Photo-isomerization upshifts the pka of the photoactive yellow protein chromophore to contribute to photocycle propagation

Article abstract of DOI:10.1016/j.jphotochem.2013.06.019

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.

Full text of DOI:10.1016/j.jphotochem.2013.06.019

Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Photo-isomerization upshifts the pKa of the Photoactive Yellow
Protein chromophore to contribute to photocycle propagation
Sadia Naseema,1, Adèle D. Laurentb,1, Elizabeth C. Carrolla, Mikas Vengrisd,
Masato Kumauchic, Wouter D. Hoffc, Anna I. Krylovb, Delmar S. Larsena,∗
a Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616 USA
b Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0482, USA
c Department of Microbiology & Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA
d Quantum Electronics Department, Faculty of Physics, Vilnius University Sauletekio 10, LT10223 Vilnius, Lithuania
a r t i c l e i n f o
a b s t r a c t
Article history:
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 pKa of the phenolate group by 0.6 units (to 10.1 0.22). Formation of the pCA thioester
reduces the pKa of the phenolic group by 0.3 units (from 9.5 0.15 to 9.2 0.16). Unfortunately, a macro-
scopic 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 pKa
upshift for the cis configuration is attributed to increased Columbic repulsion between the coumaryl tail
and the phenolate moieties. This structurally induced pKa 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.
Received 11 March 2012
Received in revised form 19 June 2013
Accepted 25 June 2013
Available online 9 July 2013
Keywords:
Photoactive Yellow Protein
Photoisomerization
Quantum Modeling
Photoreceptors
Para-coumaric acid
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
triggering the negative phototactic response of the organism to
blue light [6–8].
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 initi-
ate 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
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) [8–17] that is
covalently bound to the sole cysteine residue of the protein (Cys49)
through a thioester bond (Fig. 1A). This, in turn, initiates a com-
plicated series of reversible reactions extending over 15 decades
in time from femtoseconds to seconds [18–21] that involve chro-
mophore protonation (Fig. 1B and C) [18,22], protein unfolding, and
hydrogen-bond disruption [23,24], followed by the correspond-
ing 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 photobi-
ological activity. One relationship of particular interest is resolving
how photoexcitation initiates and propagates the PYP photocy-
cle (Fig. 2A) and specifically how chromophore isomerization
facilitates the proton transfer reactions that are critical for PYP pho-
toactivity. To understanding this, the detailed knowledge of the
∗
Corresponding author. Tel.: +1 5307526507.
E-mail address: dlarsen@ucdavis.edu (D.S. Larsen).
1
These authors contributed equally to this manuscript.
1010-6030/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2013.06.019

44
S. Naseem et al. / Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
Fig. 1. Molecular structures of studied PYP chromophores. (A) the protein-bound trans-PYP chromophore in the ground state (pG). (B) trans-pCA2− at high pH (fully
deprotonated), (C) cis-pCA2− at high pH (fully deprotonated) and (D) trans-TMpCA− at high pH (deprotonated).
interactions between structure, protonation, and electronic struc-
ture must be resolved.
Consequentially, electronic structure, chromophore deforma-
tion, and protonation intermix to activate and propagate the PYP
photocycle and are strongly modulated by the detailed interac-
tions between pCA and the surrounding protein scaffolding. The
underlying chromophore properties can be resolved by remov-
ing 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 pKa 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 pKa 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 pKa values.
When bound to the PYP protein, the pCA chromophore is ste-
rically 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). Fur-
thermore, 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 pro-
tein 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 forma-
tion of the pB signaling state (Fig. 2A) [22,27,28]. A simple scheme
describing this reaction (Fig. 2B) involves a potential energy sur-
face with the proton originally located on E46 (left well) that is
coerced to pCA (right well) over an energy barrier ꢀG‡ (blue com-
ponents) [29,30]. Multiple factors facilitate this reaction within
PYP including the making and breaking of hydrogen bonding and
evolution on the excited-state surface [31–35], 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 contri-
bution results from the electron distributions within the donor and
acceptor, which contribute to the Gibbs free energy difference (ꢀG)
for the protonation reaction and is often discussed in terms of con-
stituent protonation pKa values for the species at least for solvent
exposed species.
