(Received for publication, August 9, 1994; and in revised form, November 18, 1994)
From the
Photosystem II, the photosynthetic water oxidizing complex,
contains two well characterized redox active tyrosines, D and Z. D
forms a stable radical of unknown function. Z is an electron carrier
between the primary chlorophyll donor and the manganese catalytic site.
The vibrational difference spectra associated with the oxidation of
tyrosines Z and D have been obtained through the use of infrared
spectroscopy (MacDonald, G. M., Bixby, K. A., and Barry, B. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11024-11028). Here,
we examine the effect of deuterium exchange on these vibrational
difference spectra. While the putative C-O vibration of stable
tyrosine radical D downshifts in H
O, the
putative C-O vibration of tyrosine radical Z does not. This
result is consistent with the existence of a hydrogen bond to the
phenol oxygen of the D radical; we conclude that a hydrogen bond is not
formed to the Z radical. In an effort to identify the amino acid
residue that is the proton acceptor for Z, we have performed global
N labeling. While significant
N shifts are
observed in the vibrational difference spectrum, substitution of a
glutamine for a histidine that is predicted to lie in the environment
of tyrosine Z has little or no effect on the difference infrared
spectrum. There is also no significant change in the yield or lineshape
of the Z EPR signal under continuous illumination in this mutant. Our
results are inconsistent with the possibility that this residue,
histidine 190 of the D1 polypeptide, acts as the sole proton acceptor
for tyrosine Z.
PSII ()is one of two chlorophyll-containing reaction
centers that cooperate to transfer electrons from water to
NADP
in oxygenic photosynthesis. PSII carries out the
light-driven oxidation of water and reduction of plastoquinone; this
enzyme is made up of at least eight subunits (for review, see (1) and (2) ). The hydrophobic D1 and D2 polypeptides
bind most of the prosthetic groups that are involved in electron
transfer (3) .
To initiate electron transfer in photosystem
II, the primary chlorophyll donor, P, transfers an
electron to a pheophytin molecule after photoexcitation. The pheophytin
intermediate acceptor reduces a bound molecule of plastoquinone, the
acceptor Q
. Q
in turn reduces a second quinone,
Q
. Unlike Q
, Q
can function as a
two-electron acceptor. The chlorophyll cation radical,
P
, is reduced by a redox active
tyrosine, Z, which in turn oxidizes the manganese cluster, the
catalytic site of water oxidation. Photosystem II also contains a redox
active tyrosine, D, which forms a stable radical, D
,
and has an undetermined function (for review, see (4) and (5) ). Recently, a new signal from a redox active tyrosine, M,
has also been observed in three site-directed mutants of
PSII(6, 7, 8) . The M
tyrosine radical has an unusual structure(8) .
The
current model for the location of redox active tyrosines D and Z is
based on the assumption that they are located with approximate C symmetry with respect to the primary chlorophyll donor (9, 10) . The crystal structure of the bacterial
reaction center shows such C
symmetry in the placement of
some of its prosthetic groups(11) . Site-directed mutagenesis
experiments are consistent with this symmetric model for the location
of the redox active tyrosines in PSII. These studies suggest that
tyrosine D is located at the 160 position of the D2 subunit (12, 13) and that tyrosine Z is located at position
161 of the D1 subunit(6, 14, 15) .
In
spite of this putative C symmetry, D and Z have different
functions and physical properties. For example, the midpoint potentials
of these 2 tyrosines are predicted to differ by 240 mV. The rise and
decay kinetics of the radicals are also dramatically different. In
addition, Z
is more accessible to reduction by
exogenous donors than D
(for review, see (16) ).
The EPR signal of D is observable
for hours after illumination, and this signal was assigned to a neutral
tyrosine radical by isotopic labeling(17) . The EPR signal of
Z
is observed under illumination in manganese-depleted
preparations, and isotopic labeling has also been used to assign this
signal to a neutral tyrosine radical (18) . One spin of
D
is obtained per reaction center; up to one
Z
spin per reaction center can be observed under
illumination in manganese-depleted preparations (for review, see (16) ). The EPR lineshapes are broadened because of
immobilization of the radicals (19) and are similar, but not
identical, to each other(18) .
