Free energy profiles for H+ conduction in the D-pathway of Cytochrome c Oxidase: a study of the wild type and N98D mutant enzymes

The molecular mechanism for proton conduction in the D-pathway of Cytochrome c Oxidase (CcO) is investigated through the free energy profile, i.e., potential of mean force (PMF) calculations of both the native enzyme and the N98D mutant. The multistate empirical valence bond (MS-EVB) model was applied to simulate the interaction of an excess proton with the channel environment. In the study of the wild type enzyme, the PMF reveals the previously proposed proton trap inside the channel; it also shows a high free energy barrier against the passage of proton at the entry of the channel, where two conserved asparagines (ASN80/98) may be essential for the gating of proton uptake. We also present data from an investigation of the N98D mutant, which has been previously shown to completely eliminate proton pumping but significantly enhance the oxidase activity in Rhodobacter sphaeroides. These results suggest that mutating Asn98 to negatively charged aspartate will create an unfavorable energy barrier sufficiently high to prevent the overall proton uptake through the D-pathway, whereas with a protonated aspartic acid the proton conduction was found to be accelerated. Plausible explanations for the origin of the uncoupling of proton pumping from the oxidase activity will be discussed.     

Free energy profiles for H conduction in the D-pathway of Cytochrome c Oxidase: A study of the wild type and N98D mutant enzymes

The molecular mechanism for proton conduction in the D-pathway of Cytochrome c Oxidase (CcO) is investigated through the free energy profile, i.e., potential of mean force (PMF) calculations of both the native enzyme and the N98D mutant. The multistate empirical valence bond (MS-EVB) model was applied to simulate the interaction of an excess proton with the channel environment. In the study of the wild type enzyme, the PMF reveals the previously proposed proton trap inside the channel; it also shows a high free energy barrier against the passage of proton at the entry of the channel, where two conserved asparagines (ASN80/98) may be essential for the gating of proton uptake. We also present data from an investigation of the N98D mutant, which has been previously shown to completely eliminate proton pumping but significantly enhance the oxidase activity in Rhodobacter sphaeroides. These results suggest that mutating Asn98 to negatively charged aspartate will create an unfavorable energy barrier sufficiently high to prevent the overall proton uptake through the D-pathway, whereas with a protonated aspartic acid the proton conduction was found to be accelerated. Plausible explanations for the origin of the uncoupling of proton pumping from the oxidase activity will be discussed.     

Surface proton donors for the D-pathway of cytochrome c oxidase in the absence of subunit III

The major proton-transfer pathway into the buried active site of cytochrome c oxidase (CcO) is the D-pathway that begins with the subunit I residue Asp-132 on the inner protein surface (the cytoplasmic surface of the aa3-type CcO of Rhodobacter sphaeroides). Asp-132 is surrounded by residues from both subunits I and III. In the absence of subunit III, CcO retains activity, but the functional characteristics of the D-pathway are significantly altered such that the transfer of protons from Asp-132 into the pathway becomes the rate-limiting step. Determination of the pH-dependence of the rate constant for D-pathway proton uptake during the single-turnover of CcO indicates that the pKa of Asp-132 in the absence of subunit III is approximately 7. The removal of subunit III also allows for alternative surface proton donor/acceptors other than Asp-132. With Asp-132 altered to alanine, the rate constant for D-pathway proton uptake is very slow (5 s(-1)) in the presence of subunit III. Once subunit III is removed, the proton uptake rate constant increases 80-fold, to 400 s(-1). The pKa associated with this uptake is >10, and the initial proton donor/acceptor in D132A III (-) is proposed to be a water of the D-pathway rather than an amino acid residue. Arachidonic acid (Aa), which stimulates the activity of several D-pathway mutant CcOs, appears to become the initial proton donor/acceptor in the absence of subunit III, whether or not Asp-132 is altered. Aa shifts the pKa of the initial proton donor to 7.6 for both wild-type (WT) III (-) and D132A III (-). The results indicate that subunit III creates a barrier that helps prevent protons from donors other than Asp-132 from directly accessing the internal waters of the D-pathway, while the subunit also provides an environment that increases the rate at which Asp-132 transfers protons into the D-pathway.     

From static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility

Crystallographic structure and deuterium accessibility comparisons of CcO in different redox states have suggested conformational changes of mechanistic significance. To predict the intrinsic flexibility and low energy motions in CcO, this work has analyzed available high-resolution crystallographic structures with ProFlex and elNémo computational methods. The results identify flexible regions and potential conformational changes in CcO that correlate well with published structural and biochemical data and provide mechanistic insights. CcO is predicted to undergo rotational motions on the interior and exterior of the membrane, driven by transmembrane helical tilting and bending, coupled with rocking of the β-sheet domain. Consequently, the proton K-pathway becomes sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the pathway, associated with conserved Thr-359 and Lys-362 residues. The D-pathway helices are found to be relatively rigid, with a highly flexible entrance region involving the subunit I C-terminus, potentially regulating the uptake of protons. Constriction and dilation of hydrophobic channels in RsCcO suggest regulation of the oxygen supply to the binuclear center. This analysis points to coupled conformational changes in CcO and their potential to influence both proton and oxygen access.     