2. Experimental and computational details
Trans-pCA and HPLC grade water were purchased from Sigma-
Aldrich and used as received. The synthesis of TMpCA involved
adding 10 ml of a DMF solution of trans p-coumaric acid (3-(4-
hydroxy-phenyl) acrylic acid; Aldrich) (2.00 g) to 10 ml DMF
solution of DCC (N,Nꢀ-dicyclohexylcarbodiimide; Pierce) (1.24 g)
under magnetic stirring. The mixture was incubated at 90 ◦C for 1 h,
resulting in a p-coumaric acid anhydride solution with a character-
istic pale yellow color. Precipitated N,Nꢀ-dicyclohexylurea, which
Fig. 2. (A) PYP photocycle indicating the dark-adapted state (pG), the light-adapted state (pB), and photointermediates. The isomerization states of the p-coumaric acid
chromophore are denoted. The pB intermediates have a protonated chromophore, while pG, I0, and pR intermediates are deprotonated. (B) Free energy profile for the proton
transfer reaction involved in formation of the pBꢀ signaling state from pR.

S. Naseem et al. / Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
45
was produced during the formation of the acid anhydride, was
A
removed by centrifugation (5500 × g, 10 min) and the resulting
pale yellow solution was added to 5 ml of a DMF suspension con-
taining sodium methanethiolate (Fluka) (1.00 g) under vigorous
stirring. The resulting orange mixture was extracted with ethyl
acetate and washed three times with an aqueous solution of NH4Cl
and then washed three times with H2O. The solution was dried
over MgSO4 and evaporated in vacuo. The residual yellow oil
was dissolved in 5 ml of ethyl acetate and chromatographed on
silica-gel (Sigma–Aldrich) with hexane and ethyl acetate (from
4:1 to 1:1 (v/v)) as the eluent. After recrystallization from ether,
235 mg of pale yellow crystals was obtained with a yield of 20%.
The structure and purity of the compound was confirmed by NMR
spectroscopy: 1H-NMR (400 MHz, CD3OD, TMS) ı 2.3 (s, 3H, CH3),
6.60 and 6.64 (d, 3J = 15.6 Hz, 1H, H-C␣), 6.76 and 6.79 (d, 3J = 8.4 Hz,
2H, H-Ph3,5), 7.42 and 7.44 (d, 3J = 8.4 Hz, 2H, H-Ph2,6), 7.49 and
7.53 (d, 3J = 15.6 Hz, 1H, H-C␤).
B
Fig. 3. pH dependent absorption spectra. (A) The trans-pCA spectra of the singly-
protonated pH = 7 form (red line) and the doubly-protonated pH = 13 form (blue line)
peak at 280 nm and 330 nm, respectively. (B) trans-TMpCA in water at the same pH
values used in panel A. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)
A mercury lamp (ORIEL 68806) with either a 335-nm or 365-nm
band-pass interference filter was used to illuminate solutions of
trans-pCA and trans-TMpCA; a second illumination study of TMpCA
was also performed with a continuous-wave 446-nm LED array
(Roithner Labs, Austria L435-66-60-550). The absorption spectra
of illuminated solutions were measured at different pH values (7-
12.5) and pKa values were estimated from the decomposition into
constituent populations with different protonation states (using
static absorption spectra as a basis). The resulting cis-pCA solu-
tion exhibited no signs of degradation or thermal back conversion
to the trans conformation when kept at room temperature in the
dark (not shown). Aqueous solutions of pCA were prepared with
0.05 M KOH to ensure full deprotonatation and solutions of TMpCA
were prepared at pH 11 with a phosphate buffer to avoid hydrol-
ysis of the thioester linkage; both were prepared with an optical
density of ∼1 per mm at the peak of the absorption spectrum.
The absorption spectra were monitored as the pH of aqueous solu-
tions was adjusted drop wise with concentrated HCl and KOH stock
solutions and the same procedure was adopted with cis-pCA and
trans-TMpCA. pH values were measured with an Acorn pH Meter
(Oakton WD-35613-00) and all titration curves were repeated four
times to ensure reproducibility.
The energies, equilibrium geometries, vibrational frequencies
were computed at the ␻B97X/6-31+G(d,p) level of theory. No imag-
inary frequencies were detected for the optimized structure of
the trans pCA model at all protonation states and one imaginary
frequency was detected for mono-deprotonated cis-pCAH− that
disappeared when the fully optimized structure was allowed to
deviate from a planar geometry. For TMpCA, the basis set for the
calculations was extended to 6-31+G(2df,p) to include polariza-
tion functions for the sulfur atom in the thioester of the coumaryl
tail (Fig. 1D). The carefully re-parameterized ␻B97X functional,
which includes a long-range correction to mitigate notorious self-
interaction errors, demonstrates superior performance relative to
the B3LYP functional (e.g., as observed in the root-mean square
errors of proton affinities of 1.72 kcal/mol) [38]. The optimized
structures of all studied protonation states and isomers of TMpCA
and pCA chromophores in the gas phase were planar, with the
exception of cis-pCAH−. Charge distributions were analyzed with
Natural Bond Orbital analysis [39]. All calculations were performed
with the Q-Chem electronic structure package [40].
