BBA - Bioenergetics (v.1458, #1)

Proton-transfer reactions in bioenergetics by Peter Brzezinski (1-5).

A brief summary of the principal notions of the quantum–mechanical theory of the charge transfer reactions has been presented. In the framework of this theory, the mechanism of the proton transfer consists in the classical medium reorganization that equalizes the proton energy levels in the initial and final states, and a consequent proton transfer via a quantum–mechanical underbarrier transition. On the basis of this mechanism, factors influencing the proton transfer probability, and hence kinetic isotope effect, have been discussed; among them are the optimum tunneling distance, the involvement of the excited vibrational states, etc. Semi-classical and quantum–mechanical treatments of the Swain–Schaad relations have been compared. Some applications to enzymatic proton-transfer reactions have been described.
Keywords: Adiabatic process; Nonadiabatic process; Activation energy; Tunneling probability; Kinetic isotope effect;

Various means of calculating the effect of changing the mass of a given atom upon a chemical process are reviewed. Of particular interest is the deuterium isotope effect comparing the normal protium nucleus with its heavier deuterium congener. The replacement of the bridging protium in a neutral hydrogen bond such as the water dimer by a deuterium strengthens the interaction by a small amount via effects upon the vibrational energy. In an ionic H-bond such as the protonated water dimer, on the other hand, the reverse trend is observed in that replacement of the bridging protium by dimer weakens the interaction. In addition to the stability of a given complex, the rate at which a proton transfers from one group to another is likewise affected by deuterium substitution, viz. kinetic isotope effects (KIEs). The KIE is enlarged as the temperature drops, particularly so if the calculation of KIE includes proton tunneling. The KIE is also sensitive to any angular distortions or stretches present in the H-bond of interest. KIEs can be computed either by the standard transition state theory which is derived via only two points on the potential energy surface, or by more complete formalisms which take account of larger swaths of the surface. While more time intensive, the latter can also be applied to provide insights important in interpretation of experimental data.
Keywords: Transition state theory; Hydrogen bond; Proton transfer; Tunneling; Reaction path; Molecular dynamics;

The question of the nature of the proton bridge involved in general acid–base catalysis in both enzymic and non-enzymic systems is considered in the light of long-known but insufficiently appreciated work of Jencks and his coworkers and of more recent results from neutron-diffraction crystallography and NMR spectroscopic studies, as well as results from isotope-effect investigations. These lines of inquiry lead toward the view that the bridging proton, when between electronegative atoms, is in a stable potential at the transition state, not participating strongly in the reaction-coordinate motion. Furthermore they suggest that bond order is well-conserved at unity for bridging protons, and give rough estimates of the degree to which the proton will respond to structural changes in its bonding partners. Thus if a center involved in general-catalytic bridging becomes more basic, the proton is expected to move toward it while maintaining a unit total bond order. For a unit increase in the pK of a bridging partner, the other partner is expected to acquire about 0.06 units of negative charge. The implications are considered for charge distribution in enzymic transition states as the basicity of catalytic residues changes in the course of molecular evolution or during progress along a catalytic pathway.
Keywords: Enzyme; Catalysis; Hydrogen bond; Transition state; Active site; Charge distribution;

The coupled motion of electrons and protons occurs in many proteins. Using appropriate tools for calculation, the three-dimensional protein structure can show how each protein modulates the observed electron and proton transfer reactions. Some of the assumptions and limitations involved in calculations that rely on continuum electrostatics to calculate the energy of charges in proteins are outlined. Approaches that mix molecular mechanics and continuum electrostatics are described. Three examples of the analysis of reactions in photosynthetic reaction centers are given: comparison of the electrochemistry of hemes in different sites; analysis of the role of the protein in stabilizing the early charge separated state in photosynthesis; and calculation of the proton uptake and protein motion coupled to the electron transfer from the primary (QA) to secondary (QB) quinone. Different mechanisms for stabilizing intra-protein charged cofactors are highlighted in each reaction.
Keywords: Continuum electrostatics; pK a; Electrochemistry; Reaction center; Proton transfer; Electron transfer; Charge stabilization;

