Biochimica et Biophysica Acta 1503 (2001) 197^209
www.elsevier.com/locate/bba
Review
Oxygen ligand exchange at metal sites ^ implications for the O2 evolving
mechanism of photosystem II
Warwick Hillier 1 , Tom Wydrzynski *
Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia
Received 1 May 2000; received in revised form 16 August 2000; accepted 13 September 2000
Abstract
The mechanism for photosynthetic O2 evolution by photosystem II is currently a topic of intense debate. Important
questions remain as to what is the nature of the binding sites for the substrate water and how does the O^O bond form.
Recent measurements of the 18 O exchange between the solvent water and the photogenerated O2 as a function of the S-state
cycle have provided some surprising insights to these questions (W. Hillier, T. Wydrzynski, Biochemistry 39 (2000) 4399^
4405). The results show that one substrate water molecule is bound at the beginning of the catalytic sequence, in the S0 state,
while the second substrate water molecule binds in the S3 state or possibly earlier. It may be that the second substrate water
molecule only enters the catalytic sequence following the formation of the S3 state. Most importantly, comparison of the
observed exchange rates with oxygen ligand exchange in various metal complexes reveal that the two substrate water
molecules are most likely bound to separate MnIII ions, which do not undergo metal-centered oxidations through to the S3
state. The implication of this analysis is that in the S1 state, all four Mn ions are in the +3 oxidation state. This minireview
summarizes the arguments for this proposal. ß 2001 Elsevier Science B.V. All rights reserved.
Keywords: O2 evolution; Photosystem II; Manganese cluster; Water exchange; Substrate binding
1. Introduction
The mechanism for the oxidation of water into
molecular oxygen by photosystem II (PSII) is arguably the least understood aspect of green plant photosynthesis. The catalytic site consists of an Mn4 Ca
cluster and a redox-active tyrosine (YZ ) which are
bound to a protein sca¡old that is collectively called
the oxygen evolving complex (OEC) [1^3]. Despite
the wealth of spectroscopic and biochemical data
on the OEC, the organization of the Mn4 Ca cluster
* Corresponding author. Fax: +612-6279-8056;
E-mail: [email protected]
1
Present address: Department of Chemistry, Michigan State
University, East Lansing, MI 48824, USA.
and the mechanism for O2 evolution are still under
intense debate [4^13]. The underlying assumption in
all of the current models is that the Mn4 Ca cluster
directly forms the binding sites for the substrate
water.
Attempts to monitor substrate binding to the
Mn4 Ca cluster have been made using proton release
and magnetic resonance measurements. These studies, however, have not yet provided an unequivocal
interpretation (for a summary, see [14]). In another
approach, oxygen isotope exchange measurements
have been used. Basically, PSII-containing samples
are rapidly transferred into labelled water of known
isotopic composition (e.g. 18 O-enriched water) and
the rate of isotope incorporation into the photogenerated O2 is determined by mass spectrometric meth-
0005-2728 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 5 - 2 7 2 8 ( 0 0 ) 0 0 2 2 5 - 5
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W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
ods. The resulting exchange kinetics gives information on the binding a¤nities and hence the nature of
the substrate sites. The purpose of the present communication is to summarize some of the latest ¢ndings on the 18 O exchange measurements and their
implications for the mechanism of O2 evolution.
2.
18
O exchange measurements of photosystem II
The OEC is known to cycle through a sequence of
intermediate states, termed the Sn states (where
n = 0,1,2,3,4). Starting out in S0 , each next higher
state is generated by a single photoexcitation event
at the PSII reaction center. Upon reaching S4 , O2 is
released on a few ms time scale, S0 is regenerated,
and the cycle begins again [15,16]. Radmer and Ollinger found that following the addition of H18
2 O to
the suspension medium of dark-adapted samples, the
isotopic composition of the photogenerated O2 was
identical to the suspending medium after about 30 s
incubation [17]. These authors interpreted this result
to indicate that there were no slowly exchanging,
tightly bound forms of water in the dark stable S0
and S1 states. In a following study on the £ash-induced higher S2 and S3 states, similar results were
obtained [18]. Because they expected bound forms
of water at a metal site to undergo slow exchange,
Radmer and Ollinger concluded that the substrate
water only reacts with the OEC during the last step
of the S-state cycle, i.e. during the S3 to S4 to S0
transition.