depending on the protonation of the carboxylic acid and/or the phe-
nolic moieties [47], which can be easily identified in the electronic
absorption spectra (Fig. 3). The singly-deprotonated, neutral pH,
pCAH− state exhibits an absorption at 285 nm (4.35 eV) and the sub-
sequent deprotonation of the phenolic group at higher pHs results
in a red-shifted absorption at 332 nm (3.74 eV) for pCA2−. The sen-
sitivity of the absorption spectrum to the protonation states of the
chromophore indicates that the charge on the phenolic oxygen sig-
nificantly alters the electron density distribution in the molecule
[47–49]. Only the phenolate deprotonation reaction (pCA2−/pCAH−
and TMpCA−/TMpCA) was investigated further with pH titrations
since it is the biologically relevant protonation reaction in initiating
and propagating the PYP photocycle.
The methyl group in TMpCA acts as an inert, small capping group
that hinders the observed dynamics less than would be expected
with larger, bulkier groups [11,50]. TMpCA− in aqueous solution
has a spectrum that is appreciably red shifted (380 nm or 3.36 eV)
compared to pCA2− (335 nm or 3.70 eV) and is closer to wild-type
PYP’s absorption spectrum peaking at 446 nm (2.78 eV) [47]. Both
TMpCA and wildtype-PYP exhibit similar excited-state quenching
kinetics (∼2 ps) and TMpCA exhibits high photostability [51].
The static absorption spectra in Fig. 4 exhibit photo-stationary
equilibria established from irradiating trans-pCA2− (pH 12) and
trans-TMpCA− (pH 11) and are in agreement with the known
spectral changes associated with cis/trans isomerization for pCA2−
[52], with the cis spectrum exhibiting a blue shifted and decreased
amplitude absorption. An isosbestic point at 298 nm is observed
in the dynamics, indicative of two-population kinetics (i.e., trans
and cis isomers). Both protonation states of pCA demonstrate
photoisomerization activity with the cis isomers exhibiting a blue-
shifted absorption (Fig. 4A) relative to the trans isomer for pH = 7
and pH = 12 solutions (low pH spectra not shown). In contrast to
pCA2−, irradiation of trans-TMpCA− (at 365-nm) did not produce
a stable cis photoproduct and continued irradiation eventually
degraded the trans isomer into a final photoproduct that resembles
free phenolate ions (or phenol) in solution (Figs. S2 and S3) [53].
A 445-nm LED array was also used to excite the low energy red
tail of the trans-TMpCA− absorption spectrum and no significant
isomerization was observed (even after 10 h irradiation). Neutral
TMpCA also appeared to undergo rapid photodegradation rather
than simple photoisomerization (Fig. S2). The irradiation did
not photoconvert 100% of the trans-pCA molecules to the cis
conformation in solution, which was estimated at 84% based on
the amplitude of the red-most absorption band.
3. Experimental results
The photodynamics and static properties of several PYP chro-
mophore analogs have been previously studied outside the protein
including pCA [41], thio-methyl-p-coumaric acid (TMpCA) [42] and
others (Fig. 1) [11,43–46]. Isolated pCA (both trans and cis config-
urations) in solution can exist in three different protonation states

46
S. Naseem et al. / Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
Fig. 4. Absorption spectra after 365-nm illumination. (A) trans pCA2− acid at pH = 12.
(B) trans-TMpCA− at pH 11. Spectra for both samples are color coded to the different
exposure durations (indicated in panel A). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)
The pKa values for the trans and cis protonation reactions of
pCA2−/pCAH− and trans TMpCA−/TMpCA are directly estimated
from the titration curves in Fig. 6. The trans and cis-pCA absorp-
tion spectra (Fig. 5) are used as basis spectra to decompose
the pH-dependent absorption spectra monitored during titration
to determine the concentrations of single-deprotonated (pCAH−)
and double-deprotonated populations (pCA2−). The pKa values
are then estimated by fitting the pH-dependent concentration
profiles (e.g., ␣a for pCAH− and ␣b for pCA2−) to the sum of
Henderson-Hasselbalch approximations for the irradiated (i.e.,
cis/trans occupation ratio at 84/16%) and non-irradiated samples
(cis/trans occupation ratio at 0/100%):
Fig. 6. Occupation plots from the titration curves for trans (blue circles) and cis (red
circles) isomers of the chromophores. (A) The titration curves of aqueous pCA with
protonated pCAH− (solid lines) and deprotonated pCA2− (dashed lines) with an esti-
mated pKa of 9.5 0.18 for trans-pCA and 10.1 0.23 for cis-pCA. (B) The titration
curves of aqueous trans-TMpCA with protonated trans-TMpCAH (solid lines) and
deprotonated trans-TMpCA− (dashed lines) with an estimated pKa of 9.2 0.15 for
trans-TMpCA are estimated from the fit of the Henderson–Hasselbalch approxima-
tion to the data (Eq. (5)). For comparison, the estimated pKa of the PYP chromophore
in the solvent exposed pB state is shown (as labeled in legend) [54]. (For interpreta-
tion of the references to color in this figure legend, the reader is referred to the web
version of the article.)
