 |
INTRODUCTION |
PYP1 (1) presumably is a
blue-light photoreceptor from the purple sulfur bacterium
Ectothiorhodospira halophila (2), which shows
many similarities with the archaeal sensory rhodopsins (3, 4), although
PYP contains 4-hydroxycinnamic acid as its chromophore (5, 6) and is
water-soluble. Activation of PYP function proceeds through
light-induced trans/cis isomerization of the 7,8-vinyl bond
of its chromophore (7, 8). This ultimately leads to a modulation of the
direction of rotation of the flagella of the photoreceptor-producing
cell. The chromophore is present in the anionic form in the ground
state (pG) of PYP (9, 10). The anionic phenolate is buried within the
hydrophobic core of PYP and is stabilized via a hydrogen-bonding
network involving the amino acids Tyr-42, Thr-50, and (protonated)
Glu-46 (9). Photoactivation of PYP initiates a photocycle containing
several transient intermediate states. In a very short time,
red-shifted intermediates are formed, of which pR has the longest
lifetime. It decays bi-exponentially (with rate constants of
4·103 and 8·102 s
1 (4)) into
a blue-shifted intermediate (pB). The latter is the longest living
intermediate of this cycle (
= 0.15 s (3, 4, 11, 12)) and
is also referred to as I2 (e.g. see Refs. 3 and
12). The photocycle of PYP can be observed in a wide range of pH
values, although significant effects on the rates of the transitions
are observed (13, 14).
ApoPYP can be produced heterologously in Escherichia coli
(15) and can subsequently be converted to functional holoprotein through reconstitution with activated derivatives of its chromophore (16, 17). This capability has made PYP available in sufficient amounts
to allow detailed analysis of its photocycle using biophysical techniques like x-ray diffraction, Fourier transform infrared, Raman,
NMR, transient absorption, and fluorescence spectroscopy (see
e.g. Refs. 8, 10, 12, and 16-22), and it has led to detailed insight into the photocycle characteristics of PYP.
One aspect that has not yet been clearly resolved, however, is the
involvement of net proton uptake/release during the transitions of the
PYP photocycle. In an early study (23) it was reported that during the
lifetime of pB, PYP takes up a proton from solution. In a subsequent
paper (13) the observation of proton uptake in parallel with pB
formation was confirmed. The proton uptake was interpreted as the
result of protonation of the chromophore after its exposure to solvent
(8).
In pR, at 77 K, the hydrogen bond between the chromophore and Glu-46
remains intact, as has been demonstrated with Fourier transform
infrared (18) and x-ray diffraction (24). This finding led to the
proposal (18) that isomerization of the 4-hydroxycinnamic acid
chromophore occurs through a two-bond isomerization reaction, which is
characterized by a rotation of the carbonyl group of the chromophore
around the long axis of the 4-hydroxycinnamic acid. In addition, it was
proposed that during pB formation, proton transfer occurs from Glu-46
to the chromophore. Subsequent extension of these Fourier transform
infrared analyses, using isotope enrichment and variants of PYP
obtained through site-directed mutagenesis, provided further evidence
supporting this proposal (22). However, time-resolved x-ray
crystallography of PYP in the nanosecond time domain indicates that at
room temperature the hydrogen bond between the side chain of Glu-46 and
the phenolic oxygen of the chromophore is already disrupted in the pR
intermediate (25). Therefore, the pathway of proton transfer during the
PYP photocycle still is an unresolved issue. The two most extreme views
are that this proton transfer may take place directly, within the
chromophore binding pocket inside the protein, or indirectly, through
the bulk solvent.
As the chromophore of PYP during pB formation is protonated (8) and
Glu-46 is deprotonated (18, 22), net proton uptake by the protein while
it progresses through the photocycle is difficult to understand.