The measurements by Radmer and Ollinger on the
S3 state were subsequently con¢rmed by Bader and
coworkers [19]. However, in all of these measurements, the isotopic exchange times were limited to
V30 s or longer, because of the need for sample
equilibration in the particular experimental setups
that were used. Since it had been demonstrated
that oxygen ligands in Mn model compounds can
indeed undergo 18 O exchange [20,21], there remained
the possibility that bound forms of water in the OEC
could undergo more rapid rates of exchange. Thus,
improvement in the experimental time resolution became critical in order to determine whether substrate
water actually binds during the early S states or not.
Recently, our group was able to improve the time
resolution for the 18 O exchange measurements. By
using a closed sample chamber with a smaller volume
and a rapid injection/mixing system, 18 O exchange
times down to 6^8 ms between the addition of
H18
2 O and the photogenerated O2 could be determined, an improvement in time resolution of nearly
5000-fold over the earlier measurements. Details of
the experimental setup have been published earlier
[22] and results at m/e = 36 for the doubly labelled
18 18
O O product and at m/e = 34 for the mixed labelled 16 O18 O product were initially obtained for
the S3 state [23]. For these measurements, S1 -enriched samples suspended in H16
2 O were given two
pre£ashes to advance the OEC into the S3 state.
H18
2 O was rapidly injected and followed by a variable
delay time (vt), after which a third £ash was given to
the sample and the signals at m/e = 34 and m/e = 36
measured. The mass signals were also collected on
subsequent £ashes (after complete exchange had occurred) in order to normalize the data among di¡erent sample aliquots measured at di¡erent vt values.
Fig. 1 shows the 18 O exchange behavior for the S3
state of spinach thylakoid samples at 10³C, in which
the normalized O2 yield on the third £ash is measured at m/e = 34 (left side) and at m/e = 36 (right
side) as a function of vt. The m/e = 36 data appear
to exhibit only a single kinetic phase which is ¢t with
a simple exponential function:
36
Y ˆ …13exp…336 kt††
…1†
The ¢t is given by the solid line in the m/e = 36 data.
In contrast, the m/e = 34 data on the left side in
Fig. 3 show two distinct kinetic phases. The inset
shows an expanded time ordinate to reveal the fast
phase. The two phases, however, are unequal in amplitude, with the fast phase constituting slightly more
than half of the total signal. The basis for this di¡erence in amplitude is well explained by the enrichment
condition for two independent, exchanging sites. As
the apparent kinetics of the two phases di¡er by at
least a factor of 10, the fast phase of exchange is
virtually complete before the slow phase begins.
Thus, at short vt only one substrate water molecule
is exchanging at the catalytic site. This means at a
typical 18 O enrichment of O = 12%, the mass distribution at 32:34:36 for the two oxygen isotopes will be
88:12:0. On the other hand, at longer vt when the
second substrate water molecule is also exchanging,
the mass distribution will be 77.44:21.12:1.44 (where
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W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
199
Fig. 1. 18 O exchange measurements for spinach thylakoid samples preset in the S3 state. The normalized O2 signals of the third £ash
were determined at m/e = 34 (left side) and m/e = 36 (right side) as a function of exchange time, vt, between injection of H18
2 O and the
third £ash at 10³C. The solid lines are kinetic ¢ts to the data based on equations described in the text. The samples were suspended
in a medium consisting of 30 mM HEPES (pH 6.8), 400 mM sucrose, 15 mM NaCl, and 5 mM MgCl2 . See text for details.