1
However, for comparison, the estimated pKa of the PYP in the
solvent exposed pB state of wild-type PYP by Hellingwerf and
co-workers is shown [54].
˛
a
=
and
˛
= 1 − ˛a
(5)
b
1 + 10pH−pK
a
under the assumption that the protonation of the carboxylic group
(pKa ∼3) can be neglected in this pH range (7–12). The trans-pCA and
cis-pCA exhibit different pKa values of 9.5 0.15 (standard devia-
tion) and 10.1 0.22, respectively (Fig. 6A). Thiomethylation of the
carboxylic group in TMpCA (Fig. 1C) decreases the phenolic pKa
for the trans isomer to 9.2 0.15 (Fig. 6B). Unfortunately, the pKa
for the cis isomer of TMpCA could not be resolved since a macro-
scopic cis-TMpCA population could not be generated (Fig. 4B).
4. Quantum chemical modeling
Connecting these experimental results to pKa values computed
with ab initio methods requires accurate determination of the
Gibbs free energy difference in solution (ꢀGsol) of the underlying
deprotonation reaction:
ꢀGsol
pKa
=
(1)
2.303 RT
where T is temperature and R is the gas constant. Several method-
ologies have been developed to compute the variation of the Gibbs
free energy in solution (ꢀGsol) including MD simulations and the
free-energy perturbation approach [55], QM/MM schemes [56–58],
and ab initio calculations [59].
To model the solvent, high level ab initio theory is often com-
bined with implicit solvation models such as SS(V)PE [60,61],
COSMO [62], C-PCM [63], SM8 [64], etc., which differ in the expres-
sion of the electrostatic interaction and other empirical parameters
such as the choice of the cavity around the solute, and the dispersion
and repulsion terms of solvent-solute interaction. The thermody-
namic cycle shown in Fig. 7 is used to compute ꢀGsol with a general
deprotonation reaction (AH → A− + H+), whereby ꢀGsol is deter-
mined via the following equation:
−
+
ꢀGsol = ꢀG(AH
+ ꢀGaAq− + ꢀHaq − ꢀG
(2)
/A)
AH
aq
Fig. 5. Initial and final absorption spectra for pCA2− (blue curves, pH 13) and pCAH−
(red curves, pH 7). The trans (solid lines) and cis spectra (dashed lines) for each
protonation state are estimated from the 180 minutes of illumination with 365-nm
light. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
g
The first term on the right side of the equation is the gas phase
Gibbs energy difference between the protonated form (AH) and
deprotonated form (A−) of the solute, usually computed with the

S. Naseem et al. / Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
47
Table 1
Gas phase electronic energy (ꢀE), internal energy (ꢀU) and Gibbs free energies (ꢀG) differences between the deprotonated and protonated states of the trans and cis forms of
the two model systems. All energies are given in kcal/mol. The level of theory is ␻B97X/6-31+G(d,p) for pCA and ␻B97X/6-31+G(2df,p) for TMpCA. ꢀpKa values are computed
using gas phase ꢀE and ꢀU by Eq. (4).
Chromophore models
trans
cis
ꢀpKa (from ꢀꢀE)
ꢀpKa (from ꢀꢀU)
ꢀpKa(from ꢀꢀG)
ꢀE
ꢀU
ꢀG
ꢀE
ꢀU
ꢀG
TMpCAH/TMpCA−
pCAH−/pCA2−
338.67
402.62
329.96
394.29
330.31
393.62
339.95
410.11
331.17
401.67
331.34
401.71
−0.94
−5.49
−0.89
−5.40
−0.75
−5.92
A−
Table 2
zero-point vibrational energy correction (ZPE). ꢀG
is the dif-
aq
Gas phase energy differences between the trans and cis forms for each species in this
study. All energies are given in kcal/mol. The level of theory is ␻B97X/6-31+G(d,p)
for pCA and ␻B97X/6-31+G(2df,p) for TMpCA.
ference of the free Gibbs energy going from the gas phase to the
aqueous phase and can be computed with the implicit solvation
models described above. In particular, the SM8 model developed
by Truhlar has been shown to be reliable in computing ꢀGaq
with a mean unsigned deviation of 0.59 kcal/mol for neutral com-
pounds and 4.55 kcal/mol for anionic species [64]. Since an error of
1.36 kcal/mol in Gaq results in a one pH unit error in pKa (Eq. (1)), the
Species
Etrans − Ecis
Utrans − Ucis
Gtrans − Gcis
TMpCAH
TMpCA−
pCAH−
−4.63
−5.19
−2.01
−9.50
−5.13
−6.34
−2.34
−9.72
−5.56
−6.59
−1.61
−9.70
pCA2−
accurate determination of the solvation energies for all species is
important. For the hydrated proton (ꢀG ), the currently accepted
H+
aq
value of −264.0 kcal/mol at 1 atm and 1 M was used [65].