Therefore, we have investigated these reversible (de)protonation
reactions, using not only transient absorption spectroscopy, in
combination with pH indicator dyes, but also transient pH measurements
with a sensitive combination pH electrode. The latter technique has
been combined with simultaneous absorption measurements. Deprotonation
of bacteriorhodopsin, present in the form of purple membranes, served
as a control to test the sensitivity of the set-up for pH measurements.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant apoPYP and two variants obtained
through site-directed mutagenesis were produced heterologously in
E. coli, as described previously (15). ApoPYP was
reconstituted with the anhydride derivative of 4-hydroxycinnamic acid
according to Imamoto et al. (16). The obtained PYP was used
after removal of its polyhistidine tail. Purple membranes were kindly
provided by D. Oesterhelt (MPI, Martinsried, Germany).
4-Hydroxycinnamic acid was obtained from Sigma. All other materials
were reagent grade and were obtained from commercial sources.
Site-directed Mutagenesis--
The PYP variants E46Q and H108F
were made by site-directed mutagenesis using the mega-primer method
(26). The gene products were first screened, using restriction
analyses, and mutagenesis was subsequently confirmed by DNA sequencing.
Spectroscopy--
UV/Vis static and transient absorption spectra
were recorded with a model 8453 Hewlett Packard diode array
spectrophotometer (Portland, OR), which has a time resolution of
0.1 s. Typically UV/Vis spectra from 250 to 550 nm were recorded
every 0.1 s.
Nanosecond Time-resolved Absorption
Spectroscopy--
Laser-induced transient absorption spectra were
measured with a system composed of a Continuum Surelite I-10 YAG laser
(output intensity 140 mJ at 355 nm), a Continuum Surelite OPO (output range 410-2200 nm, set at 446 nm), and a LP900 Spectrometer, custom made by Edinburgh Instruments Ltd. (Edinburgh, UK). The spectrometer contains a 450-watt short arc Xe lamp in combination with a pulsed power supply and a Peltier cooled CCD camera (Wright Instruments). The
time resolution attainable with this set-up is 10 ns.
pH Measurements--
pH measurements were carried out with a
Mettler Toledo micro(combination)-electrode (InLab 423) connected to a
Dulas Engineering amplifier (input impedance > 1013
ohms). The amplified signal was fed into a linear strip-chart recorder
(Kipp & Zonen, Delft, The Netherlands, type BD41). A battery driven
back-off box was used to decrease the signal to the appropriate size.
pH changes were converted into moles of protons by calibration with
microliter amounts of 2.5 mM oxalic acid. Simultaneously,
the absolute pH of the solution was visualized on the display of the pH
meter. The electrode was calibrated with calibration buffers of pH
4.01, 6.98, and 9.18 (Yokogawa Europe BV, Amersfoort, The Netherlands).
The electrode signal was usually recorded at 0.025-0.1 pH units full
scale sensitivity.
Simultaneous Transient Absorption and pH
Measurements--
Absorption and pH signals were measured
simultaneously by placing a "Kraayenhof vessel" (27) in the sample
compartment of the Hewlett Packard 8453 spectrophotometer. Two of the
four available ports of the vessel were used for the measuring beam of
the spectrophotometer, and a third one was used for the combination pH
electrode. Measurements were carried out at room temperature (between
291 and 293 K). Although this vessel is equipped with Peltier
temperature control, this was not used to reduce temperature artifacts
(see "Results"). Continuous actinic illumination was provided
through the fourth port of the vessel by a Schott KL1500 light source
(containing a 150-watt halogen lamp). PYP was routinely used at a
concentration of 33 µM in a working volume of 1.8-2 ml
using an unbuffered solution containing 1 M KCl.
 |
RESULTS |
Laser-induced Transient Absorbance Measurements--
As a first
test for the occurrence of reversible (de)protonation reactions linked
to the photocycle of PYP, transient absorption spectra were recorded in
an unbuffered suspension of PYP at pH 6 in the presence of the pH
indicator dye bromcresol purple. In complete agreement with earlier
observations (23, 13), it was observed that at this pH PYP caused a
reversible alkalization of the medium upon actinic illumination, which
in time parallels the formation and decay of the pB intermediate (Fig.