32:34:36 = (13O)2 :2O(13O): O2 = 100%).
Therefore,
the relative contributions of the fast and slow phases
will be unequal, with the fast phase representing
V57% (i.e. 12/21.12) of the total amplitude and the
slow phase V43%. The 0.57:0.43 distribution between the two phases is found consistently in the
m/e = 34 data. Thus, the exchange kinetics is ¢t exactly by the sum of two exponentials in the form:
34
Y ˆ 0:43…13exp…334 k1 t†† ‡ 0:57…13exp…334 k2 t††
…2†
The ¢t for the m/e = 34 data is given by the solid line
in Fig. 3.
To measure the 18 O exchange in the other S states,
various pre£ash sequences are used and the incorporation of 18 O isotope is measured at the appropriate
S3 to S4 to S0 transition. The £ash protocols and the
data have been published earlier [11]. In all S states
biphasic kinetics are observed in the m/e = 34 data,
with an amplitude ratio for the fast phase to the slow
phase of 0.57:0.43, while only monophasic kinetics
are observed in the m/e = 36 data. The corresponding
rate constants derived from the kinetic ¢ts to the
di¡erent data sets are summarized in Table 1.
From Table 1 it can be seen that in all S states, the
slow phase kinetics in the m/e = 34 measurements
yield rate constants (34 k1 ) similar to the rate constants obtained in the m/e = 36 measurements (36 k)
but di¡erent from the rate constant for the fast phase
(34 k2 ) in the S3 state. These results show that the two
substrate water molecules undergo separate isotopic
exchange processes and that the rate of 18 O incorporation into the doubly labelled 18 O18 O product is
limited by the slow isotopic exchange process.
Thus, at least one substrate water molecule is bound
throughout the entire S-state cycle. On the other
hand, the data only show that the second substrate
water molecule is bound in the S3 state, since 34 k2
cannot be determined in the earlier S states. The
isotopic exchange re£ects koff , and not kon . Thus,
due to the 10 ms £ash spacing used in the pre£ash
protocols to probe the S0 to S1 states [11], there
remains the possibility that second substrate molecule only enters the catalytic site in the 10 to 20 ms
after the formation of the S3 state. In this case, the
Table 1
18
O isotope exchange rates measured at m/e = 36 (36 k) and at
m/e = 34 (34 k1 and 34 k2 ) in spinach thylakoid samples as a function of the S states at 10³C
S state
36
k (s31 )
S3
S2
S1
S0
2.1 þ 0.2
2.2 þ 0.1
0.022 þ 0.002
18 þ 3
34
k1 (s31 )
1.9 þ 0.2
1.9 þ 0.3
0.021 þ 0.002
8þ2
34
k2 (s31 )
36.8 þ 1.9
s 175
n.d.
n.d.
Taken from reference [11]. See text for details. N.d., not determined.
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200
W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
Fig. 2. Pictorial representation of water ligand exchange rates
for various hexa-aqua metal ions. The central gray region indicates the resolvable 18 O exchange rates determined for the substrate water exchange in the O2 evolving complex during the Sstate sequence. See text for details.
kon for substrate binding would have to be faster
than the 34 k2 for exchange in the S3 state.
In general, the 18 O exchange kinetics can be described in terms of two overall mechanisms: (1) exchange of oxygen ligands bound at a metal site (Mn
or Ca), and (2) di¡usional or isotopic equilibration
between bound water and free water.
3. Water exchange at a metal site
Rates of whole water exchange at a metal site span
a tremendous range, at least 18 orders of magnitude.
Fig. 2 presents in graphical form the water exchange
rates for a range of hexa-aqua, monomeric metal ion
complexes [24,25]. The measurable 18 O exchange
rates for the OEC all reside within the region
V1032 ^102 s31 (Table 1), which is essentially mid
range for the rate constants shown in Fig. 2. For
water exchange at a metal site, probably the most
signi¢cant factors are the charge and ionic radius
of the metal ion and any electronic occupancy of d
orbitals [24,25]. As a general rule, the water exchange
rate will decrease as the metal center (M) becomes
oxidized and the ionic radius decreases, i.e. kex (M…n† ^
OH2 ) s kex (M…n‡1† ^OH2 ). A decrease in the 18 O exchange rate would thus be consistent with an oxidation of a metal center. This will be true for Mn ions.
