Changes In Proton Affinity Research Articles

Tracing the Pathways of Waters and Protons in Photosystem II and Cytochrome c Oxidase

Photosystem II (PSII) uses water as the terminal electron donor, producing oxygen in the Mn4CaO5 oxygen evolving complex (OEC), while cytochrome c oxidase (CcO) reduces O2 to water in its heme–Cu binuclear center (BNC). Each protein is oriented in the membrane to add to the proton gradient. The OEC, which releases protons, is located near the P-side (positive, at low-pH) of the membrane. In contrast, the BNC is in the middle of CcO, so the protons needed for O2 reduction must be transferred from the N-side (negative, at high pH). In addition, CcO pumps protons from N- to P-side, coupled to the O2 reduction chemistry, to store additional energy. Thus, proton transfers are directly coupled to the OEC and BNC redox chemistry, as well as needed for CcO proton pumping. The simulations that study the changes in proton affinity of the redox active sites and the surrounding protein at different states of the reaction cycle, as well as the changes in hydration that modulate proton transfer paths, are described.

Kinetic Method Analysis of the Effect of cis- and trans-Hydroxylation on the Proton Affinity of Proline

Proton affinities of proline and hydroxyproline were measured using the Cooks’ kinetic method. The measurements show that hydroxylation increases the proton affinity, which is consistent with X3LYP computation results. This work supports findings from a previous study (S. Mezzache et al., Rapid Commun. Mass Spectrom. 2005, 19, 2279) that modification of proline increases its proton affinity, but it does not provide compelling evidence for the prediction in the same study that proton affinity of the molecule is substantially influenced by intramolecular interactions involving the proton. PBE1PBE calculations suggest that isotropic polarizability rather than intramolecular hydrogen-bonding interactions provides a more suitable diagnosis for trends in proton affinity changes associated with modifications.

Spectroscopic and Kinetic Evidence on How Bacteriorhodopsin Accomplishes Vectorial Proton Transport under Functional Conditions

Vectorial transport by pumps requires a switch in the accessibility (or affinity) of the ion binding site from the extracelullar to the cytoplamic side, or vice versa. In the proton-pump bacteriorhodopsin (bR) the nature of this switch mechanism is still controversial, although it is expected to occur during the transition between two M substates. Here, we characterized this transition by time-resolved Fourier transform infrared (FT-IR) spectroscopy under functional conditions, using a novel approach for the analysis of kinetic data: the regularized inversion of an eigenvalue problem. The use of IR spectroscopy allowed the simultaneous evaluation of the involvement of the protein backbone, retinal, amino acid side chains, and internal water molecules in the switch mechanism. We provide solid evidence that the switch is not associated with protein backbone conformational changes. On the other hand, changes in the retinal conformation (or in the orientation of the Schiff Base (SB) during the switch) are reasonably although not completely discarded. We found that the proton release group (PRG), a delocalized proton characterized by a broad continuum band in the infrared, deprotonates in the transition between two M substates. Vectorial proton transport is most likely guaranteed by the coupled proton affinity changes resulting from the PRG deprotonation, favoring an affinity-based over an accessibility-based switch mechanism.

A Role for Internal Water Molecules in Proton Affinity Changes in the Schiff Base and Asp85 for One‐way Proton Transfer in Bacteriorhodopsin†

Light-induced proton pumping in bacteriorhodospin is carried out through five proton transfer steps. We propose that the proton transfer to Asp85 from the Schiff base in the L-to-M transition is accompanied by the relocation of a water cluster on the cytoplasmic side of the Schiff base from a site close to the Schiff base in L to the Phe219-Thr46 region in M. The water cluster present in L, formed at 170 K, is more rigid than that at room temperature. This may be responsible for blocking the conversion of L to M at 170 K. In the photocycle at room temperature, this water cluster returns to the site close to the Schiff base in N, with a rigid structure similar to that of L at 170 K. The increase in the proton affinity of Asp85, which is a prerequisite for the one-way proton transfer in the M-to-N transition, is suggested to be facilitated by a structural change which disrupts interactions between Asp212 and the Schiff base, and between Asp212 and Arg82. We propose that this liberation of Asp212 is accompanied by a rearrangement of the structure of water molecules between Asp85 and Asp212, stabilizing the protonated Asp85 in M.

Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle

Bacteriorhodopsin is the smallest autonomous light-driven proton pump. Proposals as to how it achieves the directionality of its trans-membrane proton transport fall into two categories: accessibility-switch models in which proton transfer pathways in different parts of the molecule are opened and closed during the photocycle, and affinity-switch models, which focus on changes in proton affinity of groups along the transport chain during the photocycle. Using newly available structural data, and adapting current methods of protein protonation-state prediction to the non-equilibrium case, we have calculated the relative free energies of protonation microstates of groups on the transport chain during key conformational states of the photocycle. Proton flow is modeled using accessibility limitations that do not change during the photocycle. The results show that changes in affinity (microstate energy) calculable from the structural models are sufficient to drive unidirectional proton transport without invoking an accessibility switch. Modeling studies for the N state relative to late M suggest that small structural re-arrangements in the cytoplasmic side may be enough to produce the crucial affinity change of Asp96 during N that allows it to participate in the reprotonation of the Schiff base from the cytoplasmic side. Methodologically, the work represents a conceptual advance compared to the usual calculations of p K a using macroscopic electrostatic models. We operate with collective states of protonation involving all key groups, rather than the individual-group p K a values traditionally used. When combined with state-to-state transition rules based on accessibility considerations, a model for non-equilibrium proton flow is obtained. Such methods should also be applicable to other active proton-transport systems.

Predicting binding energetics from structure: looking beyond DeltaG degrees.

Structure-based design of pharmaceuticals requires the ability to predict ligand affinity based on knowledge of structure. The primary term of interest is the binding affinity constant, K, or the free energy of binding, DeltaG degrees. It is common to attempt to predict DeltaG degrees based on empirically derived terms which represent common contributions such as the hydrophobic effect, hydrogen bonding, and conformational entropy. Although these approaches have met with some success, when they fail it is difficult to know which parameter(s) need refinement. Confidence in these approaches is also limited by the fact that DeltaG degrees typically is made up of compensating enthalpic and entropic terms, DeltaH degrees and DeltaS degrees, so that accurate prediction of a DeltaG degrees value may be fortuitous and may not indicate a reasonable understanding of the underlying relationship between structure and affinity. This is further complicated by the fact that both DeltaH degrees and DeltaS degrees are strongly temperature dependent through the heat capacity change, DeltaCp. In order to avoid these difficulties, we attempt to use structural data to predict DeltaH degrees, DeltaS degrees, and DeltaCp from which DeltaG degrees can be calculated as a function of temperature. The predictions are then compared to experimentally determined values. These calculations have been applied to several systems by ourselves and others. Systems include the binding of angiotensin II to an antibody, the dimerization of interleukin-8, and the binding of inhibitors to aspartic and serine proteases. Overall the calculations are very successful, and suggest that our understanding of the contributions of the hydrophobic effect, hydrogen bonding, and conformational entropy are quite good. Several of these systems show a strong dependence of the binding energetics on pH, indicative of changes in proton affinity of ionizable groups upon binding. It is critical to account for these protonation contributions to the binding energetics in order to assess the reliability of any computational prediction of energetics from structure. Methods have been developed for determining the energetics of proton binding using isothermal titration calorimetry. The availability of these methods provides a means of understanding how protein structure can modify the pKa's of ionizable groups. This information will further add to our understanding of structural energetic relationships and our ability accurately to predict binding affinities.

Conformational Effects on the Proton Affinity of the Schiff Base in Bacteriorhodopsin: A Density Functional Study

Density functional theory (DFT) calculations have been performed on a number of Schiff base structures related to the retinal Schiff base in bacteriorhodopsin (BR). The proton affinity (PA) of the Schiff base group was calculated in species with different lengths of the conjugated double-bond system and at different cis/trans isomerization states. The results show that the length of the conjugated electronic structure has a positive effect on the PA of the system which can be related to the more delocalized electronic structures in the longer chains. Although there is no significant difference of PA between different cis/trans isomers at single or double bonds in the main chain of the polyene structure, very pronounced PA changes are predicted during the rotation of these bonds. The calculations show that the rotation of the single bond adjacent to the Schiff base group significantly decreases the PA of the Schiff base. The results of closed-shell DFT calculations indicate that the rotation of the second ...

Basicity of azoles. 1. Investigation of total energy of pyrazole and imidazole derivatives by the partitioning method

1. Calculations by the total energy partitioning method, applied to a broad group of substituted azoles, have shown that the degree of destabilization of the -π-electron system in heterocyclic compounds upon protonation can provide an explanation for only the difference in principle between the basicities of pyrazoles and imidazoles. Within each series of compounds, the proton affinity is not determined by the magnitude of destabilization of the π-electron system. 2. Among the calculated molecular orbital characteristics of pyrazoles and imidazoles — total and π-electron charges on the basic center, their changes upon protonation, the energy of the N-H bond in the cations, the magnitude of change in one-center components of the total energy upon adding a proton, and the charge characteristics of the pyrrole N atom in the ground and ionic states — there is no characteristic that can provide an adequate description of the changes in proton affinity of these compounds. 3. For practical purposes in determining the relative basicities of azoles, it is possible to use a correlation of the proton affinity with a readily calculated parameter, the charge on the proton in the cation.