1A). Because it has been
demonstrated that PYP exposes hydrophobic contact surface when it is
present in the pB state (28, 29) and pH indicators might bind to such
sites, similar measurements were performed in a solution buffered with
50 mM MES buffer (pH 6.0). Fig. 1B shows that
under these conditions bromcresol purple does not reveal any transient
absorption signal, which implies that no pH change nor any artifact
based upon nonspecific binding of the indicator to the pB form of PYP
occurs. Because E. halophila is an alkaliphilic bacterium
and therefore most probably has a slightly alkaline cytoplasm (30), we
next tested the universality of these observations by performing
similar experiments with the pH indicator dye cresol red, which has a
pK of 8. The analyses given in Fig. 1, panel C
shows, however, that at pH 8 significant pH changes are not observed,
but the photocycle intermediate is formed in an amount comparable with
the experiment performed at pH 6 (Fig. 1A).

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Fig. 1.
Reversible protonation of PYP as studied with
laser-induced transient absorption spectroscopy. PYP (12 µM) in 2 ml of an unbuffered solution containing 1 M KCl and either 100 µM bromcresol purple at
pH 6 (A and B) or 100 µM cresol red
at pH 8 (C). In B, 50 mM MES buffer
(pH 6) was added. Laser-induced transient absorbance spectra were
recorded in the indicated wavelength region after 61 µs (trace
1), 3.9 ms (trace 2), 250 ms (trace
3), and 1 s (trace 4). The
asterisk (*) refers to a calibration experiment in which the
difference (bromcresol purple, A595, cresol red A575) spectrum is
recorded, induced by the addition of 10 nmol of protons (using 2.5 mM oxalic acid).
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Simultaneous Measurements of pH and Absorbance
Transients--
Because analysis of PYP (de)protonation with the use
of pH indicator dyes is limited to the region around the pK
of such dyes, we decided to use direct measurement with a pH electrode
to investigate the pH dependence of the reversible protonation, as
observed with PYP at pH 6. Initial experiments to test the sensitivity
of the set-up were performed with unbuffered solutions of purple
membrane (containing bacteriorhodopsin at a concentration of 130 µM, in 3 M KCl). These experiments revealed
that the pH signal showed random noise (2.5·10
4 pH
units), on top of a very slowly decaying drift, and confirmed the pH
dependence of proton release by bacteriorhodopsin and the stimulation
of this proton release by increasing salt concentrations (data not
shown; see also Ref. 31).
Initial experiments with PYP revealed that pH changes could also be
observed in unbuffered solutions containing micromolar concentrations
of PYP using the pH electrode (Fig. 2).
Surprisingly, the sign of the PYP-dependent pH change was
dependent on the absolute pH (compare panels A and B of Fig.
2), whereas the sign of the drift in the pH, caused by the
light-induced heating, was not. The latter was shown in experiments in
which PYP was replaced by an equivalent amount of bovine serum albumin
(data not shown). For quantitative evaluation of these experiments, it
is important to distinguish between these heating artifacts by the
actinic illumination and actual proton uptake/release by PYP. The
Peltier temperature control of the Kraayenhof vessel responded slowly to the light-induced change in temperature of the solution in the
vessel, thus causing an oscillating signal from the pH electrode (data
not shown). Therefore, this temperature control system was switched off
in all subsequent experiments, and the PYP-mediated pH changes were
calculated from the pH recordings after correction for the
light-induced heating of the sample, which caused an apparent change in
the drift of the pH electrode (Fig. 2, A and B).
