Insights into the mechanism of proton transport in cytochrome c oxidase

Cytochrome c oxidase (CcO), known as complex IV of the electron transport chain, plays several important roles in aerobic cellular respiration. Electrons transferred from cytochrome c to CcO's catalytic site reduce molecular oxygen and produce a water molecule. These electron transfers also drive active proton pumping from the matrix (N-side) to intermembrane region (P-side) in mitochondria; the resultant proton gradient activates ATP synthase to produce ATP from ADP. Although the existence of the coupling between the electron transfer and the proton transport (PT) is established experimentally, its mechanism is not yet fully understood at the molecular level. In this work, it is shown why the reduction of heme a is essential for proton pumping. This is demonstrated via novel reactive molecular dynamics (MD) simulations that can describe the Grotthuss shuttling associated with the PT as well as the dynamic delocalization of the excess proton electronic charge defect. Moreover, the "valve" role of the Glu242 residue (bovine CcO notation) and the gate role of d-propionate of heme a(3) (PRDa3) in the explicit PT are explicitly demonstrated for the first time. These results provide conclusive evidence for the CcO proton transporting mechanism inferred from experiments, while deepening the molecular level understanding of the CcO proton switch.     

Possible mechanism of proton transfer through peptide groups in the H-pathway of the bovine cytochrome c oxidase

The peptide group connecting Tyr440 and Ser441 of the bovine cytochrome c oxidase is involved in a recently proposed proton-transfer path (H-path) where, at variance with other pathways (D- and K-paths), a usual hydrogen-bond network is interrupted, thus making this proton propagation rather unconventional. Our density-functional based molecular dynamics simulations show that, despite this anomaly and provided that a proton can reach a nearby water, a multistep proton-transfer pathway can become a viable pathway for such a reaction: a proton is initially transferred to the carbonyl oxygen of a keto form of the Tyr440-Ser441 peptide group [-CO-NH-], producing an imidic acid [-C(OH)-NH-] as a metastable state; the amide proton of the imidic acid is then transferred, spontaneously to the deprotonated carboxyl group of the Asp51 side chain, leading to the formation of an enol form [-C(OH)=N-] of the Tyr440-Ser441 peptide group. Then a subsequent enol-to-keto tautomerization occurs via a double proton-transfer path realized in the two adjacent Tyr440-Ser441 and Ser441-Asp442 peptide groups. An analysis of this multistep proton-transfer pathway shows that each elementary process occurs through the shortest distance, no permanent conformational changes are induced, thus preserving the X-ray crystal structure, and the reaction path is characterized by a reasonable activation barrier.     

The indirect coulometric titration of cytochrome c oxidase with cytochrome c

The indirect coulometric titration of cytochrome c oxidase and dioxygen using cytochrome c as a mediator is described. Results of both the indirect coulometric titrations and the cyclic voltammetric experiments reported herein verify that the reaction mechanism involves the catalytic regeneration of the electroactive species, the cytochrome c mediator, with the selective reduction of cytochrome c oxidase alone. During the indirect coulometric titrations dioxygen is reduced to water only by cytochrome c oxidase and not by either direct reduction at the electrode surface or reaction with cytochrome c. This system utilizes the electron transfer selectivity of cytochrome c for cytochrome c oxidase over dioxygen and offers a means by which the reaction of cytochrome c oxidase and dioxygen can be examined.     

Proton pumping mechanism in cytochrome c oxidase

Two different issues, important for the pumping mechanism of cyctochrome c oxidase, have been addressed in the present study. One of them concerns the nature of two key proton transfer transition states. A simple electrostatic model is used to suggest that the transition state (TS) for transfer to the pump-site should be positively charged, while the one for transfer to the binuclear center should be charge-neutral. The character of the former TS will guarantee that the protons will be pumped to the outside and not return to the inside, while the neutral character of the latter one will allow transfer with a sufficiently low barrier. In the simple electrostatic analysis, leading to this qualitative picture of the pumping process, the results from the kinetic experiments are strictly followed, but it is at least as important to follow the fundamental requirements for pumping. In this perspective, the uncertainties in the quantitative analysis should be rather unimportant for the emerging qualitative picture of the pumping mechanism. The second problem addressed concerns the purpose of the K-channel. It is argued that the reason for the presence of the K-channel could be that protons cannot pass through the binuclear center at some stage of pumping. Barriers and water binding energies were computed using hybrid density functional theory (DFT) to investigate this question.     