Fig. 2 includes the water exchange rate for the
hexa-aqua MnII ion, which is quite fast with a rate
constant of 2U107 s31 [26]. Notably absent from
Fig. 2 are the water exchange rates for MnIII and
MnIV ions, which have not been measured. Indeed,
due to their high oxidizing potential, these ions require considerable ligand ¢eld stabilization energies.
Consequently, only few MnIII and MnIV complexes
with OH or H2 O ligands have been identi¢ed [27].
Nevertheless, predictions of water exchange rates for
MnIII and MnIV ions can be made based on considerations of ligand ¢eld activation energies and trends
from other transition metal ions [23,28]. A comparison of Fe or Ru ions in di¡erent oxidation states
(Fig. 2, Table 2) reveals that the water exchange rate
decreases V104 for a formal oxidation state increase
from +2 to +3. Similarly, a comparison with CrIII
(Table 2) provides an estimate for the exchange
rate at MnIV , as the two ions are isoelectronic with
a stable d3 con¢guration. In general, therefore, the
water exchange rate for MnIV may be expected to lie
in the range of V1036 ^1034 s31 while for MnIII in
the range of V1032 ^100 s31 . (without Jahn^Teller
distortions). The slow phase of 18 O exchange in the
S0 , S1 , S2 , and S3 states and the fast phase of 18 O
exchange in the S3 state could thus indicate water
bound to either an MnIII or a MnIV ion. On the
other hand, the unresolved fast phase of 18 O exchange in the S0 , S1 , and S2 states could indicate
water bound to an MnII ion. There are, however,
other important factors to consider in water exchange at a metal site.
One of the most important of these factors is the
degree of protonation of the bound water [24,25]. In
the general case, the exchange rate will decrease as
the degree of protonation decreases, i.e. kex (M…n† ^
Table 2
Water exchange rates in various aqua complexes of di- and trivalent metal ions
Metal complex
Divalent ions
[Mn(H2 O)6 ]2‡
[Fe(H2 O)6 ]2‡
[Ru(H2 O)6 ]2‡
[Ca(H2 O)6 ]2‡
Trivalent ions
[Fe(H2 O)6 ]3‡
[Fe(H2 O)5 OH]2‡
[Ru(H2 O)6 ]3‡
[Ru(H2 O)5 OH]2‡
[Cr(H2 O)6 ]3‡
[Cr(H2 O)5 OH]2‡
BBABIO 44978 1-12-00
Water exchange rate (s31 )
Reference
2U107
4U106
2U1032
6U108
[26]
[26]
[29]
[30]
2U102
1U105
4U1036
6U1034
2U1036
2U1034
[31]
[31]
[29]
[29]
[32]
[32]
W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
OH2 ) s kex (M…n† ^OH). The degree of protonation depends on the pK of the complex, which in turn is
determined by both the oxidation state of the metal
center and the character of its ligation, and hence on
the net charge of the complex. The pK's for various
Mn complexes have been documented earlier [33]
and the following trends were noted: (1) The pK
increases as the net charge of the complex increases.
(2) Increase in the formal oxidation state of the Mn
center appears to be less important than the increase
in the net charge. In this regard, it should be noted
that an MnIII center tends to favor OH ligation while
an MnIV center, due to its poor basicity and high
oxidation potential, tends to favor fully deprotonated oxo ligands (see below). The 18 O exchange
rate for the OEC is therefore more consistent with
protonated forms of the substrate (OH2 or OH) that
are bound to MnII or MnIII sites.