5. Discussion
Useful information regarding the isomerization effects can be
gleaned from relative comparisons and ratios of calculated values,
especially of the non-solvated gas-phase species. Table 1 collects
electronic energy (ꢀE), internal energy (ꢀU), and Gibbs free energy
(ꢀG) differences for protonated and deprotonated forms of the cis
and trans PYP chromophore models in the gas phase. The differ-
ence in pKa values between the cis and trans isomers (ꢀpKa) can be
expressed as the difference of Gibbs free energy differences (ꢀꢀG)
for the respective deprotonation reactions (Fig. 2B). The dominant
contribution to ꢀꢀG is the gas phase energy difference, ꢀꢀE (or
internal energy difference, ꢀꢀU, when ZPE is included:
pKa values are important determinants of biomolecular (partic-
ularly enzymatic) function and can be used to assess functional
activity and identify active sites. However, estimating pKa val-
ues is often difficult, both experimentally and theoretically. The
experimental methods available for estimating pKa values of buried
amino acids or prosthetic groups in proteins are either indirect (e.g.,
resolving the pH dependence of kinetics such as kcat in enzymes) or
direct (following the ionization of a single residue via spectroscopy
in a pH titration) [66,67]. Unfortunately, the former is not applica-
ble in many samples (including PYP) and the latter can be greatly
obscured by the other ionizable groups in large proteins. More-
over, pKa values can be estimated with computational algorithms
from known protein X-ray structures, however, these approaches
are largely optimized for amino acid residues and are not easily
applicable to prosthetic groups like pCA in PYP [16,68]. Extending
these studies to resolve the pKa values of transient populations (e.g.,
Fig. 2A) in the protein is even more difficult to realize.
Although the pH of the buffer solution in which the PYP protein
is dissolved can be varied and its effect on the pCA chromophore can
be spectroscopically monitored, correlating such external titration
data to the internal pKa (i.e. proton affinity) of the PYP chro-
mophore is complicated by the limited accessibility of the solvent
to the chromophore. For example, the pKa estimated for the iso-
lated trans-pCA chromophore in aqueous solutions (Fig. 1B) is 9.5
[69–71]. However, in the dark-adapted pG state of wild type PYP,
the chromophore remains deprotonated in aqueous solutions with
pH values down to 3, at which the protein denatures and the chro-
mophore is exposed to the solvent [72,73]. For Io and pR, the next
intermediates in the photocycle (Fig. 2A) with an isomerized pCA,
the pKa values are unknown [74,75]. However, for the subsequent
states (pBꢀ and pB), the cis chromophore is protonated with Glu46
deprotonated and the observed pKa is 9.9 [54], which is in bet-
ter agreement the isolated trans-pCA value. Since solvent access
ꢀꢀE = ꢀEtrans − ꢀEcis
(3)
where ꢀEtrans and ꢀEcis are the energy differences between the
deprotonated and protonated phenol moiety of the trans and cis
PYP chromophore models, respectively (Table 1).
The gas phase calculations provides a useful probe into the
factors that affect ꢀpKa (without having to calculate error-prone
solvation energies), which can be evaluated as follows:
ꢀꢀG
ꢀꢀE
ꢀpKa
=
≈
(4)
2.303RT
2.303RT
As Table 1 demonstrate, ꢀG differs from ꢀU by less than
1 kcal/mol indicating that differences in ꢀG between cis and trans
isomers (and ꢀpKa) are largely dominated by the electronic energy
difference (ꢀE) and that entropic and enthalpic contributions
almost entirely cancel. Table 2 focuses on the respective energy
differences between cis and trans forms of the chromophores. The
solvation energies, which are also necessary for computing abso-
lute pKa, are given in the Supplementary Information (Table S1).
Finally, Table 3 compares the computed and experimental values
of pKa of the chromophores.
Table 3
The computed (in solution) and experimental pKa values between the trans and cis
conformations.
exp
calc
Chromophore models
Isomer
pKa
pKa
TMpCA
trans
cis
cis
9.2 0.16
n/a
13.47
13.67
n/a
pB
9.9a
pCA
trans
cis
9.5 0.15
10.1 0.22
10.20
10.82
Fig. 7. The thermodynamic cycle used to compute the Gibbs free energy changes in
a
Estimated pKa of the PYP chromophore in the solvent exposed pB state [54].
aqueous solution, ꢀGsol
.

