synthesis of ATP. The molecular details of the mechanism by which electron transfer is coupled to proton pumping in CcO is poorly understood. Recent calculations from our group indicate that His291, a ligand of the Cu(B) center of the enzyme, may play the role of the pumping element. In this paper we describe calculations in which a DFT/continuum electrostatic method is used to explore the coupling of the conformational changes of Glu242 residue, the main proton donor of both chemical and pump protons, to its pKa, and the pKa of His291, a putative proton loading site of our pumping model. The computations are done for several redox states of metal centers, different protonation states of Glu242 and His291, and two well-defined conformations of the Glu242 side chain. Thus, in addition to equilibrium redox/protonation states of the catalytic cycle, we also examine the transient and intermediate states. Different dielectric models are employed to investigate the robustness of the results, and their viability in the light of the proposed proton pumping mechanism of CcO. The main results are in agreement with the experimental measurements and support the proposed pumping mechanism. Additionally, the present calculations indicate a possibility of gating through conformational changes of Glu242; namely, in the pumping step, we find that Glu242 needs to be reprotonated before His291 can eject a proton to the P-site of membrane. As a result, the reprotonation of Glu can control proton release from the proton loading site.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 - one of the ligands of the Cu(B) redox center - which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second "chemical" proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.     

Time-resolved OH → EH transition of the aberrant ba3 oxidase from Thermus thermophilus

The kinetics of single-electron injection into the oxidized nonrelaxed state (O_H → E_H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ ∼ 17 μs) reflects oxidation of Cu_A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ ∼ 420 μs) includes reoxidation of both hemes by Cu_B. This is in significant contrast to the O_H → E_H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu_A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O_H forms also in the ba₃ oxidase from T. thermophilus.     

Modulation of the electron-proton coupling at cytochrome a by the ligation of the oxidized catalytic center in bovine cytochrome c oxidase

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a₃ (Fe_a3) and copper (Cu_B). Here, the influence of the ligation at the oxidized Fe_a3³⁺–Cu_B²⁺ center on the electron–proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (E_m(a)) for the cyanide (the low-spin Fe_a3³⁺) and the formate-ligated CcO (the high-spin Fe_a3³⁺). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of E_m(a) were found for the CN– and the formate–ligated CcO with slopes of −13 mV/pH unit and −23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron–proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron–proton coupling at the physiological pH values is also substantiated by the UV–Vis absorption and electron–paramagnetic resonance spectroscopy investigations of the cyanide–ligated oxidized CcO. It is shown that the ligand exchange at Fe_a³⁺ between His–Fe_a³⁺–His and His–Fe_a³⁺–OH⁻ occurs only at pH above 9.5 with the estimated pK >11.0.     

Electrostatic control of proton pumping in cytochrome c oxidase

As part of the mitochondrial respiratory chain, cytochrome c oxidase utilizes the energy produced by the reduction of O₂ to water to fuel vectorial proton transport. The mechanism coupling proton pumping to redox chemistry is unknown. Recent advances have provided evidence that each of the four observable transitions in the complex catalytic cycle consists of a similar sequence of events. However, the physico-chemical basis underlying this recurring sequence has not been identified. We identify this recurring pattern based on a comprehensive model of the catalytic cycle derived from the analysis of oxygen chemistry and available experimental evidence. The catalytic cycle involves the periodic repetition of a sequence of three states differing in the spatial distribution of charge in the active site: [0|1], [1|0], and [1|1], where the total charge of heme a and the binuclear center appears on the left and on the right, respectively. This sequence recurs four times per turnover despite differences in the redox chemistry. This model leads to a simple, robust, and reproducible sequence of electron and proton transfer steps and rationalizes the pumping mechanism in terms of electrostatic coupling of proton translocation to redox chemistry. Continuum electrostatic calculations support the proposed mechanism and suggest an electrostatic origin for the decoupled and inactive phenotypes of ionic mutants in the principal proton-uptake pathway.     

Ligand Trapping by Cytochrome c Oxidase: IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER*

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe_a3³⁺–Cu_B²⁺) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe_a3³⁺–CN–Cu_B²⁺ complex. Instead the cyanide-induced transition of a preformed Fe_a3³⁺–N₃–Cu_B²⁺ to the ternary complex of Fe_a3³⁺–N₃ Cu_B²⁺–CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu_B blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu_B and that, during catalysis, ligands present at this copper center control access to the iron of heme a₃ from the bulk medium.     

Cytochrome oxidase: pathways for electron tunneling and proton transfer

 Electrons from cytochrome c, the substrate of cytochrome oxidase, a redox-linked proton pump, are accepted by Cu_A in subunit II. From there they are transferred to the proton pumping machinery in subunit I, cytochrome a and cytochrome a ₃–Cu_B. The reduction of the latter site, which is the dioxygen reducing unit, is coupled to proton uptake. Dioxygen reduction involves a peroxide and a ferryl ion intermediate, and it is the transition between these and back to the resting oxidized enzyme that are coupled to proton pumping. The X-ray structures suggest electron–transfer pathways that can account for the observed rates provided that the reorganization energies are small. They also reveal two proton-transfer pathways, and mutagenesis experiments have shown that one is used for proton uptake during the initial reduction of cytochrome a ₃–Cu_B, whereas the other mediates transfer of the pumped protons. Received: 23 March 1998 / Accepted: 11 May 1998     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

The chemistry of the CuB site in cytochrome c oxidase and the importance of its unique His-Tyr bond

