Biomedical subjects

Alexei A Stuchebrukhov

Publications and source records attributed to Alexei A Stuchebrukhov.

12 recordsLinked to original sources

PubMed2006

Improved density functional theory/electrostatic calculation of the His291 protonation state in cytochrome C oxidase: self-consistent charges for solvation energy calculation.

The protonation state of His291 in cytochrome c oxidase (CcO), a ligand to the Cu(B) center of the enzyme, has been recently studied in this group by using combined density functional theory (DFT)/electrostatic (QM/MM) calculations. On the basis of these calculations, a model of the proton pumping mechanism of CcO has been proposed. Due to certain technical difficulties, the procedure used in the previous calculation to find partial atomic charges of the QM system for the solvation energy evaluation was not entirely satisfactory; i.e., it was not self-consistent. Here, we describe a procedure that resolves the problem and report on the improved calculations of the protonation state of the His residue. The new procedure fits the protein and reaction field potentials in the region of the QM system with artificial point charges placed on a surface of a sphere surrounding the QM system and a few charges inside the sphere and allows one to perform DFT calculations that involve an inhomogeneous dielectric environment in a self-consistent way. The procedure improves the accuracy of calculations in comparison with previous work. The improved results show, however, that although the absolute energies change significantly the relative energies of the protonated and deprotonated states of His291 remain close to the previously reported ones and therefore do not change significantly the pK(a) values reported earlier. Therefore, our new improved calculations support for the proposed His291 model of the CcO pump.

Electron Transport Complex IV↗

PubMed2006

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.

Animals↗

PubMed2006

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.

Electron Transport Complex IV↗

PubMed2005

DFT/electrostatic calculations of pK(a) values in cytochrome c oxidase.

Using classical electrostatic calculations, earlier we examined the dependence of the protonation state of bovine cytochrome c oxidase (CcO) on its redox state. Based on these calculations, we have proposed a model of CcO proton pumping that involves His291, one of the Cu(B) histidine ligands, which was found to respond to redox changes of the enzyme Fe(a)(3)-Cu(B) catalytic center. In this work, we employ combined density functional and continuum electrostatic calculations to evaluate the pK(a)() values of His291 and Glu242, two key residues of the model. The pK(a) values are calculated for different redox states of the enzyme, and the influence of different factors on the pK(a)'s is analyzed in detail. The calculated pK(a)() values of Glu242 are between 9.4 and 12.0, depending on the redox state of the protein, which is in excellent agreement with recent experimental measurements. Assuming the reduced state of heme a(3), His291 of the oxidized Cu(B) center possesses a pK(a)() between 2.1 and 4.0, while His291 of the reduced Cu(B) center has a pK(a) above 17. The obtained results support the proposal that the His291 ligand of the Cu(B) center in CcO is a proton pump element.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Catalysis↗

PubMed2004

On the electron tunneling in molecules: a generalized orthogonalization procedure for finding tunneling orbitals.

A simple method for finding tunneling orbitals of a long-distance electron transfer system is described. The procedure is an approximate biorthogonalization of many-electron donor and acceptor states in which the corresponding orbitals of the system are obtained; one pair of such corresponding orbitals describes the tunneling electron. Usually, the biorthogonalization is performed on the states that describe simultaneously both donor and acceptor complexes and the bridge connecting them, which make the procedure computationally demanding. In this paper a significant simplification of the procedure is proposed. In the procedure, to identify the tunneling orbitals roughly one half of the system is needed--only donor complex, or acceptor complex, plus part of the bridge. The procedure is practically equivalent to biorthogonalization, but computationally much simpler. Applications are illustrated with the results for a model of biological long-distance electron transfer in Ru-modified azurin, and electron transfer involving heme a of cytochrome c oxidase.

Journal Article↗

PubMed2004

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.

Animals↗

PubMed2004

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%.

Animals↗

PubMed2003

Computer simulation of water in cytochrome c oxidase.