The protein environmental
factors that influence the function of these redox active tyrosines are
of interest. For example, a hydrogen bond to the phenol oxygen could
influence the midpoint potential of a redox active tyrosine, since the
residue deprotonates upon oxidation(20, 21) .
D is known to be a hydrogen-bonded radical from
magnetic resonance studies(22, 23, 24) . Due
to difficulty in trapping Z
, such a study has not been
reported for tyrosine Z.
Site-directed mutagenesis has been used to
explore the effect of changes in the environments of D and Z. In the
structural models of PSII that have been developed, histidine 189 of D2 (Synechocystis numbering scheme) is located near tyrosine 160,
which is the putative location of redox active tyrosine D. A second,
C symmetry related histidine (histidine 190 of D1) is
located near tyrosine 161 of D1, which is the putative Z location (9, 10) . Recent site-directed mutagenesis studies
have shown that some substitutions at histidine 189 of D2 lead to a
loss of the characteristic 24 G D
EPR signal and to
the appearance of a narrow structureless
signal(25, 26) . However, the EPR signal of tyrosine
Z
is not affected by substitutions at histidine 190 (27) .
The site-directed mutagenesis experiments described
above provide evidence for a structural asymmetry that is imposed on
the redox active tyrosines D and Z by their protein environment. Two
lines of spectroscopic evidence also point to such a structural
difference. First, isotopic labeling and EPR spectroscopy have shown
that the EPR lineshapes of D and Z
differ when the radicals are 3,5-deuterated(18) . Second,
isotopic labeling and difference Fourier transform infrared
spectroscopy have shown that the vibrational difference spectra
associated with the oxidation of D and Z are also significantly
different from each other(21) . Through isotopic labeling of
tyrosine, vibrational modes in the difference (light minus dark)
spectrum have been assigned to Z and D and to Z
and
D
(21) . This procedure distinguishes
contributions from chlorophyll and plastoquinone from tyrosine, since
mass spectral analysis shows that these compounds are
unlabeled(17, 21) . It was suggested that the observed
spectral differences could be caused by a difference in the strength of
a hydrogen bond. A difference in hydrogen bonding to D and Z has also
been suggested in order to explain the results of the site-directed
mutagenesis experiments described above(27) .
To test the
hypothesis that Z and D differ in the strength of a hydrogen bond, we
report here the results of DO exchange and infrared
spectroscopy. Difference infrared spectroscopy offers a direct way to
determine if Z
and D
are hydrogen
bonded, since the C-O stretching vibration of a hydrogen-bonded
radical should downshift upon deuterium exchange. In addition,
difference infrared spectroscopy provides a method with which the
proton acceptor can be identified. If a residue is reversibly
protonated and deprotonated upon charge separation and recombination,
its vibrational signature should also appear in the light minus dark
difference spectrum. In order to learn more about the environment of
this redox active amino acid, we have measured the effect of
D
O exchange,
N substitution, and a mutation at
histidine 190 D1 on the difference infrared spectrum associated with
the oxidation of tyrosine Z.
Photosystem II particles were
purified from Synechocystis cells by the procedure previously
described (28) with the following modifications: a 400-ml
gradient was employed for elution from the first fast flow Q (Pharmacia
Biotech Inc.) column, and the sample was not precipitated after the
second Mono Q (Pharmacia) column. The lauryl maltoside
(dodecyl--D-maltoside) was from Anatrace (Maumee, OH).
Since the HQ190D1 mutant cells were light sensitive, all biochemical
manipulations with the HQ190D1 mutant were performed in the dark.