Another factor that is important in determining
the exchange rate is the degree of protonation of
neighboring ligands. Table 2 lists the water exchange
rates for monomeric, 6-coordinate complexes of Fe,
Ru, and Cr ions. In these cases, deprotonation of
one of the water ligands leads to an increase in the
rate of exchange for the remaining water ligands by a
factor of V100. The increase in the exchange rate is
due to the lower net charge of the deprotonated complex. Likewise a similar e¡ect is observed in multimeric metal complexes. Table 3 gives the water exchange rates for the di-W-hydroxo bridged CrIII
dimer [34]. Mono-deprotonation of this complex
also leads to an increase in the water exchange
rate, regardless of whether the water ligand is trans
or cis to the hydroxo bridges. Again, the net e¡ect is
to increase the exchange rate by a factor of V100. It
is interesting to note that the water exchange in the
Table 3
Water exchange rates in fully protonated and mono-deprotonated forms of the di-W-hydroxo Cr dimeric complex
Metal complex
[CrIII (W-OH)2 CrIII ]4‡
H2 O trans W-OH
H2 O cis W-OH
[CrIII (W-OH)2 CrIII OH]3‡
H2 O trans W-OH
H2 O cis W-OH
Taken from reference [34].
Water exchange rate (s31 )
4U1034
7U1035
1U1032
5U1033
201
CrIII dimer is overall faster compared to the monomeric CrIII ion (Table 2), by a factor of V10. A
similar di¡erence in exchange rates is found between
RhIII ions [35] and di-hydroxo bridged RhIII dimers
[36,37]. Thus, in the organization of the Mn4 cluster
in the OEC, deprotonation of neighboring ligands
could well have an in£uence on the 18 O exchange
rates.
Still other important factors that may in£uence the
rate of water exchange at a metal site are the coordination geometry (axial vs. equatorial) and spin
state of the metal center (high spin vs. low spin).
MnIII ions, for example, are in a d4 con¢guration
and, if in 6-coordinate octahedral geometry, may exhibit Jahn^Teller distortions due to degeneracy in the
eg orbital. These distortions result in a lengthening of
the axial bonds in which the rate of ligand exchange
will be increased. Conversely, the shorter equatorial
bonds will undergo slower ligand exchange. Likewise, high spin metal complexes are expected to
have faster exchange rates than low spin complexes.
The structure of the Mn4 cluster in the OEC is yet to
be de¢ned but it is believed to be mainly coordinated
to carboxyl ligands [38,39], which will in£uence the
overall water exchange rates.
4. Other oxygen ligand exchange at a metal site
The oxidation of water to molecular oxygen may
occur via a number of pathways and involve a range
of intermediates. It is important to appreciate that
the 18 O exchange measurements could re£ect the exchange of oxygen ligands other than water, such as
W-oxo (^O^), peroxo (^O^O^) or oxo (NO) ligands.
Indeed, various models for O2 evolution have invoked such intermediates. The critical question for
these models is during which step in the S-state cycle
does the O^O bond form.
4.1. W-oxo bridging ligands
It is generally agreed that the Mn4 cluster in the
OEC is arranged as two, coupled di-W-oxo bridged
Mn dimers. In one proposal, it is suggested that Woxo bridges condense via a W-R2 :R2 peroxo intermediate to generate O2 [12,38], similar to a mechanism that was suggested for certain binuclear Cu
BBABIO 44978 1-12-00
202
W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
complexes [40]. In another proposal, the condensation of the W-oxo bridges in one Mn dimer forms a
peroxo intermediate, which is chemically oxidized to
O2 by the second Mn dimer [41]. The key question
for these proposals is whether or not symmetrical Woxo bridges can undergo 18 O exchange in the times
that have been measured for the OEC (i.e. less than a
second, see Table 1).