Effects of electric fields on proton transport through water chains

Molecular dynamics simulations on quantum energy surfaces are carried out to study the effects of perturbing electric fields on proton transport (PT) in protonated water chains. As an idealized model of a hydrophobic cavity in the interior of a protein the water molecules are confined into a carbon nanotube (CNT). The water chain connects a hydrated hydronium ion (H3O+) at one end of the CNT and an imidazole molecule at the other end. Without perturbing electric fields PT from the hydronium proton donor to the imidazole acceptor occurs on a picosecond time scale. External perturbations to PT are created by electric fields of varying intensities, normal to the CNT axis, generated by a neutral pair of charges on the nanotube wall. For fields above approximately 0.5 VA, the hydronium ion is effectively trapped at the CNT center, and PT blocked. Fields of comparable strength are generated inside proteins by nearby polar/charged amino acids. At lower fields the system displays a rich dynamic behavior, where the excess charge shuttles back and forth along the water chain before reaching the acceptor group on the picosecond time scale. The effects of the perturbing field on the proton movement are analyzed in terms of structural and dynamic properties of the water chain. The implications of these observations on PT in biomolecular systems and its control by external perturbing fields are discussed.     

Calculated reaction cycle of cytochrome c oxidase

The catalytic cycle of cytochrome c oxidase has been simulated by means of quantum mechanical calculations. The experimental energetics of the catalytic cycle is nearly reproduced. The atomic structures of the intermediates are suggested. In particular, the structures of nonactive "resting" intermediates are proposed.     

Proton exit channels in bovine cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal transmembrane enzyme of the respiratory electron transport chain in aerobic cells. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping, a process which results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping and proton exit pathways remain unknown. In our previous work, the continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine cytochrome c oxidase for different redox states of the enzyme. A possible mechanism of oxygen reduction and proton pumping via His291 was proposed. In this paper, using electrostatic calculations, we examine the proton exit pathways in the enzyme. By monitoring the changes of the protonation states, proton affinities, and energies of electrostatic interactions between the titratable groups in different redox states of CcO, we identified the clusters of strongly interacting residues. Using these data, we detected four possible proton exit points on the periplasmic side of the membrane (Lys171B/Asp173B, His24B/Asp25B, Asp51, and Asp300). We then were able to trace the proton exit pathways and to evaluate the energy profiles along the paths. On the basis of energetic considerations and the conservation of the residues in a protein sequence, the most likely exit pathway is one via the Lys171B/Asp173B site. The obtained results are fully consistent with our His291 model of proton pumping, and provide a rationale for the absence of proton leaking in CcO between the pumping strokes.     

Iron porphyrins as models of cytochrome c oxidase

A series of iron porphyrins has been synthesized as models of cytochrome c oxidase; their activity as 4e catalysts in the reduction of dioxygen has been studied at pH 7. These compounds have been obtained by grafting very different residues onto the same iron complex, namely tripodal tetraamines, pickets, and straps, in order to change the environment of the metal center. In the case of porphyrins bearing a tripodal cap, the secondary amines have been alkylated with different substituents so as to modify the electronic environment of the distal pocket. Surprisingly, when the iron porphyrin is functionalized with four identical acrylamido pickets, the resulting complex exhibits biomimetic activity in that it catalyzes oxygen reduction with almost no production of hydrogen peroxide. The crystal structure of the redox-inactive zinc(II) analogue is reported; this shows how the metal influences the spatial arrangement of the four pickets through axial coordination and hydrogen bonding. Even a bis-strapped iron porphyrin, for which no dimerization or self-aggregation can occur at the electrode surface, acts as a 4e catalyst for O2 reduction. It is thus demonstrated that at pH close to physiological values, the iron porphyrin is an intrinsically efficient catalyst for the reduction of oxygen to water.     