The response time of the electrode (t0.9) to
additions of small amounts of oxalic acid is ~10 s, because of the
mixing time in the vessel. The pH response upon illumination, however,
is immediate (i.e. within 1 s). The contribution of PYP
to the light-induced pH change can therefore be calculated from the
measured pH changes by back extrapolation to the time of switching on
the light, as shown in Fig. 2, A and B. Under
most conditions, however, very little correction for this light-induced
heating was necessary (Fig. 2C). The minimal amount of
actinic illumination required for the maximal extent of pB formation
was determined at pH 4.5 by parallel absorbance measurements (see
below). The intensity of the actinic illumination was adjusted by
variation of the voltage on the Schott KL1500 light source.

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Fig. 2.
Typical recordings in the set-up for
simultaneous pH and absorbance measurements. Details of the set-up
are described under "Experimental Procedures." Panels A
and B show a typical recording of the pH signal at pH 6.07 (A) and 9.95 (B). In panel C, the pH
signal is complemented with a recording of the absorbance signal. The
series of spectra from which the latter recording was derived shows an
authentic isosbestic point at 386 nm (and shows zero absorbance > 550 nm). 1 and 2, actinic light on and off,
respectively; 3, graphical method to determine the extent of
(de)protonation of PYP upon illumination by back-extrapolation;
4a and 4b, addition of 10 and 20 nmol,
respectively, of H+.
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Absorbance changes of PYP (from 250 to 550 nm) were recorded every
0.1 s, simultaneously with the pH measurements. There was no
measurable interference of the actinic illumination (as we concluded
from experiments at slightly alkaline pH (pH ± 8) with wild type
PYP and in the alkaline pH range with the E46Q mutant protein (see
Figs. 3 and 5)). With this set-up, rates
of (de)protonation and pG bleaching and recovery can be measured that
are slower than ~5 s
1. Fig. 2C shows the
result of a typical simultaneous recording of the pH and the absorbance
(of which only the value at 446 nm is plotted).

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Fig. 3.
Reversible (de)protonation of PYP at acidic,
neutral, and alkaline pH as measured with a combination pH
electrode. The pH of the unbuffered solution of PYP was adjusted
with small aliquots of concentrated HCl and/or KOH. Transient pH
changes were analyzed as described under "Experimental Procedures"
and in the legend to Fig. 2. , extent of pB accumulation as
calculated from the absorbance signal; , extent of the light-induced
change in the degree of protonation of PYP. Positive numbers
refer to proton uptake by PYP.
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The pB intermediate is by far the longest living intermediate of PYP at
all pH values tested so far. Therefore, to a good approximation
(compare Refs. 32 and 33), the photocycle of PYP can be simplified to a
two-state photocycle in which only the ground state pG and the
intermediate pB are taken into consideration. The amount of pB
formation can then be calculated from these transient absorbance
measurements by taking
in which the superscripts l and d refer to
the steady state reading of the absorbance at 446 nm in the light and
the dark, respectively.
By analyzing the changes in pH induced by illumination of PYP in the
range of pH 4-11, it was revealed that PYP transiently takes
up protons at acidic pH but releases protons at
alkaline pH (Fig. 3). In addition, in a wide pH range at slightly
alkaline pH only a very small (de)protonation was observed. The
inversion of the signal of the light-induced pH change could even be
effected by having the sample (making use of the slight pH drift) go
through the pH transition range (~pH 7.8). Changes in the protonation level of PYP cannot be measured outside of the pH range from 4 to 11, because at a pH < 4.0 significant amounts of pBdark
are formed (14), and at pH > 11 the thiol ester linkage in PYP
starts to hydrolyze at a significant rate (34). The noise on the signal from the pH electrode increases slightly at alkaline pH, but it is
always less than 0.05 H+/PYP in the set-up used.
Calculation of the amount of pB formed under these conditions shows
that it varies between 85 (at acidic pH) and 20% (at neutral pH). The
variation in this amount is primarily because of the pH dependence of
the rate of the pG recovery reaction (13, 14). These data can then be
used to calculate the number of protons taken up or released per
molecule of pB formed. This calculation leads to the conclusion that
this number is ~1 at low pH and >1 at high pH. The calculations at
alkaline pH, however, are complicated by changes in the spectral
characteristics of pB (see below).