48
S. Naseem et al. / Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
is important for titration studies, we use isolated model chro-
mophores in solution instead to explore the pKa values and how
the chromophore structure influences them.
readily generated and exhibits a pKa of 9.9 [54]. The inability to
form a stable cis-TMpCA− in solution from the deprotonated con-
former suggests the cis configuration of the chromophore may be
unstable when deprotonated and does not require “help” from the
protein environment to return to the trans-configuration for the
thermally activated photocycle. Hence, the primary job of the pGꢀ
intermediate (Fig. 2) may be to maintain the chromophore in a
deprotonated state long enough to allow it to re-isomerise to the
trans-configuration in pG.
Although photo-isomerization is a commonly observed mech-
anism in the primary and secondary responses of many
photosensory proteins [2,3], its role in initiating and propagat-
ing the photocycle dynamics is not yet fully understood. The
interplay between isomerization, protonation and photoreceptor
activity was discussed by Warshel in his seminal work on the bac-
teriorhodpsin (bR) photoactive protein [76], who proposed that
the photo-isomerization of the retinal chromophore bound to bR
induces a pKa shift to drive the proton pumping activity of the sys-
tem. This hypothesis was recently revised to argue that site-specific
protein–retinal interactions between the different isomers must
also be considered [77]. Later, Ottolenghi and Sheves suggested
that only a charge redistribution or repolarization is required to
initiate the bR photocycle and that the isomerization of the retinal
chromophore is only a secondary effect [78]. A similar observation
was demonstrated by Hellingwerf and co-workers who showed
that the PYP photocycle can be weakly activated when the pCA
chromophore is substituted with a fully locked analog, whereby
isomerization is completely blocked [79].
The titration measurements in Fig. 6 estimate a pKa for trans-
pCA (9.5 0.15) that is in agreement with previous measurements
(9.35) [69–71]. Upon removal of all substitutions to the benzene
ring (i.e., for bare phenol), the pKa upshifts to 9.89 due to the
absence of the inductive withdrawing capacity of the coumaryl
tail [69,80]. To understand why the coumaryl tail preferentially
upshifts the pKa for the cis vs. trans conformer (by 0.6 pH units),
we turn to quantum chemical calculations. A 0.6 pH upshift in pKa
equates to a less than 1 kcal/mol energy in the Gibbs free energy
difference (Eq. (1)), which is difficult to reproduce quantitatively
(especially due to errors in solvation energies) with typical error
bars of electronic structure calculations [64].
5.1. Interpretation of electronic structure calculations
The gas phase deprotonation energies (ꢀE and ꢀU) for the trans
and cis isomer for each possible protonation pair of TMpCA and
pCA are contrasted in Table 1 and Fig. 8. For the biologically rel-
evant phenolate-deprotonation reaction for TMpCA/TMpCA− and
pCAH−/pCA2− systems, ꢀEtrans is smaller than ꢀEcis, which results
in a negative ꢀpKa (cis isomer is more basic) in agreement with the
titration measurements of Fig. 6 (Eqs. (3) and (4)). While TMpCA−,
TMpCA, and pCA2− are planar in both trans and cis isomer con-
figurations, pCAH− is non-planar in its cis form, with the oxygen
atoms of COO− out of plane of the phenol ring and double bond
by 53◦, likely due to steric and Coulombic repulsion (Table S2).
To compare ꢀpKa in TMpCA and in pCA, the energy differences
between trans and cis forms for each species are given in Table 2.
The energetics suggest that ꢀpKa in TMpCA is smaller than in pCA
due to the gas phase energy differences. Morevoer, the energy gap
between deprotonated and protonated TMpCA chromophores will
be always lower in energy than the one for pCAH− and fully depro-
tonated pCA2−, owing to a larger energy gap between the trans and
cis forms of pCA2− (9.5 kcal/mol). Since a stable cis-TMpCA popula-
tion could not be generated in solution, this could not be confirmed
experimentally.
The above gas-phase analysis was extended to include the sur-
rounding solvent (water) via four nonspecific continuum models:
IEF-PCM or SS(V)PE [60,61] (which are identical), COSMO [62], C-
PCM [63], and SM8 [64]. The best agreement for the computed pKa
values with the experimental results was obtained for the SM8
method, especially with the Truhlar’s M06-2X functional (Table 3).