Protein-protein interactions studied by EPR relaxation measurements: cytochrome c and cytochrome c oxidase

The complex formed between cytochrome c oxidase from Paracoccus denitrificans and its electron-transfer partner cytochrome c has been studied by multi-frequency pulse electron paramagnetic resonance spectroscopy. The dipolar relaxation of a fast-relaxing paramagnetic center induced on a more slowly relaxing center can be used to measure their distance in the range of 1-4 nm. This method has been used here for the first time to study transient protein-protein complex formation, employing soluble fragments for both interacting species. We observed significantly enhanced transversal relaxation of the CuA center in cytochrome c oxidase due to the fast-relaxing iron of cytochrome c upon complex formation. The possibility to measure cytochrome c oxidase in the presence and absence of cytochrome c permitted us to separate the dipolar relaxation from other relaxation contributions. This allowed a quantitative simulation and interpretation of the relaxation data. The specific temperature dependence of the dipolar relaxation together with the high orientational selectivity achieved at high magnetic field values may provide detailed information on distance and relative orientation of the two proteins with respect to each other in the complex. Our experimental results cannot be explained by any single well-defined structure of the complex of cytochrome c oxidase with cytochrome c, but rather suggest that a broad distribution in distances and relative orientations between the two proteins exist within this complex.     

Conformational transitions in the regulation of cytochrome c oxidase activity

Oxidation of cytochrome c, catalyzed by cytochrome oxidase embedded in artificial liposomes of high respiratory control ratio (between 5 and 9.5), has been studied by rapid mixing techniques, under which conditions the enzyme undergoes a limited number of turnovers (from 1 to 5). The time course of the reaction could be satisfactorily simulated by a procedure derived from the concerted two-state model of Monod-Wyman-Changeux.The bulk of data and the novel analytical approach confirm the proposal that cytochrome oxidase undergoes a transition from a fast-reacting to a slow-reacting form as a consequence of the electrochemical gradient built up across the phospholipidic bilayer, and substantiate the idea that the conformational change:

• •occurs as an all-or-none process after about one turnover irrespective of the molar ratio between substrate and enzyme, and
• •is not immediately correlated to the other well known transition from the resting to the pulsed form of the enzyme.

     

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]¹⁵⁺ ion being the most intense peak. With the addition of H₂O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.     

Rapid purification of cytochrome c oxidase from Paracoccus denitrificans

Two methods are described for the purification of cytochrome c oxidase from Triton X-100 extracts of the periplasma membrane of Paracoccus denitrificans. The first is a large-scale procedure for the preparation of 100-250 nmol of cytochrome c oxidase (10-20 mg) in 1 week. The second is a rapid procedure for isolating up to 25 nmol in 2-3 days. Owing to the high yields given by fast protein liquid chromatography (FPLC) on Mono Q columns, the overall yield is about 20%, whereas the yield in many other previously published procedures does not exceed 10%. The use of FPLC on Mono Q also offers a considerable saving of time.     

Thermodynamic properties of internal water molecules in the hydrophobic cavity around the catalytic center of cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump that creates a membrane proton gradient responsible for driving ATP synthesis in aerobic cells. The crystal structure of the enzyme has been recently solved; however, the details of the mechanism of its proton pumping remain unknown. The enzyme internal water molecules play a key role in proton translocation through the enzyme. Here, we examine the thermodynamic properties of internal water in a hydrophobic cavity around the catalytic center of the enzyme. The crystal structure does not show any water molecules in this region; it is believed, however, that, since protons are delivered to the catalytic center, where the reduction of molecular oxygen occurs, at least some water molecules must be present there. The goal of the present study was to examine how many water molecules are present in the catalytic center cavity and why these water molecules are not observed in the crystal structure of the enzyme. The behavior of water molecules is discussed in the context of redox-coupled proton translocation in the enzyme.     