Statistical mechanics and molecular dynamics simulations have been carried out to study the distribution and dynamics of internal water molecules in bovine heart cytochrome c oxidase (CcO). CcO is found to be capable of holding plenty of water, which in subunit I alone amounts to about 165 molecules. The dynamic characterization of these water molecules is carried out. The nascent water molecules produced in the redox reaction at the heme a(3)-CuB binuclear site form an intriguing chain structure. The chain begins at the position of Glu242 at the end of the D channel, and has a fork structure, one branch of which leads to the binuclear center, and the other to the propionate d of heme a(3). The branch that leads to the binuclear center has dynamic access both to the site where the formation of water occurs, and to delta-nitrogen of His291. From the binuclear center, the chain continues to run into the K channel. The stability of this hydrogen bond network is examined dynamically. The catalytic site is located at the hydrophobic region, and the nascent water molecules are produced at the top of the energy hill. The energy gradient is utilized as the mechanism of water removal from the protein. The water exit channels are explored using high-temperature dynamics simulations. Two putative channels for water exit from the catalytic site have been identified. One is leading directly toward Mg(2+) site. However, this channel is only open when His291 is dissociated from CuB. If His291 is bound to CuB, the only channel for water exit is the one that originates at E242 and leads toward the middle of the membrane. This is the same channel that is presumably used for oxygen supply.

Animals↗

PubMed2002

Proton transport via the membrane surface.

Some proton pumps, such as cytochrome c oxidase (C(c)O), translocate protons across biological membranes at a rate that considerably exceeds the rate of proton transport to the entrance of the proton-conducting channel via bulk diffusion. This effect is usually ascribed to a proton-collecting antenna surrounding the channel entrance. In this paper, we consider a realistic phenomenological model of such an antenna. In our model, a homogeneous membrane surface, which can mediate proton diffusion toward the channel entrance, is populated with protolytic groups that are in dynamic equilibrium with the solution. Equations that describe coupled surface-bulk proton diffusion are derived and analyzed. A general expression for the rate constant of proton transport via such a coupled surface-bulk diffusion mechanism is obtained. A rigorous criterion is formulated of when proton diffusion along the surface enhances the transport. The enhancement factor is found to depend on the ratio of the surface and bulk diffusional constants, pK(a) values of surface protolytic groups, and their concentration. A capture radius for a proton on the surface and an effective size of the antenna are found. The theory also predicts the effective distance that a proton can migrate on the membrane surface between a source (such as CcO) and a sink (such as ATP synthase) without fully equilibrating with the bulk. In pure aqueous solutions, protons can travel over long distances (microns). In buffered solutions, the travel distance is much shorter (nanometers); still the enhancement effect of the surface diffusion on the proton flow to a target on the surface can be tens to hundreds at physiological buffer concentrations. These results are discussed in a general context of chemiosmotic theory.

Biophysical Phenomena↗

PubMed2002

Electron transfer in ferredoxin: are tunneling pathways evolutionarily conserved?

A theoretical study of electron transfer (ET) pathways in a recently crystallized Clostridium acidurici ferredoxin is reported. The electronic structure of the protein complex is treated at the semiempirical extended Hückel level, and the tunneling pathways are calculated with the rigorous quantum mechanical method of tunneling currents. The model predicts two pathways between the two [4Fe-4S] cubanes: a strong one running directly from Cys(14) to Cys(43) and a weaker one from Cys(14) via Ile(23) to Cys(18), whereas other amino acids do not play a significant role in the electron tunneling. The cysteine ligands conduct almost all of the current when Ile(23) is mutated to valine in silico, so that there is no appreciable change in the ET rate. The calculated value of the transfer matrix element is consistent with the experimentally determined rate of transfer. Results of the sequence analysis performed on this ferredoxin reveal that Ile(23) is a highly variable amino acid compared with the cubane-ligating cysteine amino acids, even though Ile(23) lies directly between the donor and acceptor complexes. We further argue that the homologous proteins with a [3Fe-4S] cofactor, which does not have one of the four cysteine ligands, use the same tunneling pathways as those in this ferredoxin, on the basis of the high homology as well as the absolute conservation of Cys(14) and Cys(43) which serve as the main tunneling conduit. Our results explain why mutation of amino acids around and between the donor and acceptor cubane clusters, including that of Ile(23), does not appreciably affect the rate of transfer and add support to the proposal that there exist evolutionarily conserved electron tunneling pathways in biological ET reactions.

Amino Acid Sequence↗