Proton transfers in the bacteriorhodopsin photocycle

The steps in the mechanism of proton transport in bacteriorhodopsin include examples for most kinds of proton transfer reactions that might occur in a transmembrane pump: proton transfer via a bridging water molecule, coupled protonation/deprotonation of two buried groups separated by a considerable distance, long-range proton migration over a hydrogen-bonded aqueous chain, and capture as well as release of protons at the membrane-water interface. The conceptual and technical advantages of this system have allowed close examination of many of these model reactions, some at an atomic level.     

Proton transfers in the bacteriorhodopsin photocycle

The steps in the mechanism of proton transport in bacteriorhodopsin include examples for most kinds of proton transfer reactions that might occur in a transmembrane pump: proton transfer via a bridging water molecule, coupled protonation/deprotonation of two buried groups separated by a considerable distance, long-range proton migration over a hydrogen-bonded aqueous chain, and capture as well as release of protons at the membrane-water interface. The conceptual and technical advantages of this system have allowed close examination of many of these model reactions, some at an atomic level.     

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.     

Theory and simulation of proton-coupled electron transfer, hydrogen-atom transfer, and proton translocation in proteins

A theory of proton coupled electron transfer (PCET) is reviewed with application to charge transfer steps in the photosystem II oxygen-evolving complex (PSII/OEC). The relation between PCET when it is a concerted electron proton transfer (ETPT) process and hydrogen-atom transfer (HAT) reactions is discussed. Signatures expected for HAT reactions in terms of the size of the kinetic isotope effect and overall magnitude of the rate constant are discussed in the context of PSII/OEC. The formal similarity of ETPT to proton transfer and translocation is used to introduce a combined quantum mechanical (for the transferring protons) and molecular dynamics for the heavy-atom degrees of freedom approach. The method is used to examine double proton transfer in cytochrome c oxidase where two waters and a glutamate (Glu286) that is implicated in the proton translocation mechanism form a cyclic hydrogen bonded structure. Protonation of the glutamate is found to occur in agreement with experimental results.     

Barrier Compression and Its Contribution to Both Classical and Quantum Mechanical Aspects of Enzyme Catalysis

It is generally accepted that enzymes catalyze reactions by lowering the apparent activation energy by transition state stabilization or through destabilization of ground states. A more controversial proposal is that enzymes can also accelerate reactions through barrier compression—an idea that has emerged from studies of H-tunneling reactions in enzyme systems. The effects of barrier compression on classical (over-the-barrier) reactions, and the partitioning between tunneling and classical reaction paths, have largely been ignored. We performed theoretical and computational studies on the effects of barrier compression on the shape of potential energy surfaces/reaction barriers for model (malonaldehyde and methane/methyl radical anion) and enzymatic (aromatic amine dehydrogenase) proton transfer systems. In all cases, we find that barrier compression is associated with an approximately linear decrease in the activation energy. For partially nonadiabatic proton transfers, we show that barrier compression enhances, to similar extents, the rate of classical and proton tunneling reactions. Our analysis suggests that barrier compression—through fast promoting vibrations, or other means—could be a general mechanism for enhancing the rate of not only tunneling, but also classical, proton transfers in enzyme catalysis.     

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

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

Excited state intramolecular proton transfer in 3-hydroxy flavone and 5-hydroxy flavone: A DFT based comparative study

Potential energy (PE) curves for the intramolecular proton transfer in the ground (GSIPT) and excited (ESIPT) states of 3-hydroxy-flavone (3HF) and 5-hydroxy-flavone (5HF) were studied using DFT/B3LYP (6-31G (d,p)) and TD-DFT/B3LYP (6-31G (d,p)) level of theory respectively. Our calculations suggest the non-viability of ground state intramolecular proton transfer for both the compounds. Calculated PE curves of 3HF for the ground and excited singlet states proton transfer process explain its four state laser diagram. Excited states PE calculations support the ESIPT process to both 5HF and 3HF. The difference in ESIPT emission process of 3HF and 5HF have been explained in terms of HOMO and LUMO electron distribution of the enol and keto tautomer of these two compounds.     

Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn(4)CaO(5) cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S(i) states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S(3) → S(0) transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn(4)CaO(5) cluster that leads to O(2) formation. Early proton movement was also detected during the S(2) → S(3) transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn(4)CaO(5) cluster in the S(2) and S(3) states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S(0) → S(1) transition, which could contribute to the stabilization of the S(1) state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.     

Excited state intramolecular proton transfer in 3-hydroxychromone: a DFT-based computational study

Potential energy (PE) curves for intramolecular proton transfer in the ground (GSIPT) and intramolecular proton transfer in the excited (ESIPT) states of 3-hydroxychromone (3HC) have been studied using DFT-B3LYP/6-31G(d,p) and TD-DFT/6-31G(d,p) level of theory, respectively. Our calculations suggest the non-viability of GSIPT in 3HC. Excited states PE calculations show the existence of ESIPT process in 3HC. ESIPT in 3HC has also been explained in terms of HOMO and LUMO electron densities of the enol and keto tautomers of 3HC.     

Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic haem b centre of cytochrome b561: a unique machinery for the biological transmembrane electron transfer

Cytochromes b(561) are a family of transmembrane proteins found in most eukaryotic cells and contain two haem b prosthetic groups per molecule being coordinated with four His residues from four different transmembrane alpha-helices. Although cytochromes b(561) residing in the chromaffin vesicles has long been known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate, newly found members were apparently lacking in the sequence for putative ascorbate-binding site but exhibiting a transmembrane ferrireductase activity. We propose that cytochrome b(561) has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) atom of the axial His residue at the extravesicular haem center, as an initial step of the transmembrane electron transfer. This mechanism utilizes the well-known electrochemistry of ascorbate for a biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants.     

Ultrafast catalytic processes in enzymes

The study of biocatalysis and biotransformation in the transition-state region has been challenging and difficult, but recent advances on two important photoenzymes in nature, DNA photolyase and protochlorophyllide oxidoreductase, have enabled the investigation of their catalytic processes in real time. By following the entire evolution of substrate transformation, the functional dynamics constituting a series of elementary reactions have been mapped out. The five fundamental reactions in the enzymes, namely electron transfer, bond breaking and making, proton and hydride transfer, all occur ultrafast within subnanosecond. The direct clocking of catalytic transition states probes central, unmasked chemical processes and provides mechanistic insights into the role of the dynamics in enzyme function, which not only facilitates the formation of the enzyme-substrate complex in the transition-state configurations, but also modulates the subsequent catalytic reactions for maximum biotransformation efficiency.     

The proton pump of the mitochondrial bc1 complex

The molecular mechanism of the proton pump activity by the respiratory chain bc1 complex is still unknown. This group has proposed since long time that protonation/deprotonation events in the apoproteins of the complex are cooperatively linked to the oxido-reduction reactions at the quinone catalytic centre. Protolytic residues in the apoproteins can thus provide proton transfer pathways between the bulk aqueons phases and the redox centre. A series of experiments has been carried out aimed at demonstrating a role of particular complex subunits in the pump process. In this paper recent results are reviewed which have evidenced a definite role of polypeptide carboxyl residues in the proton pump mechanism. In particular, experiments carried out with both the bovine and P. denitrificans purified enzymes have indicated a specific involvement of aspartic residue(s) in the Rieske Fe/S protein in the proton pump function.     

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.     

Dynamics of proton transfer and enzymatic activity

The rate-determining elementary reaction step, i.e. proton transfer from the chymotrypsin active centre to the scissile substrate bond has been studied in the present work. On the basis of our theoretical results a hypothesis was formulated to explain chymotrypsin enzymatic efficiency. After ES complex formation excited vibrational states are populated in the enzyme molecule. In the rate-determining elementary reaction step, the proton transfer takes place from the first excited vibrational state of the N-H bond in the imidazole group of His57. This proton transfer is realised by quantum mechanical tunneling mechanism.     