It has been shown that the W-oxo bridges in a
binuclear MnIII dimer can undergo exchange with
solvent water [20,21], though the relevant exchange
rates were not determined. Typically, however, W-oxo
bridge exchange is a slow process. Table 4 lists the
18
O exchange rates for the bridging oxygen in some
W-oxo bridged complexes. The de¢ning in£uence for
W-oxo exchange appears to be the type of pathway
for exchange. The [MoV O3 ]4‡ complex proceeds via
an intramolecular rearrangement [45] while the
CrIII (W-OH)2 CrIII complex proceeds via a ring opening mechanism [34]. In contrast, the RhIII (W-OH)2 RhIII complex is inert to W-oxo exchange [37].
As discussed above, deprotonation of monomeric
complexes can in£uence the rate of water ligand exchange. This is also true for the exchange of W-oxo
bridges. For example, the mono-deprotonated
CrIII (W-oxo)CrII OH complex has a bridge exchange
rate V50 times faster than the fully protonated form
[34]. Nevertheless, the 18 O exchange rates for the
OEC all appear to be too fast to be due to exchange
of W-oxo bridges, at least until de¢nitive exchange
rates of appropriate Mn di-W-oxo model compounds
are measured.
4.2. Peroxo ligands
A concerted 4-electron pathway for water oxida-
tion is generally considered to be more di¤cult thermodynamically than a two 2-electron oxidation pathway involving a bound peroxo intermediate [46]. As
such, bound peroxo intermediates have often been
proposed to be involved in the O2 evolving reaction
[5,47]. Indeed, various assays for light-induced H2 O2
generation by PSII have yielded signals interpreted to
arise from bound peroxide [48]. Similarly, peroxo
intermediates have been proposed in various Mn
model compounds, some of which oxidize water
[49^51]. In a recent re¢nement of the peroxide hypothesis for the OEC, it has been suggested that in
the S3 state there exists an enantiomeric intermediate
between an MnIV /MnIV site having terminal water
ligands and an MnIII /MIII bound peroxide, which
are in rapid (V1 ms) redox isomerization [5]. However, this model cannot account for the independent
kinetic behavior of the two sites through the S-state
cycle (Table 1). Rather, the H2 O2 produced by PSII
samples under some conditions may only arise from
a side reaction in a perturbed OEC [52].
4.3. Terminal oxo ligands
Condensation of two oxo ligands on opposing
MnIV ions has been proposed as the mechanism for
O^O bond formation in the OEC [4]. This mechanism provides a thermodynamically viable means to
couple a 4-electron oxidation with chemical bond
formation. There are, however, only a limited number of measurements of oxo exchange in non-porphyrin metal complexes that can be used for comparison. Table 5 lists some examples and, generally
speaking, oxo exchange tends to be quite slow. The
important issue for oxo exchange with solvent water
is whether or not the oxo ligand can be protonated.
Table 4
W-Oxo bridge exchange rates in various metal complexes
Metal complex
III
(Fe )2 W-oxo in Ribonucleotide reductase
CrIII (W-OH)2 CrIII
RhIII (W-OH)2 RhIII
RuIII ^O^RuIII
4‡
[MoIV
3 O4 (H2 O)9 ]
V
4‡
[Mo O3 ]
W-Oxo bridge exchange rate (s31 )
34a
8U10
1U1035
substitution inert
6U1036b
t1=2 s 10 years
V1036b
At ambient room temperature except for:
a
At 4³C.
b
At 40³C.
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Reference
[42]
[34]
[37]
[43]
[44]
[45]
W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
Table 5
Oxo exchange rates in various metal complexes
Metal complex
Oxo exchange rate (s31 )
Reference
TiIV NO
VIV NO
[MoV O3 ]4‡
2U104
2U1035
4U1033a
[53]
[54]
[45]
At ambient room temperature except for:
a
At 0³C.
The TiNO complex is an example in which the oxo
ligand is believed to be easily protonated to account
for its very fast exchange compared to the VNO
complex [53,54]. In an alternate mechanism, oxo exchange may occur via the generation of a cation at
the metal center and a relocation of the electron
density [55], analogous to organic carbonyls. In all
cases, oxo exchange involves complex intermolecular
rearrangements.
