Multiple Protonation States Research Articles

Heterogeneity of the P +Q −A recombination kinetics in reaction centers from Rhodopseudomonas viridis: the effects of pH and temperature

The decay of the flash-induced absorbance changes related to the primary electron donor, P + , was measured in reaction centers from Rhodopseudomonas viridis . The decay, which occurs by the recombination or back-reaction of P + Q − A , was found to be not asingle exponential and could be decomposed into two components. At pH 9,in the presence of o -phenanthroline, the two phases exhibited rate constants of 350 ± 25 s −1 ( k slow ) and 1400 ± 100 s −1 ( k fast ). In the absence of o -phenanthroline, in Q B -depleted reaction centers, the rate constants were 370 ± 25 and 1700 ± 100 s −1 , respectively. k slow and k fast display temperature-dependences similar to those previously described by Shopes and Wraight ((1987) Biochim. Biophys. Acta 893, 409–425) for the total decay component. Down to 240 K, the temperature-dependences of k slow and k fast indicate thermally activated processes, whichare presumed to proceed by repopulation of the P + I − statefrom P + Q − A . At temperatures below 240 K, an activationless process dominates with rate constants for the two phases whichdepend somewhat on the concentration of glycerol. In aqueous buffer, the limiting values at low temperatures, k T slow and k T fast , were determined to be 155 ± 10 and 460 ± 60 s −1 , respectively. After correction for these limiting rates, the ambient temperature-dependences were linear in an Arrhenius plot. At pH 9, the activation energies (enthalpies, Δ H ) were 0.258 ± 0.009 and 0.278 ± 0.021 eV for the slow and fast phases, respectively. The entropies of activation were also quite large. Consequently the activation free energies (Δ G ) were inverted compared to the activation enthalpies, i.e., Δ G slow = 0.292 ± 0.011 eV and Δ G fast = 0.260 ± 0.022 eV. k slow and k fast were also significantly pH-dependent. As the pH was raised from pH 6, both rate constants showed a shallow minimum at pH 7.5–8, and accelerated significantly as the pH was raised further. The pH-dependence of both components was more marked in the absence than in the presence of o -phenanthroline. The relative proportions of k slow and k fast were also strongly pH-dependent in the range pH 5.5–11.5. The amplitude curves displayed two waves that were dependent on ionic conditions and on the presence of o -phenanthroline. The pH-and temperature-dependences of the rates and amplitudes of the recombination components are interpreted in terms of multiple protonation states of the reaction center affecting the energetics of the thermally activated recombination pathway. The pH-dependence of the rates can be understood as arising from the electrostatic stabilization of the charge-separation states, P + Q − A and P + I − , due to the interaction of at leasttwo protonation sites with the species Q − A and I − . The pH-dependence of the recombination rates, in the range pH 5.5–10, couldbe accounted for in this way, with two distinct values for p K Q A : one with p K Q A about 6, for which protonation resulted in the relative stabilization of P + I − , decreasing Δ G and leading to acceleration of the back-reaction, and one with p K Q A about 9, which relatively stabilized P + Q − A in the protonated state, increasing Δ G and causing slower recombination. The biphasicity of the recombination kinetics in this pH range was ascribed to the comparable rates of the back-reaction and of protonation of the reaction center at most pH values. Thus, the distribution of protonation states established in the dark cannot fully reequilibrate with the new p K values of the charge-separated state, on the timescale of the back-reaction. The behavior described here for the P + QA charge recombination in Rps. viridis is in contrast to that in Rhodobacter sphaeroides , which exhibits monophasic back reaction kinetics at ambient temperatures. It is suggested that in the latter species the back-reaction is sufficiently slow ( k = 10 s −1 ) that protonation states present after the flash can equilibrate prior to the charge recombination.

Use of polylysine-coated slides in preparation of cell samples for diagnostic cytology with special reference to urine sample.

<h3>Abstract</h3> The prediction of acid dissociation constants (p<i>K</i>_a) is a prerequisite for predicting many other properties of a small molecule, such as its protein-ligand binding affinity, distribution coefficient (log <i>D</i>), membrane permeability, and solubility. The prediction of each of these properties requires knowledge of the relevant protonation states and solution free energy penalties of each state. The SAMPL6 p<i>K</i>_a Challenge was the first time that a separate challenge was conducted for evaluating p<i>K</i>_a predictions as part of the Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) exercises. This challenge was motivated by significant inaccuracies observed in prior physical property prediction challenges, such as the SAMPL5 log <i>D</i> Challenge, caused by protonation state and p<i>K</i>_a prediction issues. The goal of the p<i>K</i>_a challenge was to assess the performance of contemporary p<i>K</i>_a prediction methods for drug-like molecules. The challenge set was composed of 24 small molecules that resembled fragments of kinase inhibitors, a number of which were multiprotic. Eleven research groups contributed blind predictions for a total of 37 p<i>K</i>_a distinct prediction methods. In addition to blinded submissions, four widely used p<i>K</i>_a prediction methods were included in the analysis as reference methods. Collecting both microscopic and macroscopic p<i>K</i>_a predictions allowed in-depth evaluation of p<i>K</i>_a prediction performance. This article highlights deficiencies of typical p<i>K</i>_a prediction evaluation approaches when the distinction between microscopic and macroscopic p<i>K</i>_as is ignored; in particular, we suggest more stringent evaluation criteria for microscopic and macroscopic p<i>K</i>_a predictions guided by the available experimental data. Top-performing submissions for macroscopic p<i>K</i>_a predictions achieved RMSE of 0.7-1.0 p<i>K</i>_a units and included both quantum chemical and empirical approaches, where the total number of extra or missing macroscopic p<i>K</i>_as predicted by these submissions were fewer than 8 for 24 molecules. A large number of submissions had RMSE spanning 1-3 p<i>K</i>_a units. Molecules with sulfur-containing heterocycles or iodo and bromo groups were less accurately predicted on average considering all methods evaluated. For a subset of molecules, we utilized experimentally-determined microstates based on NMR to evaluate the dominant tautomer predictions for each macroscopic state. Prediction of dominant tautomers was a major source of error for microscopic p<i>K</i>_a predictions, especially errors in charged tautomers. The degree of inaccuracy in p<i>K</i>_a predictions observed in this challenge is detrimental to the protein-ligand binding affinity predictions due to errors in dominant protonation state predictions and the calculation of free energy corrections for multiple protonation states. Underestimation of ligand p<i>K</i>_a by 1 unit can lead to errors in binding free energy errors up to 1.2 kcal/mol. The SAMPL6 p<i>K</i>_a Challenge demonstrated the need for improving p<i>K</i>_a prediction methods for drug-like molecules, especially for challenging moieties and multiprotic molecules.