A proton motive force transducer and its role in proton pumps, proton engines, tobacco mosaic virus assembly and hemoglobin allosterism.

In this paper I am proposing a new, conformationally dependent basic site in proteins. The initial formulation of this proposal was based on the following: (1) bacteriorhodopsin is a light-driven proton pump and as such is a prototype for understanding proton-mediated energy transduction in biological systems; (2) current evidence suggests about 2 protons are pumped for each photon absorbed; (3) given the usual role of prolines as helix breakers, it is surprising to find about 2 prolines deeply embedded in the membrane-spanning, probably α-helical, portion of the bacteriorhodopsin molecule; (4) another presumptive proton translocator, the F0 proteolipid, is also helical and has a critical proline in its structure; (5) workers interested in protein folding have explained the existence of fast and slow folding subgroups of the same protein molecule as being due to cis : trans isomerization about the proline imide group; (6) the cis : trans isomerization is acid catalyzed; (7) simple chemical considerations predict that the proton affinity of the proline nitrogen should increase dramatically as the imide group is distorted away from planarity and should be a maximum midway between the cis and trans forms; thus, stabilization of the intermediate by protonation accounts for the acid catalysis of the proline cis : trans isomerization. Linking these observations together suggests that proline-containing α-helices may play a role in proton motive energy transduction. Due to the absence of a proton on the proline nitrogen, a proline-containing helix has a “proton hole” between the proline nitrogen and the carbonyl oxygen four residues earlier in the sequence. Here I propose a model in which the paramount feature is the change in pKa associated with a change in geometry of the “proton hole.” Order of magnitude calculations suggest that the proton hole should change its pKa by about 8 units, corresponding to a 108 change in proton affinity, for every 10 kcal of distortion energy, V. Calculations also show that it is energetically feasible to modulate the pKa of this site over the dynamic range of pKa = 2–14. Such a large value for ΔpKaΔV and such a dynamic range makes this site an ideal basis for an “integral proton injector,” an abstract model for proton pumping suggested on purely theoretical grounds by Nagle & Mille (J. chem. Phys.74, 1367–1372, 1981). Finally, two well studied proteins, the α-chain of hemoglobin and tobacco mossaic virus coat protein, both show features in their X-ray determined structures suggesting the possibility of protonation and deprotonation of the proton hole in α-helices containing proline. For TMV coat protein, there is a proline-containing α-helix that is located precisely in the region of the protein which undergoes an acid-induced conformational rearrangement. Structural changes at this locus have been singled out in comparisons of the X-ray structures of the TMV protein in its two conformations. For the α-chain of horse hemoglobin, there are two concurrent sites that are likely protonated and one contrary site that likely becomes deprotonated as hemoglobin converts from the liganded to the deoxy form. The contrary proline is proposed to help maintain co-operative oxygen binding over a wide pH range. The absence of one of the concurrent proline site in marsupial hemoglobin accounts for the small Bohr effect exhibited by these hemoglobins. The absence of the contrary proline site in carp hemoglobin accounts in a very logical way for the large Bohr effect and the lack of cooperative oxygen binding at both low and high pH by this hemoglobin.

Ionic solvation in water + co-solvent mixtures. Part 5.—Free energies of transfer of large single ions from water into water + methanol with the “neutral” component removed

Values for the change in proton affinity, ΔPa, of large molecules on transferring them from water into mixtures of water with methanol have been calculated. –ΔPa for anions and ΔPa for cations represent an elimination of the non-electrostatic or “neutral” component from the free energy of transfer of the ion i, ΔG°t(i). Although plots of –ΔPa against reciprocal of dielectric constant are linear for the anions, the values of their slopes show that there is a structural component of this “electrostatic” free energy, ΔG°t(i)e, in addition to the Born contribution. ΔG°t(F–) is calculated from data newly available, and ΔG°t(halide) values are compared with ΔG°t(i)e values for the large ions. The negative values for ΔG°t(i)e for large cations confirm that there is a contribution from structural changes in the solvent; these are compared with the negative values for ΔG°t(i) for small cations.