The pKa shifts are well reproduced with the computed ꢀpKa val-
ues for TMpCA and pCA are −0.20 (Exp. −0.7) and −0.62 (Exp. −0.6),
respectively; however, absolute values are less accurate. The best
agreement is observed for pCA. Comparing ꢀpKa obtained from gas
phase ꢀꢀG (Table 1) with ꢀpKa that includes the solvation effect
(Table 3), we observe that the solvent has little effect on TMpCA
(−0.75 vs. −0.20) due to the nearly identical ꢀꢀGsol for the trans
and cis isomers, while for pCA ꢀpKa decreases by 5 pH units (−5.92
to −0.6) due to the 6 kcal/mol solvent effect discussed above (Table
S1 for ꢀꢀGsol).
To rationalize why the energy penalty for deprotonation is
higher for cis isomers of TMpCA and pCAH−, the electron charge
distribution patterns were analyzed. Fig. 9 shows the Natural
Bond Orbital charge distribution for TMpCA with three primary
structural components: (1) phenol, (2) the methylene bridge and
(3) the thiomethyl group (or carboxylate group for pCA). Some-
what surprisingly, isomerization does not considerably affect the
charge distribution pattern with similar net charges of the three
moieties observed (Table 4). However, the extra negative charge
in the deprotonated species resides more on the phenolate and
thiomethyl (or carboxylate) groups than on the bridge, hence the
Coulomb repulsion between these two moieties increases upon
deprotonation. Since these two groups are closer in the cis forms,
we attribute the energy penalty for deprotonation to the larger
Coulomb repulsion between the groups hosting excess charge in
the cis relative to the trans isomers.
The
calculated
ꢀGsol
and
ꢀꢀGsol
(i.e.,
ꢀGsol(AH)–ꢀGsol(A−)–ꢀGsol (H+)) values for each com-
pound/conformer are given in Table S1. While the solvation
energy is important for reproducing absolute pKa values, the
solvent stabilization for cis and trans isomers of TMpCA appear
to be near identical. Thus, neglecting the solvent contributions
does not strongly affect ꢀpKa of TMpCA. However, ꢀꢀGsol for
depronation reaction between the cis and trans forms of pCA
is significant (6 kcal/mol). This provides a basis for explaining
the observed pKa difference between the cis and trans isomers
of pCA with gas phase energy differences (ꢀE or ꢀU) that can
be computed far more accurately because they do not depend
on solvent models (Table 1). Although such calculations are not
expected to give quantitatively accurate pKa values, they provide
a good starting point for interpreting the experimental data.
While TMpCA is arguably a better model for the PYP chro-
mophore in solution than pCA, as it features the pCA-cysteine
thioester moeity found in PYP protein, its poor photoconversion
yield in solution does not allow for the determination of pKa of
the cis form like with pCA. This may result from two mecha-
nisms: (1) a low or no photoisomerization quantum yield and
(2) the cis-TMpCA− conformation is unstable and rapidly con-
verts back to the trans form. Very weak photoisomerization is
observed at high pH = 12 (Fig. 4B), which is in agreement with the
ultrafast pump–dump–probe signals for TMpCA− that also exhibit
weak isomerization yields (<2%) [81]. In contrast, pCA within PYP
has a significantly higher photo-isomerization yield of ∼33% [82].
Attempting to generate cis-TMpCAH at pH 7 is more successful
(Figure S2) in forming the stable cis-form, although secondary
photodegradation reactions occur to also generate free phenol
absorbing at ∼265 nm. In contrast to the solution phase attempts,
the cis isomerized chromophore within PYP in the pB state can be

S. Naseem et al. / Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
49
Fig. 8. Relative gas phase energies of the cis (red lines) and trans forms (blue lines) of TMpCA (left) and pCA (right) in different protonation states. Energy difference between
trans and cis forms for each species is indicated as is the deprotonation energy (red vertical line for cis and blue vertical line for trans) for the TMpCAH/TMpCA− reaction
(Table 2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 9. Partial natural bond orbital charges of the three moieties (phenol, methylene bridge and thiomethyl) of the trans and cis isomers of TMpCA under neutral and
deprotonated conditions.
5.2. Application to the PYP photocycle
the experimental comparison of the solvent relaxed trans and
cis-pCA molecules in solution to the pCA protein and provides a
means to explore the electronic energy contribution to different
conformations. Groenhof et al. proposed a similar pKa upshift upon
photo-isomerization of the PYP chromophore, which they predict
to be appreciably twisted with a full break in the electronic conju-
gation between the phenolate moiety and the coumaryl tail [25].
This would upshift the pKa to a value comparable to bare phenolate
(9.89) [69]. However, our measurements and calculations clearly
indicate that such a significant twist of pCA is unneeded to upshift
the pKa. This is supported by the near planar chromophore struc-
tures extracted from transient X-ray measurements on PYP by Ihee
and co-workers demonstrating that the pCA is largely planar for the
transient structures prior to protonation (Fig. 10 and Table S2) [84].
