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.     

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.     

An IR study of protonation changes associated with heme-heme electron transfer in bovine cytochrome c oxidase

IR changes caused by photolysis of CO from the mixed valence form of bovine cytochrome c oxidase have been investigated over the pH/pD range 6-9.8. Band assignments were based on effects of H2O/D2O exchange and by comparisons with published IR data and crystallographic data. Changes arise both from CO photolysis and from subsequent reversed electron transfer from heme a3 to heme a. This reversed electron transfer is known to have pH-independent and, above pH 8, pH-dependent components. The pH-independent component is associated with a trough around the 1742 cm(-1) band attributable to one or more protonated carboxylic acids. Its peak position, but not extent, is pH-dependent, indicative of a titratable group with a pK of 8.2 whose acid form causes increased hydrogen bonding to the IR-detectable carboxylic group. A different protonatable group with pK above 9 controls the extent of the pH-dependent component. This phase is associated with perturbation of an arginine guanidinium that is most clearly observed as a trough at 1592 cm(-1) after H/D exchange. It is suggested that this group, probably Arg-438 that is in close contact with propionate groups of both hemes and already proposed to be of functional significance, lowers the energy of the transient charge-uncompensated electron-transfer intermediate by changing the charge distribution in response to heme-heme electron transfer. No other IR signature of a titratable group that controls the extent of the pH-dependent phase is present, and it most likely arises from a nonphysiological deprotonation of the proximal water ligand of ferric heme a3 at high pH that has been reported to exhibit a similar pK.     

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.     

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.     

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.     

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.     

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.

     

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.     

Intricate role of water in proton transport through cytochrome c oxidase

Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O(2) to H(2)O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from Rhodobacter sphaeroides . Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules halfway up the D pathway, were replaced by more bulky hydrophobic residues (Ser200Ile, Ser200Val/Ser201Val, and Ser200Val/Ser201Tyr) to query the effects of changing the local structure on enzyme activity as well as proton uptake, release, and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcO's were lowered, both the Ser200Ile and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P(R) → F and F → O transitions (P(R) is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O(2), F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the P(R) → F transition is shown to be essentially pH independent up to pH 12 (i.e., the apparent pK(a) of Glu286 is increased from 9.4 by at least 3 pK(a) units) in the Ser200Val/Ser201Val mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO.     

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.     

Mutation scanning analysis of mitochondrial cytochrome c oxidase subunit 1 reveals limited gene flow among bovine lungworm subpopulations in Sweden

A mutation scanning approach was employed to investigate the population genetic structure of the bovine lungworm, Dictyocaulus viviparus (Nematoda: Trichostrongyloidea), in southern Sweden. A total of 252 individual nematodes were collected from cattle representing 17 farms. A portion of the mitochondrial cytochrome c oxidase subunit 1 gene (pcox1) was amplified from genomic DNA isolated from individual lungworms by the polymerase chain reaction (PCR), and then subjected to single-strand conformation polymorphism (SSCP). Samples with distinct SSCP profiles were then sequenced. In total, 12 distinct pcox1 haplotypes (393 bp) were defined for the 252 individuals, and pairwise sequence differences among the haplotypes ranged from 0.3-2.3%. Average haplotype diversity and nucleotide diversity values were 0.16 and 0.002, respectively. There was no particular correlation between pcox1 haplotypes and their geographical origin. The "overall fixation" indices F(ST) and N(ST) were calculated to be 0.77 and 0.65, respectively. The results of this study revealed that both the mitochondrial DNA sequence diversity within populations and the gene flow among populations of D. viviparus were low. This is similar to findings for some parasitic nematodes of plants and insects, but distinctly different from gastrointestinal trichostrongyloid nematodes of domesticated ruminants considered to have relatively high levels of genetic diversity and gene flow. Such differences were interpreted to relate mainly to differences in host movement as well as parasite biology, population sizes and transmission patterns, and should therefore be of epidemiological relevance.*     

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.     

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%.     

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.     

Reconstitution of cytochrome c oxidase into phospholipid vesicles: Effect of detergents

Cytochrome c oxidase vesicles prepared using enzyme preparations subjected to cycles of freezing and thawing (+20 to −20°C) before reconstitution, display a decrease in respiratory control ratio (RCR); if the protein is incubated with detergents before reconstitution, a higher RCR value is restored. This effect is attributed to a detergent-mediated optimization of the structural assembly of the proteo-membrane unit occurring at the early stages of reconstitution. The same type of experiment carried out at different temperatures showed that incubation at 35°C for 30 min leads to a severe, irreversible loss of RCR.