Identifying the proton loading site cluster in the ba3 cytochrome c oxidase that loads and traps protons

Cytochrome c Oxidase (CcO) is the terminal electron acceptor in aerobic respiratory chain, reducing O₂ to water. The released free energy is stored by pumping protons through the protein, maintaining the transmembrane electrochemical gradient. Protons are held transiently in a proton loading site (PLS) that binds and releases protons driven by the electron transfer reaction cycle. Multi-Conformation Continuum Electrostatics (MCCE) was applied to crystal structures and Molecular Dynamics snapshots of the B-type Thermus thermophilus CcO. Six residues are identified as the PLS, binding and releasing protons as the charges on heme b and the binuclear center are changed: the heme a₃ propionic acids, Asp287, Asp372, His376 and Glu126B. The unloaded state has one proton and the loaded state two protons on these six residues. Different input structures, modifying the PLS conformation, show different proton distributions and result in different proton pumping behaviors. One loaded and one unloaded protonation states have the loaded/unloaded states close in energy so the PLS binds and releases a proton through the reaction cycle. The alternative proton distributions have state energies too far apart to be shifted by the electron transfers so are locked in loaded or unloaded states. Here the protein can use active states to load and unload protons, but has nearby trapped states, which stabilize PLS protonation state, providing new ideas about the CcO proton pumping mechanism. The distance between the PLS residues Asp287 and His376 correlates with the energy difference between loaded and unloaded states.     

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.     

Network analysis of a proposed exit pathway for protons to the P-side of cytochrome c oxidase

Cytochrome c Oxidase (CcO) reduces O₂, the terminal electron acceptor, to water in the aerobic, respiratory electron transport chain. The energy released by O₂ reductions is stored by removing eight protons from the high pH, N-side, of the membrane with four used for chemistry in the active site and four pumped to the low pH, P-side. The proton transfers must occur along controllable proton pathways that prevent energy dissipating movement towards the N-side. The CcO N-side has well established D- and K-channels to deliver protons to the protein interior. The P-side has a buried core of hydrogen-bonded protonatable residues designated the Proton Loading Site cluster (PLS cluster) and many protonatable residues on the P-side surface, providing no obvious unique exit. Hydrogen bond pathways were identified in Molecular Dynamics (MD) trajectories of Rb. sphaeroides CcO prepared in the P_R state with the heme a₃ propionate and Glu286 in different protonation states. Grand Canonical Monte Carlo sampling of water locations, polar proton positions and residue protonation states in trajectory snapshots identify a limited number of water mediated, proton paths from PLS cluster to the surface via a (P-exit) cluster of residues. Key P-exit residues include His93, Ser168, Thr100 and Asn96. The hydrogen bonds between PLS cluster and P-exit clusters are mediated by a water wire in a cavity centered near Thr100, whose hydration can be interrupted by a hydrophobic pair, Leu255B (near Cu_A) and Ile99. Connections between the D channel and PLS via Glu286 are controlled by a second, variably hydrated cavity.

Glu-286 rotation and water wire reorientation are unlikely the gating elements for proton pumping in cytochrome C oxidase

One of the key unresolved issues regarding proton pumping in cytochrome c oxidase (CcO) is the identity of the gating element that prevents the backflow of protons. In this study, we analyze two popular proposals for this element: isomerization of the key branching residue (Glu-286) and (re)orientation of water molecules in the hydrophobic cavity. Using a multifaceted set of computational analyses that involve CcO embedded in either an implicit or explicit treatment of lipid membrane, we show that neither Glu-286 nor active-site water likely constitutes the gating element. Detailed energetic and structural analyses of the simulation results indicate that the gating-relevant properties of these structural motifs observed in previous work are likely a result of the simplified computational models employed in those studies.     