Manganese was removed from all PSII samples (except the HQ190D1 mutant,
see below) as described(20) , except that incubation in
NH
OH was only 40 min. The HQ19OD1 PSII samples were not
manganese-depleted, since without this treatment the yield of
Z
under steady state illumination was similar to the
yield obtained in control manganese-depleted PSII samples. This argues
that this mutant cannot assemble a functional manganese cluster, in
agreement with an earlier characterization of the HF190 and HY190
mutants in a eukaryotic green algae, Chlamydomonous
reinhardtii(27) .
For most experiments, purified PSII
particles were concentrated by binding the preparation to a Mono Q 5/5
column(31) . The column was then washed with 15 ml of buffer A
(50 mM MES-NaOH, pH 6.5, 20 mM CaCl,
0.05% lauryl maltoside, 15 mM NaCl). The column was
inverted(31) , and the sample eluted in a concentrated form
with high salt buffer (buffer A with 275 mM NaCl). The sample
was dialyzed overnight against 5 mM MES-NaOH, pH
6.0(20) , and was frozen at -80 °C before use.
For
deuterium exchange, the concentration, dialysis, and freezing of the
PSII sample were carried out in the same manner as described in the
paragraph above except that DO buffers were used (99.9%,
Cambridge Isotopes). The fraction of amide groups exchanged by this
procedure is approximately 20% based on the deuterium-induced change in
ratio of the amide II and amide I bands in the infrared absorption
spectrum(32) . This number is in good agreement with the extent
of exchange achieved for bacteriorhodopsin by dehydration of the sample
and rehydration with D
O(32) . The pD of the
D
O buffers was the same as the pH values reported above.
The pD is reported here as the uncorrected pH meter reading (33) and was adjusted using sodium deuteroxide (99.5%,
Cambridge Isotopes). The D
O and H
O samples were
different halves of the same pooled PSII preparation.
For some experiments, samples were concentrated in Centricon 100 concentrators (Amicon, Beverly, MA).
DO exchange has predictable effects on the
infrared absorption spectrum of proteins(34) . As expected for
an extensively
helical protein, the PSII amide I band (1654
cm
), which arises mainly from the C=O stretch
of the peptide bond(34) , is not appreciably affected by
exchange (data not shown). However, the amide II vibration (1549
cm
) decreases in intensity after H-N
deuteration due to the uncoupling of the C-N stretching and
N-H bending modes(34) . New intensity, due to the
C-N vibration, appears at 1456 cm
(amide II`).
In Fig. 1A, we present the difference infrared
spectrum associated with the oxidation of tyrosine Z in control (dashedline) and deuterium exchanged (solidline) PSII samples. The dark adaptation time between
illumination and accumulation of the dark spectrum is 8 min. Such a
short dark delay permits the decay of Z, but
D
will not significantly decay and will not contribute
to these spectra. The data presented in Fig. 1A differ
slightly from our earlier reported spectra because the 10% PSI
contamination previously obtained (21) has been reduced to an
undetectable level. As expected, only small frequency shifts are
observed in the amide I region after exchange (compare solid and dashedlines). In contrast, a negative line
at 1559 cm
and a positive line at 1535
cm
downshift approximately 90 cm
.
This is the approximate downshift of the amide II band in the infrared
absorption spectrum after deuterium exchange, and this result provides
evidence for the assignment of these positive and negative lines in the difference infrared spectrum to amide II. Deuterium exchange
experiments on
N-labeled samples confirm that there is a
negative contribution from amide II` in the 1470 cm
region after D
O treatment. The new shifted amide II`
lines are broader than the corresponding amide II modes. This is
possible since H-N deuteration changes the normal mode
description(34) .
Figure 1:
Difference (light minus dark) infrared
spectra of purified PSII samples from the cyanobacterium, Synechocystis sp. PCC 6803. In each panel, the dashedline is the control spectrum. In A, PSII was
DO-exchanged (solidline); in B,
PSII was globally labeled with
N (solidline); in C, PSII was isolated from the HQ190D1
mutant (solidline). There was an 8-min dark
adaptation time between illumination and accumulation of the
``dark'' spectrum. Samples were dehydrated and contained 1.5
mM potassium ferricyanide and 1.5 mM potassium
ferrocyanide, which do not contribute to the spectrum in the region
shown here(20, 21) . Spectra were recorded at -9
± 1 °C. The data presented are the average of 5-23
difference spectra. The tickmarks on the yaxis correspond to 1
10
absorbance units. The data are normalized on the basis of amide I
band intensity in the infrared absorption spectrum, that is, on the
basis of the total amount of protein in the
sample.