The Spectrum of pB at High pH--
One of the groups that may
contribute to the inversion from reversible proton uptake (at low pH)
to reversible proton release at high pH in PYP is the phenolate moiety
of the chromophore. Evidence suggests that the chromophore of PYP is
exposed to solvent when the protein is in the pB state (8, 23). Because
phenolates generally have a pK around 9, one might
anticipate that the chromophore in the pB state could not be protonated
at pH values above 9. We therefore analyzed in detail the light-induced
absorbance difference spectra of PYP in the alkaline pH range. The
results obtained indeed show a transition from the known pB difference
spectrum at neutral pH (with an absorbance maximum at 355 nm (4)) to a
difference spectrum with a component that absorbs maximally at 420 nm,
which is indicative of the presence of a phenolate anion (Fig.
4 and Ref. 34). A similar transition in
the spectrum of pB was also detected with laser-induced transient
absorbance measurements (data not shown). The latter experiments,
however, are more complicated because they require (for technical
reasons) that the sample be kept at the very alkaline pH for a longer
period, which causes interference with the measurements through thiol ester hydrolysis (5, 6, 33) of PYP. The pH dependence of this presumed
phenol/phenolate transition has a surprising feature. When the
absorbance difference data at 416 nm are fitted with the
Henderson-Hasselbalch equation, a pK > 10 is
calculated, significantly higher than the pK of 8.8 measured
for the phenolic group of 4-hydroxycinnamic acid and related model
compounds (17). We did obtain this latter value upon titration of the
chromophore of PYP when the protein was dissolved in 8 M
guanidinium-HCl (data not shown).

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Fig. 4.
PH dependence of the spectral characteristics
of the long-lived (blue-shifted) photocycle intermediate of PYP.
Absorbance difference spectra ("after-minus-before" actinic
illumination) of wild type PYP at pH 7.47 ( ) and 10.91( ). From
the absolute spectrum of the illuminated PYP sample, a scaled spectrum
of the ground state (i.e. pG) of PYP was subtracted, to
remove the contribution of this state from the difference
spectrum.
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Analysis of Site-directed Mutants of PYP--
To obtain more
insight into the nature of the specific groups of PYP that contribute
to the light-induced reversible (de)protonation, variants were
constructed through site-directed mutagenesis. The pH dependence of the
pH changes caused by illumination of PYP suggests that (a)
histidine(s) is involved in the light-induced proton uptake at acidic
pH. PYP contains two histidines, His-3 and His-108. As His-3 is
positioned close to the relatively exposed N terminus of the protein
(see also under "Discussion"), we selected His-108 and changed it
into phenylalanine. The H108F protein indeed shows a similar pH
dependence of the reversible protonation at alkaline pH and a
decreased reversible proton uptake at acidic pH (Fig.
5).

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Fig. 5.
PH dependence of the light-induced reversible
(de)protonation of wild type PYP and the H108F and E46Q mutant
forms. Data as shown in Fig. 3 for wild type PYP were recorded for
the H108F and E46Q proteins as well and plotted for all three as the
calculated H+/pB molar ratio: , wild type PYP; ,
E46Q; , H108F. The lines through the data points for wild
type PYP and H108F are fitted curves, based on the
Henderson-Hasselbalch equation, using n (the number of
protons involved in the transition) = 1. The pK values
obtained for wild type PYP and H108F are 6.6 and 5.5 for the titration
between pH 8 and 5, respectively. For the titration between pH 8 and
10.5, these values are >10.2 and >10, respectively. When the data
points for wild type PYP between pH 5 and 8 are fitted without fixing
the n value, the fit yields n = 1.2 and
pK = 6.6.