Many factors influence the condensed phase proton transfer
reactions within the highly organized protein scaffold. For pCA,
important aspects include specific distance between residue and
chromophore, fluctuation dynamics needed to promote energy
barrier crossing (ꢀG‡ in Fig. 2B), and the inherent thermody-
namics originating from the electronic structure (ꢀG or pKa) of
the chromophore [25,37,83]. For PYP, the limited solvent access
to pCA in the pG state complicates the traditional pKa determi-
nation approaches, and the characterization of pKa values for
transient populations, i.e., pR is significantly more difficult to
realize. To address this for the pre-protonation populations in PYP,
we chose to study isolated model chromophores, which allows
Table 4
Natural bond orbital (NBO) charge distributions for the pCA and TMpCA in gas phase. The differences between charges of the cis and trans forms are indicated in parentheses.
Chromophore models
Phenol(ate)
Bridge
C( O)SCH3/C( O)O(H)
TMpCA
trans-TMpCAH
cis-TMpCAH
trans-TMpCA−
cis-TMpCA−
0.02 (+0.02)
0.04
0.03 (−0.02)
0.01
−0.05 (0.00)
−0.05
−0.70 (+0.06)
−0.08 (−0.05)
−0.22 (0.00)
−0.22
−0.64
−0.13
pCA
trans-pCAH−
cis-pCAH−
trans-pCA2−
cis-pCA2−
−0.09 (+0.02)
−0.07
−0.07 (−0.04)
−0.11
−0.84 (+0.03)
−0.81
−0.97 (+0.03)
−0.94
−0.13 (−0.05)
−0.18
−0.90 (+0.02)
−0.88

50
S. Naseem et al. / Journal of Photochemistry and Photobiology A: Chemistry 270 (2013) 43–52
6. Concluding remarks
We explored the role that cis and trans configurations plays on
manipulating the pKa of the chromophore of Photoactive Yellow
Protein (PYP) by investigating isolated para-coumaric acid (pCA)
and thiomethyl-para-coumaric acid (TMpCA) models PYP chro-
mophores in solution. UV illumination converts pCA from the trans
to the cis form and the pKa of the both conformers are resolved
via pH titration coupled with electronic spectroscopy. The cis con-
former exhibits a 0.6 pH unit higher pKa than the trans form, which
is directly compared to electronic structure calculations, which
demonstrated how geometries and charge distributions. We ratio-
nalize that this higher pKa originates from Columbic interactions
between the phenolate and tail moieties, rather than changes in the
conjugation backbone of the chromophore. Future work involves
modeling the solvent explicitly using QM/MM(EFP) to compute pKa
values within Warshel’s Linear response approximation in addition
to computing ionization energies and excitation energies.
Acknowledgements
This work was supported with a Career Development Award
(CDA0016/2007-C) from the Human Frontiers Science Program
Organization to DSL and support from NSF grant: MCB-1051590.
The computational part of this study was conducted under
the auspices of the iOpenShell Center for Computational Stud-
ies of Electronic Structure and Spectroscopy of Open-Shell and
Electronically Excited Species (http://iopenshell.usc.edu) sup-
ported by the National Science Foundation through the CRIF:CRF
CHE-0625419+0624602+0625237 grant, as well as through the
CHE-0951634 grant to AIK. We like to thank Dr. Scott Corley for
aid in figures preparation.
Fig. 10. First four deprotonated structures from transient X-ray diffraction experi-
ments (PDB codes: 1TS0, 1TS7, 1TS8). In all four structures, the phenolate moiety is
near planar with the double bond in the coumaryl tail indicating that conjugation
has not been broken between the phenol conjugation system and the double bond
bridge components (Table S2).
Braslavsky and coworkers estimated from photoacoustic mea-
surements that the energy content of pR is 28 kcal/mol, which is
approximately half of the nergy from 446-nm photons at the peak of
PYP absorption spectrum [85]. The measured and calculated 0.6 pH
upshift of the pKa for the cis isomer pKa corresponds to 1 kcal/mol
energy difference (Eq. (1)), which is small compared to typical sta-
bilities of proteins. However, Groenhof estimate a ꢀG of for the
protonation of pCA at 5 kcal/mol, which would be facilitated by the
20% decrease due to the cis conformation [25]. Photoreceptor pro-
teins and signal transduction proteins, in general, are designed to
take a stimulus (i.e., photon absorption) and couple it to a macro-
scopic deformation capable of propagation signal transduction in
vivo; these proteins cannot be too strongly stabilized in deep energy
wells.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jphotochem.
2013.06.019.
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