Photo-isomerization upshifts the pK_a of the Photoactive Yellow Protein chromophore to contribute to photocycle propagation

Highlights

• Isolated para-coumaric acid (pCA) and thiomethyl-para-coumaric acid (TMpCA) PYP models were studied.

• UV illumination converts pCA from the trans to the cis conformer.

• The pK_a of the both conformers are resolved via pH titration coupled with electronic spectroscopy.

• The cis conformer of pCA exhibits a 0.6 pH unit higher pK_a than the trans form.

• Quantum calculations show how geometry modifies charge distributions.

• The higher pK_a originates from Columbic interactions between phenolate and tail moieties.

Abstract

The influence of chromophore structure on the protonation constant of the Photoactive Yellow Protein chromophore is explored with isolated para-coumaric acid (pCA) and thiomethyl-para-coumaric acid model chromophores in solution. pH titration coupled with visible absorption spectra of the trans and photogenerated cis conformer of isolated pCA demonstrates that the isomerization of the chromophore increases the pK_a of the phenolate group by 0.6 units (to 10.1 ± 0.22). Formation of the pCA thioester reduces the pK_a of the phenolic group by 0.3 units (from 9.5 ± 0.15 to 9.2 ± 0.16). Unfortunately, a macroscopic cis-TMpCA population was not achieved via photoexcitation. Both trends were explained with electronic structure calculations including a Natural Bond Orbital analysis that resolves that the pK_a upshift for the cis configuration is attributed to increased Columbic repulsion between the coumaryl tail and the phenolate moieties. This structurally induced pK_a upshift after isomerization is argued to aid in the protonation of the chromophore within the PYP protein environment and the subsequent propagation of the photocycle response and in vivo photo-activity.

Introduction

Photosensory receptor proteins transduce photon energy into physiological function and play essential roles for most organisms to adjust their behavior and metabolism in response to the quantity and quality of light in their environment [1]. These proteins operate by the photoexcitation of small molecular chromophores bound within complex protein scaffoldings; the excitation of which initiate light-dependent structural changes in the surrounding protein and ultimately trigger signal phototransduction pathways [2], [3]. Photoreceptors are also ideal systems to study the molecular-level basis for structure-function relationships since they share similar structural motifs with more ubiquitous non-light activating signal transducion proteins and can be triggered with short pulses of light to synchronously observe their signaling activity [2], [4], [5]. One such photoreceptor is the Photoactive Yellow Protein (PYP), which is a small 125 amino acid-containing water-soluble protein found in the bacterium Halorhodospira halophila and is responsible for triggering the negative phototactic response of the organism to blue light [6], [7], [8].

The underlying photophysics of PYP is complex with a broad range of chemical reactions participating in its photoresponse. Photo-excitation of the dark-adapted pG state of PYP induces a rapid (<2 ps) trans/cis isomerization around a double bond of the internally bound p-coumaric acid molecule (pCA) [8], [9], [10], [11], [12], [13], [14], [15], [16], [17] that is covalently bound to the sole cysteine residue of the protein (Cys49) through a thioester bond (Fig. 1A). This, in turn, initiates a complicated series of reversible reactions extending over 15 decades in time from femtoseconds to seconds [18], [19], [20], [21] that involve chromophore protonation (Fig. 1B and C) [18], [22], protein unfolding, and hydrogen-bond disruption [23], [24], followed by the corresponding recovery reactions to complete a photocycle by reforming pG. Due to the simplicity of PYP's single-domain protein structure, it is an excellent system for studying the complex relationship between protein dynamics and chemical reaction dynamics and more specifically how nature has tuned both to generate photobiological activity. One relationship of particular interest is resolving how photoexcitation initiates and propagates the PYP photocycle (Fig. 2A) and specifically how chromophore isomerization facilitates the proton transfer reactions that are critical for PYP photoactivity. To understanding this, the detailed knowledge of the interactions between structure, protonation, and electronic structure must be resolved.

When bound to the PYP protein, the pCA chromophore is sterically constrained by the surrounding protein nanospace, which includes hydrogen bonds with several amino acids (Glu46 and Tyr42 on the phenolate side and Cys49 on the carbonyl side). Furthermore, the pCA chromophore is also influenced by electrostatic interactions with nearby residues, especially the guanidinium cation of Arg52 (Fig. 1A) [25]. Protonation of pCA within the protein occurs via the donation of a labile proton from Glu46, although Tyr42 also participates in the hydrogen bonding to pCA [18], [22], [26], which induces a partial unfolding (ms) of the protein and the formation of the pB signaling state (Fig. 2A) [22], [27], [28]. A simple scheme describing this reaction (Fig. 2B) involves a potential energy surface with the proton originally located on E46 (left well) that is coerced to pCA (right well) over an energy barrier ΔG^‡ (blue components) [29], [30]. Multiple factors facilitate this reaction within PYP including the making and breaking of hydrogen bonding and evolution on the excited-state surface [31], [32], [33], [34], [35], which also involve individual bond angles and distances, fluctuations, energetics (both local and long range), and the intrinsic proton affinities of Glu46 (donor) and pCA (acceptor) molecules [25], [36], [37]. The last contribution results from the electron distributions within the donor and acceptor, which contribute to the Gibbs free energy difference (ΔG) for the protonation reaction and is often discussed in terms of constituent protonation pK_a values for the species at least for solvent exposed species.

Consequentially, electronic structure, chromophore deformation, and protonation intermix to activate and propagate the PYP photocycle and are strongly modulated by the detailed interactions between pCA and the surrounding protein scaffolding. The underlying chromophore properties can be resolved by removing the complexities of the protein environment and studying the isolated chromophore properties in solution. Here, we investigate the effect of chromophore conformation on the the pK_a of the PYP chromophore by exploring the structure- induced protonation properties of two model chromophores outside of the PYP protein environment: pCA and its thiomethyl ester analog, TMpCA (Fig. 1D). The pK_a values of the trans and cis forms of pCA were resolved in vitro after photo-irradiation and were compared to high-level ab initio electron structure calculations to provide insight into the electron charge distributions for the different conformations and how they affect the pK_a values.