Unfortunately, to date no oxo exchange has been
demonstrated in a non-porphyrin MnIV NO complex.
One kinetic constraint of the MnIV ion is its d3 con¢guration and the half ¢lled t2g orbitals: dxy , dyz and
dxz . Such an electronic con¢guration tends to make
any MnIV complex inherently stable as the electron
distribution repels incoming ligands. An example of
this situation is the high spin CrIII ion, which has
only a very slow water ligand exchange (Fig. 2, Table
2). On the other hand, a 5-coordinate MnIV with a
di¡erent ligand ¢eld splitting may exhibit an altered
¢lling of the d orbitals, which could facilitate ligand
exchange.
The oxo exchange in high valence porphyrin complexes represents a very di¡erent situation in which
redox tautomerism plays an important role [56,57].
There are biological examples of oxo exchange involving FeIV porphyrins. Both horseradish peroxidase (HRP) compound II and the cytochrome c peroxidase (CCP) undergo FeIV NO oxo exchange with
bulk water, but only when H bonding is present
[58,59]. Without H bonding there is no oxo exchange
[60]. The HRP enzyme can undergo Mn substitution
but, although catalytically active [61], does not
undergo 18 O exchange with solvent water [62]. To
account for the absence of exchange, it was suggested
that a weaker MnIV NO bond and the enhanced basicity of the Mn complex alters the H bond donor
group in the HRP enzyme [62,63]. This interpretation
203
appears to be supported by studies of a model
MnIV NO porphyrin compound [64,65], which exhibits a weaker oxo bond compared with the FeIV or
CrIV complexes.
Taken together, the above arguments suggest that
any oxo exchange at an MnIV ion in the OEC will
strongly depend upon its coordination geometry as
well as the involvement of a strong basic group in
close proximity to impose an H bond. Nevertheless,
oxo exchange at an Mn site is expected to be relatively slow.
5. Involvement of calcium
There is a single, tightly bound, CaII ion associated
with PSII [66] that can be reversibly depleted in correlation with the O2 evolving activity [67]. A CaII ion
is also known to be required for the photoassembly
of the Mn4 cluster [68,69] and may reside close by
the Mn [70,71]. Early suggestions for the function of
Ca have included a role as a substrate holding site
[72] or as a `gatekeeper' which controls accessibility/
reactivity of the OEC [73].
However, the resolvable 18 O exchange rates for the
OEC appear too slow for a water ligand to a CaII
site. Water exchange at a CaII ion is usually very fast
(i.e. with an exchange rate approaching V109 s31 ,
see Fig. 2, Table 2). Although within a protein there
may be a number of ways to slow down ligand exchange (e.g. H bonding or activation barriers imposed by ligand access, see below), we note that in
the S3 state both exchange rates would have to slow
down by 7 or 8 orders of magnitude. On the other
hand, since the fast phase of exchange cannot be
determined in the S0 , S1 , or S2 states, it may be
that a CaII ion is involved in the substrate binding
associated with these states. But it seems unlikely
that a CaII ion would be involved in substrate binding in the earlier S states and not in the S3 state.
Furthermore, when the 18 O exchange was measured
in samples in which Ca was functionally replaced
with Sr, it was found that both kinetic phases in
the m/e = 34 data for the S3 state are altered, and
surprisingly in opposite directions (Hillier and Wydrzynski, unpublished results). This result would seem
to indicate that Ca has an important structural role
in the OEC rather than a catalytic role.