The effect of DO exchange on
the vibrational modes of Z
will now be considered. To
summarize some of the relevant previous assignments (Table 1), a
component of the positive line at 1477 cm
has been
assigned to Z
, and a component of the negative line at
1657 cm
has been assigned to Z(21) .
Negative lines at 1522 cm
and in the spectral region
from 1264 to 1231 cm
were also assigned to Z in this
earlier work. Furthermore, the component of the 1477 cm
line previously assigned to Z
was tentatively
attributed to the C-O stretching vibration (
7a), based on a
comparison with vibrational studies of phenoxyl radicals (35) and with the tyrosyl radical in ribonucleotide
reductase(36) . Our signal-to-noise was insufficient to
identify any ring-stretching modes (for example,
8a) of
Z
. There is also a higher frequency component of the
``1477 cm
'' feature that downshifts upon
N labeling (see Fig. 1B) and is unaffected
by
H or
C labeling of tyrosine (refer to Fig. 3A in (21) ). Thus, there are at least two
components of the spectral feature at 1477 cm
in Fig. 1A. The component at higher frequency does not
originate from tyrosine, and the component at slightly lower frequency
is a Z
vibrational mode.
Figure 3:
Hydrogen bonding to the neutral tyrosine
radicals, D and Z. Note that the protonation state and
hydrogen bonding characteristics of the tyrosine residues, D and Z, are
not as yet known.
The 1450 cm region of Fig. 1A is expanded in Fig. 2A. The difference spectra in H
O (dottedline) and D
O (solidline) have been scaled to give a 1477 cm
line with the same amplitude in order to facilitate comparison.
This accounts for the apparent base line displacement caused by the
downshift of the amide II vibration in D
O (see
``Discussion''). It seems unlikely that both components of
the 1477 cm
feature would shift to the same extent
in D
O. Therefore, any downshift of a 1477 cm
component would cause a lineshape change and the appearance of a
new shifted line. Fig. 2A shows that there is no
lineshape change in the 1477 cm
feature and no new
shifted line in this spectral region. There is a small
amplitude change at 1477 cm
when control (Fig. 1A, dashedline) and deuterium
exchanged (Fig. 1B, solidline)
samples are compared. However, deuterium exchange experiments on
N-labeled samples show a much smaller H/D-induced
alteration in the amplitude of the 1477 cm
feature
when compared with a deuterium-exchanged
N sample (data
not shown) since
N-labeling has the effect of downshifting
an amide II or II` line (Fig. 1B). Therefore, this
experiment demonstrates that the H/D-induced difference in the
amplitude of the 1477 cm
feature in Fig. 1A is caused by the negative amide II`
contribution. Our data demonstrate that there is no significant
downshift of the C-O vibrational mode of Z
upon
deuterium exchange.
Figure 2:
Comparison of the effects of
DO exchange on the vibrational difference spectrum
associated with the oxidation of Z (A) and with the oxidation
of Z and D (B). The tickmarks on both the yaxes correspond to 2
10
absorbance units. In both A and B, the dashedline is the control spectrum and was plotted
on the scale shown on the left-handaxis,
and the solidline is the spectrum obtained after
deuterium exchange and was plotted on the scale shown on the right-handaxis. The small expansion of all
the spectra obtained after deuterium exchange (solidlines) accounts for the base line displacement caused by
downshift of the amide II band into this region (see text). The data
shown in A are repeated from Fig. 1A and are
the average of 23 (dashedline) and 11 (solidline) difference spectra. In B, the sample was
dark adapted at 5 ± 2 °C for 5 h and contained no acceptors.
The data in Fig. 2B are the average of 3 (dashedline) and 4 (solidline) difference
spectra.