Solvent-assisted excited state proton transfer and photoacidity of 2-hydroxypyridine. A nonadiabatic dynamics study

Reactions involving proton transfer, especially those taking place in excited states (ESPT), received considerable attention. These reactions underlie many biological and biochemical processes that are vital to life. DNA mutations excited state funnelling and transport phenomena are just examples. Among many molecules undergoing this type of photoreactions, phenols show a unique property, the so-called photoacidity. In the present communication, solvent (methanol and ammonia) assisted excited state proton transfer and photo acidity of 2-hydroxypyridine (2HP) are investigated at the DFT M06-2X / def2pvv level of theory. Excited states of 2HP and its complexes with methanol and ammonia were examined at two levels. First, sampling a Wigner distribution of 300 points, the photoabsorption spectra were simulated within the nuclear ensemble approximation. Second, simulation of the dynamics of the excited states with the surface hopping method. Several separate spectral windows with three hundred trajectories each, were considered. Lifetime of excited states and photochemical channels observed were discussed.     

Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase

This review describes the development and application of photoactive ruthenium complexes to study electron transfer and proton pumping reactions in cytochrome c oxidase (CcO). CcO uses four electrons from Cc to reduce O(2) to two waters, and pumps four protons across the membrane. The electron transfer reactions in cytochrome oxidase are very rapid, and cannot be resolved by stopped-flow mixing techniques. Methods have been developed to covalently attach a photoactive tris(bipyridine)ruthenium group [Ru(II)] to Cc to form Ru-39-Cc. Photoexcitation of Ru(II) to the excited state Ru(II*), a strong reductant, leads to rapid electron transfer to the ferric heme group in Cc, followed by electron transfer to Cu(A) in CcO with a rate constant of 60,000s(-1). Ruthenium kinetics and mutagenesis studies have been used to define the domain for the interaction between Cc and CcO. New ruthenium dimers have also been developed to rapidly inject electrons into Cu(A) of CcO with yields as high as 60%, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism.     

Design of molecular switching and signaling based on proton transfer in 2-hydroxy Schiff bases: a computational study

The present work aims to exploit the possibility of using the tautomerism in 2-hydroxy Schiff bases for molecular switching. The enol imine (E)? enaminone (K) tautomerization in a series of 2-hydroxy Schiff bases have been investigated theoretically at the DFT/B3LYP/6-311G** level of theory. The intramolecular proton transfer processes have been explored, transition structures have been located and characterized. The kinetics and thermodynamics of the proton transfer process, and its time scale have been computed and discussed in the framework of the suitability as molecular switches. Substituent effects have been computed and its effect on the enthalpy changes (?H*) and activation energies (?G*) have been analyzed and discussed. Nonspecific solvent effects have also been taken into account by using the polarized continuum model (IPCM) of two different solvent. The tautomerization energies are decreased and hence the endothermic nature of the enol imine ? enaminone tautomerization. The potential energy barriers, on the other hand, are increased due to the relative destabilization of the transition states. The NBO charge populations show that there is a high positive charge on the hydrogen atom during the process in all cases, which confirms that the proton transfer proceeds through a three-center interaction. The proton transfer processes, in all cases studied are kinetically allowed. The low potential energy barrier suggests that interconversion between the two tautomeric forms is spontaneous and the two forms may coexist.     

Studies of the bacteriorhodopsin photocycle without the use of light: clues to proton transfer coupled reactions

In the photochemical cycle of bacteriorhodopsin, the light-driven proton pump of halobacteria, only the first step, the isomerization of the all-trans retinal to 13-cis, is dependent on illumination. Because the steps that accomplish the translocation of a proton during the ensuing reaction sequence of intermediate states are thermal reactions, they have direct analogies with such steps in other ion pumps. In a surprisingly large number of cases, the reactions of the photocycle could be studied without using light. This review recounts experiments of this kind, and what they contribute to understanding the transport mechanism of this pump, and perhaps indirectly other ion pumps as well.     

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.