Oxygen and proton pathways in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump, which couples the reduction of oxygen to water to the translocation of protons across the membrane. The recently solved x-ray structures of cytochrome c oxidase permit molecular dynamics simulations of the underlying transport processes. To eventually establish the proton pump mechanism, we investigate the transport of the substrates, oxygen and protons, through the enzyme. Molecular dynamics simulations of oxygen diffusion through the protein reveal a well-defined pathway to the oxygen-binding site starting at a hydrophobic cavity near the membrane-exposed surface of subunit I, close to the interface to subunit III. A large number of water sites are predicted within the protein, which could play an essential role for the transfer of protons in cytochrome c oxidase. The water molecules form two channels along which protons can enter from the cytoplasmic (matrix) side of the protein and reach the binuclear center. A possible pumping mechanism is proposed that involves a shuttling motion of a glutamic acid side chain, which could then transfer a proton to a propionate group of heme α₃. Proteins 30:100–107, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Molecular dynamics simulation of proton transport through the influenza A virus M2 channel

The structural and dynamical properties of a solvated proton in the influenza A virus M2 channel are studied using a molecular dynamics (MD) simulation technique. The second-generation multi-state empirical valence bond (MS-EVB2) model was used to describe the interaction between the excess proton and the channel environment. Solvation structures of the excess proton and its mobility characteristics along the channel were determined. It was found that the excess proton is capable of crossing the channel gate formed by the ring of four histidine residues even though the gate was only partially open. Although the hydronium ion itself did not cross the channel gate by traditional diffusion, the excess proton was able to transport through the ring of histidine residues by hopping between two water molecules located at the opposite sides of the gate. Our data also indicate that the proton diffusion through the channel may be correlated with the changes in channel conformations. To validate this observation, a separate simulation of the proton in a "frozen" channel has been conducted, which showed that the proton mobility becomes inhibited.     

The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase

In cytochrome c oxidase (CcO), exergonic electron transfer reactions from cytochrome c to oxygen drive proton pumping across the membrane. Elucidation of the proton pumping mechanism requires identification of the molecular components involved in the proton transfer reactions and investigation of the coupling between internal electron and proton transfer reactions in CcO. While the proton-input trajectory in CcO is relatively well characterized, the components of the output pathway have not been identified in detail. In this study, we have investigated the pH dependence of electron transfer reactions that are linked to proton translocation in a structural variant of CcO in which Arg481, which interacts with the heme D-ring propionates in a proposed proton output pathway, was replaced with Lys (RK481 CcO). The results show that in RK481 CcO the midpoint potentials of hemes a and a(3) were lowered by approximately 40 and approximately 15 mV, respectively, which stabilizes the reduced state of Cu(A) during reaction of the reduced CcO with O(2). In addition, while the pH dependence of the F --> O rate in wild-type CcO is determined by the protonation state of two protonatable groups with pK(a) values of 6.3 and 9.4, only the high-pK(a) group influences this rate in RK481 CcO. The results indicate that the protonation state of the Arg481 heme a(3) D-ring propionate cluster having a pK(a) of approximately 6.3 modulates the rate of internal electron transfer and may act as an acceptor of pumped protons.     

Heme-copper oxidases with modified D- and K-pathways are yet efficient proton pumps

The cytochrome aa(3)-type quinol oxidase from the archaeon Acidianus ambivalens and the ba(3)-type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme-copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H(+)/electron in a heme-copper oxidase that lacks most residues of the K- and D-channels. Also, the structure of the ba(3) oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D-pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton-pumping mechanism.     

Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is a problem of great current interest. Despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. In addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent pK(a) values associated with the pH dependencies of specific proton transfer (PT) reactions in CcO. This work takes a key step in resolving the above problems, by considering mutations, such as the Asn139Asp replacement, that blocks proton pumping without affecting PT to the catalytic site. We first introduce a formulation that makes it possible to relate the apparent pK(a) of Glu286 to different conformational states of this residue. We then use the new formulation along with the calculated pK(a) values of Glu286 at these different conformations to reproduce the experimentally observed apparent pK(a) of the residue. Next, we take the X-ray structures of the native and Asn139Asp mutant of the Paracoccus denitrificans CcO (N131D in this system) and reproduce for the first time the change in the primary PT pathways (and other key features) based on simulations that start with the observed structural changes. We also consider the competition between proton transport to the catalytic site and the pump site, as a function of the bulk pH, as well as the H/D isotope effect, and use this information to explore the relative height of the two barriers. The paper emphasizes the crucial role of energy-based considerations that include the PT process, and the delicate control of PT in CcO.     

Fluorescence quenching of reconstituted NCD-4-labeled cytochrome c oxidase complex by DOXYL-stearic acids.