The Cu_B metal center is at the core of the active site of the heme-copper oxidases, comprising a copper atom ligating three histidine residues one of which is covalently bonded to a tyrosine residue. Using quantum chemical methodology, we have studied the Cu_B site in several redox and ligand states proposed to be intermediates of the catalytic cycle. The importance of the His-Tyr crosslink was investigated by comparing energetics, charge, and spin distributions between systems with and without the crosslink. The His-Tyr bond was shown to decrease the proton affinity and increase the electron affinity of both Tyr-244 and the copper. A previously unnoticed internal electronic equilibrium between the copper atom and the tyrosine was observed, which seems to be coupled to the unique structure of the system. In certain states the copper and Tyr-244 compete for the unpaired electron, the localization of which is determined by the oxygenous ligand of the copper. This electronic equilibrium was found to be sensitive to the presence of a positive charge 10 Å away from the center, simulating the effect of Lys-319 in the K-pathway of proton transfer. The combined results provide an explanation for why the heme-copper oxidases need two pathways of proton uptake, and why the K-pathway is active only in the second half of the reaction cycle.     

Critical roles of the CuB site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates

Mammalian cytochrome c oxidase (CcO) reduces O₂ to water in a bimetallic site including Fe_a3 and Cu_B giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at ∼1.8 Å resolution of fully reduced CcO crystals treated with O₂ for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained ∼45% of the O-form structure, ∼45% of the E-form structure, and ∼20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long Cu_B²⁺-OH⁻ distance and Cu_B¹⁺-H₂O structure keeping Fe_a3³⁺-OH⁻ state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O→E and E→R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping.     

Redox-coupled proton pumping in cytochrome c oxidase: Further insights from computer simulation

The membrane-bound enzyme cytochrome c oxidase, the terminal member in the respiratory chain, converts oxygen into water and generates an electrochemical gradient by coupling the electron transfer to proton pumping across the membrane. Here we have investigated the dynamics of an excess proton and the surrounding protein environment near the active sites. The multi-state empirical valence bond (MS-EVB) molecular dynamics method was used to simulate the explicit dynamics of proton transfer through the critically important hydrophobic channel between Glu242 (bovine notation) and the D-propionate of heme a₃ (PRDa₃) for the first time. The results from these molecular dynamics simulations indicate that the PRDa₃ can indeed re-orientate and dissociate from Arg438, despite the high stability of such an ion pair, and has the ability to accept protons via bound water molecules. Any large conformational change of the adjacent heme a D-propionate group is, however, sterically blocked directly by the protein. Free energy calculations of the PRDa₃ side chain isomerization and the proton translocation between Glu242 and the PRDa₃ site have also been performed. The results exhibit a redox state-dependent dynamical behavior and indicate that reduction of the low-spin heme a may initiate internal transfer of the pumped proton from Glu242 to the PRDa₃ site.     

Exploring pathways and barriers for coupled ET/PT in cytochrome c oxidase: A general framework for examining energetics and mechanistic alternatives

Gaining a detailed understanding of the energetics of the proton pumping process in cytochrome c oxidase (CcO) is one of the challenges of modern biophysics. Although there are several current mechanistic proposals, most of these ideas have not been subjected to consistent structure-function considerations. In particular most works have not related the activation barriers for different mechanistic proposals to the protein structure. The present work describes a general approach for exploring the energetics of different feasible models of the action of CcO, using the observed protein structure, established simulation methods and a modified Marcus' formulation. We start by reviewing our methods for evaluation of the energy diagrams for different proton translocation paths and then present a systematic analysis of various constraints that should be imposed on any energy diagram for the pumping process. After the general analysis we turn to the actual computational study, where we construct energy diagrams for forward and backward paths, using the estimated calculated reduction potentials and pK_a values of all the relevant sites (including internal water molecules). We then explore the relationship between the calculated energy diagrams and key experimental constraints. This comparison allows us to identify some barriers that are not fully consistent with the overall requirement for an efficient pumping. In particular we identify back leakage channels, which are hard to block without stopping the forward channels. This helps to identify open problems that will require further experimental and theoretical studies. We also consider reasonable adjustments of the calculated barriers that may lead to a working pump. Although the present analysis does not establish a unique and workable model for the mechanism of CcO, it presents what is probably the most consistent current analysis of the barriers for different feasible pathways. Perhaps more importantly, the framework developed here should provide a general way for examining any proposal for the action of CcO as well as for the analysis of further experimental findings about the action of this fascinating system.     

Proton transfer function of carbonic anhydrase: Insights from QM/MM simulations

Recent QM/MM analyses of proton transfer function of human carbonic anhydrase II (CAII) are briefly reviewed. The topics include a preliminary analysis of nuclear quadrupole coupling constant calculations for the zinc ion and more detailed analyses of microscopic pK_a of the zinc-bound water and free energy profile for the proton transfer. From a methodological perspective, our results emphasize that performing sufficient sampling is essential to the calculation of all these quantities, which reflects the well solvated nature of CAII active site. From a mechanistic perspective, our analyses highlight the importance of electrostatics in shaping the energetics and kinetics of proton transfer in CAII for its function. We argue that once the pK_a for the zinc-bound water is modulated to be in the proper range (~ 7.0), proton transfer through a relatively well solvated cavity towards/from the protein surface (His64) does not require any major acceleration. Therefore, although structural details like the length of the water wire between the donor and acceptor groups still may make a non-negligible contribution, our computational results and the framework of analysis suggest that the significance of such “fine-tuning” is likely secondary to the modulation of pK_a of the zinc-bound water. We encourage further experimental analysis with mutation of (charged) residues not in the immediate neighborhood of the zinc ion to quantitatively test this electrostatics based framework; in particular, Φ analysis based on these mutations may shed further light into the relative importance of the classical Grotthus mechanism and the “proton hole” pathway that we have proposed recently for CAII.