The hydration of CO2 and the dehydration of HCO3 catalyzed by the carbonic anhydrases is accompanied by the transfer of protons between solution and the zinc-bound water molecule in the active site. This transfer is facilitated by amino acid residues of the enzyme which act as intramolecular proton shuttles; variants of carbonic anhydrase lacking such shuttle residues are enhanced or rescued in catalysis by intermolecular proton transfer from donors such as imidazole in solution. The resulting rate constants for proton transfer when compared with the values of the pK a of the donor and acceptor give Brønsted plots of high curvature. These data are described by Marcus theory which shows an intrinsic barrier for proton transfer from 1 to 2 kcal/mol and work terms or thermodynamic contributions to the free energy of reaction from 4 to10 kcal/mol. The interpretation of these Marcus parameters is discussed in terms of the well-studied pathway of the catalysis and structure of the enzymes.
Keywords: Proton transfer; Marcus theory; Carbonic anhydrase; Carbon dioxide; Enzyme kinetics;

Common themes and problems of bioenergetics and voltage-gated proton channels by Thomas E. DeCoursey; Vladimir V. Cherny (104-119).
The existence of a proton-selective pathway through a protein is a common feature of voltage-gated proton channels and a number of molecules that play pivotal roles in bioenergetics. Although the functions and structures of these molecules are quite diverse, the proton conducting pathways share a number of fundamental properties. Conceptual parallels include the translocation by hydrogen-bonded chain mechanisms, problems of supply and demand, equivalence of chemical and electrical proton gradients, proton wells, alternating access sites, pK a changes induced by protein conformational change, and heavy metal participation in proton transfer processes. An archetypal mechanism involves input and output proton pathways (hydrogen-bonded chains) joined by a regulatory site that switches the accessibility of the bound proton from one ‘channel’ to the other, by means of a pK a change, molecular movement, or both. Although little is known about the structure of voltage-gated proton channels, they appear to share many of these features. Evidently, nature has devised a limited number of mechanisms to accomplish various design strategies, and these fundamental mechanisms are repeated with minor variation in many superficially disparate molecules.
Keywords: Proton; Proton channel; Proton transport; pH regulation; Hydrogen ion; Proton permeability;

Biophysical aspects of intra-protein proton transfer by Sharron Brandsburg-Zabary; Orit Fried; Yael Marantz; Esther Nachliel; Menachem Gutman (120-134).
The passage of proton trough proteins is common to all membranal energy conserving enzymes. While the routes differ among the various proteins, the mechanism of proton propagation is based on the same chemical–physical principles. The proton progresses through a sequence of dissociation association steps where the protein and water molecules function as a solvent that lowers the energy penalty associated with the generation of ions in the protein. The propagation of the proton in the protein is a random walk, between the temporary proton binding sites that make the conducting path, that is biased by the intra-protein electrostatic potential. Kinetic measurements of proton transfer reactions, in the sub-ns up to μs time frame, allow to monitor the dynamics of the partial reactions of an overall proton transfer through a protein.
Keywords: Proton transfer; Diffusion; Electrostatic potential; Ion conductive channel; Microcavity;

Bacteriorhodopsin is probably the best understood proton pump so far and is considered to be a model system for proton translocating membrane proteins. The basis of a molecular description of proton translocation is set by having the luxury of six highly resolved structural models at hand. Details of the mechanism and reaction dynamics were elucidated by a whole variety of biophysical techniques. The current molecular picture of catalysis by BR will be presented with examples from time-resolved spectroscopy. FT-IR spectroscopy monitors single proton transfer events within bacteriorhodopsin and judiciously positioned pH indicators detect proton migration at the membrane surface. Emerging properties are briefly outlined that underlie the efficient proton transfer across and along biological membranes.
Keywords: Retinal protein; Membrane; Electron crystallography; X-Ray structure; Infrared spectroscopy; pH probe;

Proton and electron transfer in bacterial reaction centers by M.Y Okamura; M.L Paddock; M.S Graige; G Feher (148-163).
The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule QB that binds two electrons and two protons at the active site. This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the QB site. The mechanism of the first electron transfer, k (1) AB, Q AQB→QAQ B, was shown to be rate limited by conformational gating. The mechanism of the second electron transfer, k (2) AB, was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H++Q AQ B⇔Q AQBH→QA(QBH). The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.