A hydrogen bonded radical would show a downshift
of the C-O vibrational mode after deuterium exchange. Therefore,
there are two possible explanations of our findings: first, deuterium
exchange has not occurred in the vicinity of Z or,
second, there is no hydrogen bond to Z
.
First, we
consider the possibility that deuterium exchange has not occurred in
the vicinity of Z. Although an extensive amount of
washing and dialysis of samples against D
O buffers was
performed (see ``Experimental Procedures'') and although
samples were frozen and thawed in D
O, the percentage of
exchanged amide groups is approximately 20%. This low number of
exchangeable H-N groups is typical of membrane proteins (for
example, see (32) ). However, low amounts of global exchange in
membrane proteins can be combined with an extensive amount of exchange
in solvent-exposed regions(32) . As a control for exchange on
the donor side of PSII in the samples used above, we consider the
effect of exchange on the vibrational modes of tyrosine radical
D
in the same samples. D
is known to
be a hydrogen-bonded neutral radical (Fig. 3) from magnetic
resonance results(17, 22, 23, 24) .
In Fig. 2B, we present the vibrational spectrum
associated with the oxidation of both tyrosine Z and D. These data were
obtained by allowing a 5-h dark incubation time so that both the
D and Z
radicals decay and contribute
to the spectra. The same control (dashedline) and
exchanged (solidline) samples were used to generate Fig. 2, A and B. In each panel, the spectra
obtained after deuterium exchange are plotted on the same slightly
expanded vertical scale to account for the ``base line
displacement'' observed in Fig. 1A. We have
previously assigned a line at approximately 1473 cm
to D
(21) (Table 1). Comparison of Fig. 2, A and B (control spectra, dashedlines) shows that, upon long dark adaptation, there is a
change in lineshape in the 1477 cm
region. This
broadening on the low frequency side of the 1477 cm
line is consistent with the appearance of a new vibrational mode
at approximately 1473 cm
.
In Fig. 2A, to which Z alone contributes,
there is no significant spectral change at 1477 cm
after deuterium exchange (solidline). However, Fig. 2B shows that, after D
O exchange, the
1473 cm
line of stable radical D
downshifts, probably to 1460 cm
. An additional
base line displacement occurs due to a larger contribution from the
negative amide II` vibration. The data in Fig. 2B are
consistent with the idea that the D
radical is
involved in a hydrogen bond and that the environment around D has
exchanged with solvent (Fig. 3). Since Z
is
known to be more accessible to exogenous reductants than
D
(37) , this control experiment provides
evidence that the lack of D
O effect on the vibrational
lines of Z
is not caused by lack of exchange. We
conclude that Z
is not hydrogen bonded (Fig. 3). This conclusion is based on the assignment of the 1477
cm
line to the C-O stretching vibration.
However, we see no other significant H/D shifts in the region between
1530 and 1477 cm
; this is a reasonable spectral
range for observation of the C-O stretch of
Z
(21) . Also, note that deuteration effects on
the exchange-sensitive modes of the neutral residue, Z, are observed.
Substantial intensity and frequency shifts are seen in the 1250
cm
spectral region, as expected if tyrosine Z is
protonated (Fig. 3) and if deuterium exchange has occurred (38) (data not shown). However, our detailed analysis of these
spectral changes awaits a firm set of normal mode assignments.
Since
Z is a neutral tyrosine radical(18) , and Z is
likely to be protonated in the reduced form (Fig. 3), the
tyrosine must deprotonate when it is oxidized. The nature of the proton
acceptor is of importance for understanding the effect of the
environment on the physical properties of redox active tyrosine, Z. The
vibrational spectrum associated with the reversible protonation and
deprotonation of this acceptor should appear in our difference infrared
spectrum (Fig. 3). Since structural models of PSII predict that
a histidine is in the environment of redox active tyrosine Z (9, 10) and since a histidine has been predicted to be
the proton acceptor(12, 39) , we performed global
N labeling to assess whether significant
N/
N shifts are observed in the infrared
spectrum. A comparison of control
N- (dashedline) and
N-labeled (solidline) PSII particles is shown in Fig. 1B.