Electrostatic study of the proton pumping mechanism in bovine heart cytochrome C oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the cell respiratory chain in mitochondria and aerobic bacteria. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process that results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping remains unknown. In the present paper, continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine heart cytochrome c oxidase for different redox states of the enzyme along its catalytic cycle. Three different computational models of the enzyme were employed to test the stability of the results. The energetics and pH dependence of the P-->F, F-->O, and O-->E steps of the cycle have been investigated. On the basis of electrostatic calculations, two possible schemes of redox-linked proton pumping are discussed. The first scheme involves His291 as a pump element, whereas the second scheme involves a group linked to propionate D of heme a(3). In both schemes, loading of the pump site is coupled to ET between the two hemes of the enzyme, while transfer of a chemical proton is accompanied by ejection of the pumped H(+). The two models, as well as the energetics results are compared with recent experimental kinetic data. The proton pumping across the membrane is an endergonic process, which requires a sufficient amount of energy to be provided by the chemical reaction in the active site. In our calculations, the conversion of OH(-) to H(2)O provides 520 meV of energy to displace pump protons from a loading site and overall about 635 meV for each electron passing through the system. Assuming that the two charges are translocated per electron against the membrane potential of 200 meV, the model predicts an overall efficiency of 63%.     

Oriented immobilization and electron transfer to the cytochrome c oxidase

Direct electron transfer to cytochrome c oxidase (CcO) is investigated as a function of packing density of the surface layer. This is varied by the surface concentration of chelator molecules when the enzyme is immobilized on the electrode using the his-tag technology. Chelator molecules with a terminal nitrilotriacetic acid group are synthesized ex situ in contrast to in situ synthesis used in a previous work. Self-assembled monolayers of the chelator mixed at different mole fractions with a dilution molecule are prepared to bind the CcO after complex formation with Ni²⁺ ions. The CcO, which is immobilized in the solubilized form, is then reconstituted into a protein-tethered bilayer lipid membrane (ptBLM). Varying the mixing ratio of chelator to dilution molecules enabled us to control the packing density of CcO residing in the ptBLM. Subtle differences in the architecture of the protein/lipid layers revealed by surface-enhanced IR absorption spectroscopy are considered to be essential for an effective electron transfer. Cyclic voltammograms are measured under anaerobic conditions at different scan rates and analyzed by means of a model which describes the transfer of four electrons to CcO in the ptBLM. The rate constants thus obtained show a marked dependence on the packing density.     

Direct detection of formate ligation in cytochrome c oxidase by ATR-FTIR spectroscopy

The IR signature of binding of formate to the heme a(3-)Cu(B) binuclear site of bovine cytochrome c oxidase has been obtained by perfusion ATR-FTIR spectroscopy. The data show unequivocally that formate binds in its anionic form despite its binding being electroneutral overall. The bound formate can be distinguished from free ligand by the binding-induced sharpening and downshifting of vibrational bands. Formate ligation also causes shifts of vibrational modes of heme a(3) and its substituents and perturbation of histidine residues. The association of the accompanying protonation change with a carboxylate or tyrosine can be ruled out and may involve a histidine metal ligand or, more likely, a simple displacement into the bulk phase of a hydroxide ligand to heme a(3) or CU(B), a reaction which would account for stoichiometric proton uptake and maintenance of net charge within the binuclear center domain.     

Synthesis of cytochrome c oxidase models bearing a Tyr244 mimic

A close structural analogue of the metal-free cytochrome c oxidase active site has been synthesized. This model has a proximal imidazole tail and three distal imidazole pickets attached to a porphyrin. One distal imidazole is cross-linked to a phenol, mimicking Tyr(244). The strategy behind the successful synthesis of this regioisomerically pure model involved discovering the best sequence to introduce the phenol-substituted imidazole and employing a fluorinated substituent.