Proton pumping by cytochrome oxidase: progress, problems and postulates by Dmitry Zaslavsky; Robert B. Gennis (164-179).
The current status of our knowledge about the mechanism of proton pumping by cytochrome oxidase is discussed. Significant progress has resulted from the study of site-directed mutants within the proton-conducting pathways of the bacterial oxidases. There appear to be two channels to facilitate proton translocation within the enzyme and they are important at different parts of the catalytic cycle. The use of hydrogen peroxide as an alternative substrate provides a very useful experimental tool to explore the enzymology of this system, and insights gained from this approach are described. Proton transfer is coupled to and appears to regulate the rate of electron transfer steps during turnover. It is proposed that the initial step in the reaction involves a proton transfer to the active site that is important to convert metal-ligated hydroxide to water, which can more rapidly dissociate from the metals and allow the reaction with dioxygen which, we propose, can bind the one-electron reduced heme-copper center. Coordinated movement of protons and electrons over both short and long distances within the enzyme appear to be important at different parts of the catalytic cycle. During the initial reduction of dioxygen, direct hydrogen transfer to form a tyrosyl radical at the active site seems likely. Subsequent steps can be effectively blocked by mutation of a residue at the surface of the protein, apparently preventing the entry of protons.
Keywords: Oxidase; Cytochrome; Proton; Channel; Oxygen; Enzyme; Peroxidase;

Where is ‘outside’ in cytochrome c oxidase and how and when do protons get there? by Denise A Mills; Laurence Florens; Carrie Hiser; Jie Qian; Shelagh Ferguson-Miller (180-187).
Cytochrome c oxidase moves both electrons and protons in its dual role as a terminal electron acceptor and a contributor to the proton motive force which drives the formation of ATP. Although the sequence of electron transfer events is well-defined, the correlated mechanism and routes by which protons are translocated across the membrane are not. A recent model [Michel, Proc. Natl. Acad. Sci. USA 95 (1998) 12819] offers a detailed molecular description of when and how protons are translocated through the protein to the outside, which contrasts with previous models in several respects. This article reviews the behavior of site-directed mutants of Rhodobacter sphaeroides cytochrome c oxidase in the context of these different models. Studies of the internally located lysine 362 on the K channel and aspartate 132 on the D channel, indicate that D132, but not K362, is connected to the exterior region. Analysis of the externally located arginine pair, 481 and 482, and the Mg/Mn ligands, histidine 411 and aspartate 412, which are part of the hydrogen-bonded network that includes the heme propionates, indicates that alterations in this region do not strongly compromise proton pumping, but do influence the pH dependence of overall activity and the control of activity by the pH gradient. The results are suggestive of a region of ‘sequestered’ protons: beyond a major energetic gate, but selectively responsive to the external environment.
Keywords: Cytochrome c oxidase; Magnesium; Proton; Arginine pair; Respiratory control;

Proton and hydrogen currents in photosynthetic water oxidation by Cecilia Tommos; Gerald T. Babcock (199-219).
The photosynthetic processes that lead to water oxidation involve an evolution in time from photon dynamics to photochemically-driven electron transfer to coupled electron/proton chemistry. The redox-active tyrosine, YZ, is the component at which the proton currents necessary for water oxidation are switched on. The thermodynamic and kinetic implications of this function for YZ are discussed. These considerations also provide insight into the related roles of YZ in preserving the high photochemical quantum efficiency in Photosystem II (PSII) and of conserving the highly oxidizing conditions generated by the photochemistry in the PSII reaction center. The oxidation of YZ by P680 + can be described well by a treatment that invokes proton coupling within the context of non-adiabatic electron transfer. The reduction of YZ , however, appears to proceed by an adiabatic process that may have hydrogen-atom transfer character.
Keywords: Photosystem II; Oxygen-evolving complex; Water-oxidizing complex; Oxygen evolution; Water oxidation; Tyrosine radical; Redox-active amino acid;