A 15 cm
shift is observed for the amide II vibration
in the infrared absorption spectrum of
N-labeled samples
(data not shown). Therefore, we conclude that the negative line at 1559
cm
and the positive line at 1535
cm
, assigned above to amide II vibrations, shift to
1545 and 1519 cm
, respectively, upon
N
labeling.
Aside from the changes in the amide II lines, there are
other complex changes upon N labeling. For example, a
positive line at 1549 cm
downshifts, probably to
give the positive shoulder at 1524 cm
, and a
negative mode at 1540 cm
shifts to 1510
cm
. We also observe a positive line at 1456
cm
that downshifts upon
N incorporation
and intensity changes at approximately 1660 and 1630
cm
. The difference infrared spectrum associated with
the protonation of the imidazole group of histidine in vitro has been presented previously(20) . These in vitro spectra show positive lines at approximately 1650, 1630, 1540,
1390, and 1370 cm
and negative lines at 1590, 1490,
and 1410 cm
. Also, the
N induced shifts
of an imidazole group range from 8 to 16
cm
(40) . Therefore, the
N
shifts that we have observed in Fig. 1B may be
consistent with a contribution to the spectrum from a histidine residue
and, therefore, with a change in the structure or environment of a
histidine upon charge separation. However, since substantial frequency
changes can occur when in vitro data are compared with
vibrational spectra of proteins, we cannot rule out a contribution from
another nitrogen containing amino acid residue. Also, note that the
structural change could be an alteration in the environment of the
nitrogen containing amino acid and is not necessarily a protonation
change. A shift in the high frequency component of the 1477
cm
line is observed upon
N labeling.
Previously, the high frequency component of this line was tentatively
attributed to Q
(21) on the
basis of the work described in (41) . However, the
N shift observed here appears to be inconsistent with this
Q
assignment.
Since the result of our N labeling experiment is consistent with a histidine
contribution to the difference spectrum, we generated a site-directed
mutation at a histidine residue predicted to be in the environment of
Z. Site-directed mutagenesis can distinguish changes on the donor and
acceptor side, which isotopic labeling alone cannot do. Histidine 190
of the D1 polypeptide is predicted to lie close to tyrosine Z in
structural models of PSII (9, 10) and has been
predicted to be the proton acceptor for tyrosine Z(39) . If
this hypothesis is correct, this residue should be reversibly
protonated and deprotonated upon oxidation of Z by the primary
chlorophyll donor. To test this hypothesis, a glutamine was substituted
for this histidine, and the effects of this mutation were assessed.
This mutant is inactive in oxygen evolution (see ``Experimental
Procedures''). Also, it has been reported to have a low quantum
yield for Z
Q
formation (39) .
Fig. 4presents EPR spectra
recorded on control (Fig. 4A) and HQ190D1 (Fig. 4B) PSII preparations. The EPR spectra were
recorded under the conditions used for infrared spectroscopy. In each
panel, the spectrum recorded under illumination (solidline) is the composite of the D and
Z
EPR spectra, while the spectrum recorded after 8 min
in the dark (dottedline) is the D
spectrum. Thus, the observed light-induced increase (compare solid and dottedlines) is due to the
accumulation of Z
in the absence of a functional
manganese cluster. The data in Fig. 4show that, under steady
state illumination in the presence of acceptors, HQ190D1 PSII
preparations exhibit Z
and D
EPR
lineshapes that are similar to the lineshapes observed in the control
preparation. A similar result has been obtained by Roffey et al.(27) for the HF190 and HY190 mutants in the eukaroytic
green algae, C. reinhardtii(27) . Fig. 4also
shows that the yields of Z
and D
under continuous illumination are similar when the control (Fig. 4A) and mutant (Fig. 4B)
preparations are compared. This is in contrast to the observations of
Roffey et al.(27) who noted a low D
yield in their mutants. Also, due to photosensitivity, Roffey et al.(27) could not measure a Z
EPR
spectrum under continuous illumination in the HF190D1 or HY190D1
mutants of C. reinhardtii. This observation is in contrast to
our results on the cyanobacterial HQ190D1 mutant.