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In the E46Q mutant of PYP (13, 22), direct protonation of the
chromophore by its hydrogen-bonding partner in the chromophore-binding pocket cannot take place. It may therefore be anticipated that in this
derivative, upon formation of pB a proton has to be taken up from the
bulk aqueous phase. The results presented in Fig. 5 confirm that the
extent of proton uptake per molecule of pB has indeed increased in E46Q
(Fig. 5). Unfortunately, however, the reduced stability and altered
photocycle kinetics of this protein preclude analyses over the same pH
range as with wild type PYP, because with the E46Q protein significant
amounts of a pB-like intermediate are formed already at pH values below
6. A pK of 4.2 was measured for the transition between pG
and this pB-like intermediate of the E46Q protein. This latter protein also shows very little pB formation at alkaline pH because of the
accelerated rate of recovery of pG (data not shown).
 |
DISCUSSION |
The results obtained fully confirm the reported protonation of PYP
at slightly acidic pH (13, 23). Binding of the pH indicator dye
bromcresol purple to the hydrophobic contact surface that is exposed by
PYP in its pB state (28, 29, 35) can not explain the results obtained.
This conclusion can be reached from Fig. 1B, which shows
that the reversible pH changes disappear upon buffering of the PYP
solution. The extent of protonation (~1 H+/pB, measured
at 100 µM bromcresol purple) also agrees with the value
reported by Meyer et al. (13).
The sensitivity of the pH electrode and the stability of PYP against
extreme pH values allowed us to record light-induced pH changes in a
wide pH range. Parallel measurement of absorbance transitions then
allow one to calculate the extent of this (de)protonation in terms of
the H+/pB ratio, if a simplified photocycle scheme is used
(32, 33), in which only two intermediates (i.e. pG and pB)
are considered. This simplification is supported by steady state
"light-minus-dark" absorbance difference spectra of these samples.
Significant amounts of intermediates other than pG and pB were not
detected (note, however, that at very high pH the spectrum of the pB
intermediate changes (see below)). The amount of pB intermediate formed
is calculated from the extent of pG bleaching, as the latter
intermediate can be detected more accurately. The pG intermediate has a
2-3-fold higher extinction coefficient (3, 4) and absorbs in a more favorable wavelength region with respect to the signal to noise ratio
of our detection systems (see e.g. Fig. 1).
Analysis of the steady state pH changes revealed that PYP in the pB
state can show reversible net proton uptake from as well as
net proton release to the solvent. At slightly alkaline pH (i.e. 7.5-8.5), however, neither significant proton uptake
nor proton release occurs in the pB state of PYP. This absence of photocycle associated pH changes is in line with simultaneous proton
uptake by the chromophore and proton release by Glu-46. Because these
counteracting processes are also expected to occur at and below pH 6 (e.g. see Refs. 13, 18, and 22), most likely an additional
group(s) is involved in this net proton uptake at low pH. A fit of the
data for wild type PYP revealed that the pK of the
transition from "absence of proton uptake/release" at pH 8 to
"proton uptake" at pH < 5 has an apparent pK of
6.6 (assuming n = 1 in the fit). Because of the
numerical value of this apparent pK, the two histidines of
PYP (His-3 and His-108) are candidates to provide the side chain to
mediate this proton uptake. His-3 is located close to the protein
surface, and in the crystal structure it appears not to be involved in
the hydrogen bonding responsible for the closure of the N-terminal
helical lariat (9). Furthermore, in solution the five residues at the N
terminus of PYP are in a disordered state (36). Therefore, in the pG
state of PYP, His-3 is already exposed to solvent and thus is not
likely to contribute to the observed proton uptake at acidic pH.
His-108 is buried inside the hydrophobic core of PYP at a considerable distance from the chromophore (>15 Å). Analysis of the H108F
derivative (Fig. 5) showed that His-108 does indeed contribute to the
light-induced pH changes at acidic pH. Analysis of the titration curve
of the H108F mutant with the Henderson-Hasselbalch equation gives an apparent pK of 5.5. However, because of the uncertainty
about the end point of this titration, we can not unambiguously
interpret this latter value.