It has been known for some time that dicyclohexylcarbodiimide (DCCD) inhibits the proton translocation function of the cytochrome c oxidase complex (CcO) and that there is one major site in subunit III which is modified upon reaction with DCCD (Glu-90 for the bovine enzyme). We have examined the reaction of bovine CcO with N-cyclohexyl-N'-(4-dimethylamino-alpha-napthyl)carbodiimide (NCD-4), a fluorescent analog of DCCD. NCD-4 labeling of CcO is strongly inhibited by DCCD implicating Glu-90 of subunit III as the site of chemical modification by NCD-4. The fluorescence of reconstituted NCD-4-labeled bovine CcO is strongly quenched by hydrophobic nitroxides, whereas hydrophilic nitroxides and iodide ions have a reduced quenching ability. It is concluded that the Glu-90 of subunit III resides near the protein-lipid interface of the membrane spanning region of the enzyme. Different quenching abilities of 5-, 7-, 10-, 12-, and 16-4,4-dimethyl-3-oxazolinyloxy-stearic acids suggest that the NCD-4 label is located in the membrane bilayer in the region near the middle of the hydrocarbon tail of stearic acid. In light of these results, it is unlikely that Glu-90 is part of a proton channel that is associated with the proton pumping machinery of the enzyme but the outcome of this study does not eliminate an allosteric regulatory role for this residue.     

Tracing the D-pathway in reconstituted site-directed mutants of cytochrome c oxidase from Paracoccus denitrificans

Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.     

Proton transport in carbonic anhydrase: Insights from molecular simulation

This article reviews the insights gained from molecular simulations of human carbonic anhydrase II (HCA II) utilizing non-reactive and reactive force fields. The simulations with a reactive force field explore protein transfer and transport via Grotthuss shuttling, while the non-reactive simulations probe the larger conformational dynamics that underpin the various contributions to the rate-limiting proton transfer event. Specific attention is given to the orientational stability of the His64 group and the characteristics of the active site water cluster, in an effort to determine both of their impact on the maximal catalytic rate. The explicit proton transfer and transport events are described by the multistate empirical valence bond (MS-EVB) method, as are alternative pathways for the excess proton charge defect to enter/leave the active site. The simulation results are interpreted in light of experimental results on the wild-type enzyme and various site-specific mutations of HCA II in order to better elucidate the key factors that contribute to its exceptional efficiency.     

Dynamics of the glutamic acid 242 side chain in cytochrome c oxidase

In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a(3)/Cu(B) site, and for proton pumping. When present, the side chain of Glu-242 is orientated "down" towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity "above" Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an "up" position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the "up" position is preferred energetically when the cavity contains four water molecules, but the "down" position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the "resting" state of the enzyme, and that the "up/down" equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O(2) reduction.     

Understanding the cytochrome c oxidase proton pump: thermodynamics of redox linkage.

The cytochrome c oxidase complex (CcO) catalyzes the four-electron reduction of dioxygen to water by using electrons from ferrocytochrome c. Redox free energy released in this highly exergonic process is utilized to drive the translocation of protons across a transmembrane electrochemical gradient. Although numerous chemical models of proton pumping have been developed, few attempts have been made to explain the stepwise transfer of energy in the context of proposed protein conformational changes. A model is described that seeks to clarify the thermodynamics of the proton pumping function of CcO and that illustrates the importance of electron and proton gating to prevent the occurrence of the more exergonic electron leak and proton slip reactions. The redox energy of the CcO-membrane system is formulated in terms of a multidimensional energy surface projected into two dimensions, a nuclear coordinate associated with electron transfer and a nuclear coordinate associated with elements of the proton pump. This model provides an understanding of how a transmembrane electrochemical gradient affects the efficiency of the proton pumping process. Specifically, electron leak and proton slip reactions become kinetically viable as a result of the greater energy barriers that develop for the desired reactions in the presence of a transmembrane potential.     

Proton transport behavior through the influenza A M2 channel: insights from molecular simulation

The structural properties of the influenza A virus M2 transmembrane channel in dimyristoylphosphatidylcholine bilayer for each of the four protonation states of the proton-gating His-37 tetrad and their effects on proton transport for this low-pH activated, highly proton-selective channel are studied by classical molecular dynamics with the multistate empirical valence-bond (MS-EVB) methodology. The excess proton permeation free energy profile and maximum ion conductance calculated from the MS-EVB simulation data combined with the Poisson-Nernst-Planck theory indicates that the triply protonated His-37 state is the most likely open state via a significant side-chain conformational change of the His-37 tetrad. This proposed open state of M2 has a calculated proton permeation free energy barrier of 7 kcal/mol and a maximum conductance of 53 pS compared to the experimental value of 6 pS. By contrast, the maximum conductance for Na(+) is calculated to be four orders of magnitude lower, in reasonable agreement with the experimentally observed proton selectivity. The pH value to activate the channel opening is estimated to be 5.5 from dielectric continuum theory, which is also consistent with experimental results. This study further reveals that the Ala-29 residue region is the primary binding site for the antiflu drug amantadine (AMT), probably because that domain is relatively spacious and hydrophobic. The presence of AMT is calculated to reduce the proton conductance by 99.8% due to a significant dehydration penalty of the excess proton in the vicinity of the channel-bound AMT.