Figure 4:
EPR spectra of PSII from control (A) and HQ190D1 (B) PSII samples from the
cyanobacterium, Synechocystis sp. PCC 6803. The samples
contained 90 µg (A) and 60 µg (B) of
chlorophyll. In each panel, the solidline was
recorded under illumination, the dottedline was
recorded after an 8-min dark adaptation, and the dashedline was recorded after a 90-min dark adaptation. The
conditions were as follows: gain, 2.0 10
; microwave
frequency, 9.1 GHz; modulation amplitude, 3.2 G; microwave power, 0.67
milliwatts; scan time, 4 min; time constant, 2 s. Samples were
dehydrated and contained 1.5 mM potassium ferricyanide and 1.5
mM potassium ferrocyanide. Spectra were recorded at -9
± 1 °C. The tickmarks on the xaxis correspond to 25 G. The g value of D
in the control preparation is known to be
2.0046(44) .
The spectral
shifts that result from N labeling (Fig. 1B) are quite dramatic, indicating that if
protonation of a histidine is contributing to the difference infrared
spectrum its impact on the difference spectrum is relatively large.
Therefore, we would expect that if histidine 190 is the proton acceptor
for Z, removal of this residue and substitution with a glutamine will
have a similarly dramatic impact on the vibrational spectrum. In Fig. 1C, we present a comparison of difference infrared
data recorded on purified PSII preparations from the HQ190D1 mutant (solidline) with infrared data recorded on a control
preparation (dashedline). The HQ190D1 preparation
contains a small amount of PSI contamination due to the decreased PSII
content in this mutant (note the differential feature at 1750
cm
). In spite of this, the control and mutant
difference spectra are similar in frequency and relative intensity in
every spectral region. Construction of a mutant-PSI double difference
spectrum shows that most of the small differences in frequency that do
exist arise from PSI (data not shown). If substitution of a glutamine
for this histidine has no large influence on the vibrational difference
spectrum, then it is unlikely that this mutation causes a significant
alteration in the structural changes that occur upon charge separation.
This result is inconsistent with the possibility that histidine 190
acts as the sole proton acceptor for tyrosine Z and is reversibly
protonated and deprotonated upon charge separation. Therefore, the
molecular basis of the photoheterotrophic phenotype in this mutant is
still undetermined.
To summarize, our infrared results are
consistent with a hydrogen bond to tyrosine radical D that is not formed to the Z
radical (Fig. 3). This hydrogen bond downshifts the C-O frequency
of D
relative to Z
and may account
for some of the functional differences observed between the two redox
active amino acids. This change in hydrogen bonding would be expected
to increase spin density at the phenol oxygen in Z
and
to increase the g
component of the g tensor of Z
with respect to D
(see (42) and
references therein). Other structural differences, such as a change in
the geometry at the C1-C
bond, may also exist. For example,
reconciliation of the differences observed in the EPR spectra of
3,5-deuterated D
and Z
with the
results reported here require such a change in geometry (18) in
addition to the spin density redistribution predicted by our infrared
studies. Although our data are consistent with the conclusion that
Z
is not hydrogen bonded, its C-O vibrational
frequency is downshifted approximately 20 cm
from
the C-O vibration of the stable tyrosine radical in the enzyme,
ribonucleotide reductase (36) . This spectral difference could
be caused by an electrostatic perturbation, since the tyrosyl radical
in ribonucleotide reductase is near an iron center(43) . We
have also obtained evidence that structural changes occur in
nitrogen-containing amino acid residues and/or prosthetic groups upon
charge separation in PSII. These structural changes have a large impact
on the difference infrared spectrum. Finally, a mutation at histidine
190 of D1 has little influence on the detailed structural changes that
occur in PSII upon charge separation.