Multiple groups with a high pK (>10) must be involved in
the reversible deprotonation of PYP. For these Tyr-42, Tyr-118, and Arg-110 are candidates, as they are buried inside the hydrophobic core
of PYP in the pG state and are expected to have a pK of
~10 (37). However, because the protein environment may significantly modulate the pK of functional groups within a protein, other
groups may be involved as well.
In this study we have used steady state accumulation of the pB
intermediate by continuous illumination to measure the net change in
protonation level of this intermediate rather than selecting a range of
pH indicators that could be used for transient absorption measurements
of pH changes from pH 4 to 11. The results obtained with the E46Q
protein, however, show the limitations in this approach. Because of the
altered recovery kinetics that are displayed by this protein (see also
Ref. 13), no significant amount of pB intermediate accumulates during
actinic illumination of this protein at alkaline pH values. The use of
monochromatic actinic light of 450 nm to prevent interference by the
light-induced branching reaction from pB back to pG (32) did not
significantly increase the amount of pB of the E46Q protein in the
steady state in the light.2
Kinetic analyses of the simultaneously measured pH and absorbance
signals are possible only in a limited time domain. These analyses,
however, have not revealed discrepancies in the rate of dark recovery
of the two signals. The latter would have provided evidence for
heterogeneity of the pB state. Such heterogeneity may be anticipated
because in the recovery reaction of the ground state of PYP, several
partial reactions have to take place, like re-isomerization of the
chromophore, refolding of the protein to the pG conformation, and
re-formation of the hydrogen-bonding network between the chromophore
and Glu-46 and Tyr-42. The kinetics of the recovery reaction of wild
type PYP and the E46Q protein were in general agreement with previously
obtained results (e.g. Refs. 3, 4, 11, 13, and 14).
Whereas the pK of the chromophore of PYP is tuned to a very
low value in its pG state (i.e. pK 2.7 (14)), in
the pB state it is tuned to a very high value (i.e.
pK > 10). In agreement with this, it was observed that
the position of the absorbance maximum of this presumed anionic form of
the chromophore in pB, is tuned measurably from its position in aqueous
solvents (i.e. 400 nm (34)) to ~425 nm. Hydrogen bonding
between the phenolate moiety and Arg-52 (8) may contribute to this
tuning of pB. Furthermore, the results of this study are in line with
kinetic, mass spectrometric, and NMR analyses on PYP, which also
suggests that this protein shows a partial unfolding upon formation of its presumed signaling state pB (28, 29, 35).
Assuming that the (de)protonation reactions in PYP in a crystalline
matrix are the same as in solution, we must conclude that Glu-46 in the
pB state at neutral pH will be ionized and present in a relatively
hydrophobic pocket. In view of the differences noted between the
structure of the pB intermediate as measured with NMR and with Laue
diffraction (8, 35), this assumption is not necessarily true; but if it
is true, it may be a significant factor in the free energy that drives
pB back to the ground state pG.
The current results, recent data on PYP (e.g. Refs. 24, 25,
and 35), and the effects of high pressure on protein structure (38)
lead to the following view of the photocycle of PYP. Light initiates a
two-bond isomerization of the p-coumaryl chromophore in PYP,
which then leads to proton transfer from Glu-46 to its phenolate
moiety. This process in turn leads to significant unfolding of the
protein and exposure of titratable groups to the solvent, all of which
is reversed in the recovery reaction from pB to pG. Tuning of the
pK of the functional groups involved, possibly via reversible (de)hydration of the chromophore in the binding pocket, may
be at the heart of this mechanism.
 |
CONCLUSION |
The results presented in this study demonstrate that transient
(de)protonation of PYP in its pB state is not observed at neutral to
slightly alkaline pH. This conclusion provides further support for the
assumption that the chromophore in the pB state is most likely
protonated by the proton from Glu46.
Extending this interpretation to the data obtained at acidic and
alkaline pH then leads to the conclusion that the formation of pB is
paralleled by a significant solvent exposure of functional groups that
are buried inside the protein in the pG state.