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W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
6. Limitations in substrate accessibility
In general, the residence times for integral water in
proteins tend to be quite short. NMR measurements
indicate very fast exchange (108 ^109 s31 ) for surface
bound water and a rather broad range of exchange
for internally bound water (103 ^109 s31 ), where the
exchange is thought to be mostly determined by protein structural dynamics [74,75]. For water exchange
into the OEC, we know that water di¡usion across
the membrane is not involved. PSII-enriched membrane fragments and puri¢ed core complexes from
both spinach and cyanobacteria, which do not maintain a vesicular structure, exhibit biphasic exchange
kinetics and rate constants very similar to what is
measured for intact spinach thylakoid samples (Hillier and Wydrzynski, unpublished results). On the
other hand, the water exchange into the OEC may
still be in£uenced by an accessibility barrier since the
catalytic site is generally believed to be sequestered
away from the bulk solvent water. As such a phenomenological `water channel' must exist, something
akin to what has been proposed for the cytochrome c
oxidase [76,77]. A water channel in the OEC may
provide an important role in optimizing the O2
evolving reaction [52]. If this is the case, then solvent
accessibility to the catalytic site might at least govern
the fast phase of exchange, unless there is a separate
water channel for each of the substrate molecules.
The possible existence of a water channel in the
OEC and its role in the O2 evolving mechanism
must await de¢nitive structural determinations of
the OEC.
7. On the nature of substrate binding to
photosystem II
The discussion up to this point has covered some
aspects of water and oxygen ligand exchange at metal sites in relation to the 18 O exchange measurements
of the OEC. In general, it is di¤cult to be precise
about the nature of the binding site based only on
the 18 O exchange of a single S state. However, comparisons of the 18 O exchange as a function of the
di¡erent S states are highly valuable and begin to
de¢ne the types of mechanisms that may be involved.
Three important ¢ndings from the 18 O exchange
measurements of the OEC can be summarized as
follows: (1) For all S states (S0 , S1 , S2 and S3 ), the
two substrate molecules exhibit di¡erent exchange
behavior. This observation strongly suggests that
the O^O bond is formed only after the S3 state, during the last step of the S-state cycle. (2) There is not a
sequential slowing in the exchange rates from S0 to
S1 to S2 that can be interpreted in terms of a coupled
Mn cluster undergoing sequential oxidation reactions. (3) The rate for the slow phase of exchange
in the S0 state is remarkably similar to the rate of
slow phase of exchange in the S3 state, indicating
that the nature of the binding site in these two states
is not hugely di¡erent. In the following sections the
18
O exchange behavior during each S-state transition
is discussed in terms of the possible nature of the
binding sites.
7.1. S0 to S1 transition
In the S0 state, the slow phase of exchange is determined to be V10 s31 while in the S1 state it is
V0.02 s31 (Table 1). The net e¡ect upon the S0 to S1
transition is a slowing down in the slow phase of
exchange by a factor of V1000. Such a slowing
down in the exchange rate may be consistent with
a formal oxidation state increase at an Mn binding
site. Indeed, current UV, XANES, and EPR measurements do suggest that an MnII ion is oxidized
to MnIII on the S0 to S1 transition [78^82].
However, based on the magnitude of the rate constant of the slow phase of exchange in the S0 state,
we feel that the substrate water molecule is unlikely
to be bound to an MnII ion. The rate of whole water
exchange at an MnII site is generally expected to be
much more rapid (Fig. 2), particularly if the MnII
site is bound within a multimeric metal cluster (Tables 2 and 3). Although there may be ways that the
protein environment could slow down the rate of
exchange (see above), it is unlikely that such mechanisms could slow down the exchange so much to
account for our measurements. Rather, in comparison with other hydrated metal complexes (Fig. 2), we
believe the magnitude of the slow exchange in the S0
state is best interpreted as water bound to an MnIII
ion. If this is the case, then the slowing down of
exchange rate upon the S0 to S1 transition would
have to be due to another mechanism. One possibil-
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W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
ity is a pK shift in the complex. Based on Mn model
compounds, an increase in charge of the complex
can lower the pK by as much as 8 pH units [83].
Thus, if a neighboring MnII ion in the OEC is oxidized to MnIII , the resulting decrease in the pK could
lead to a deprotonation of the substrate water at
another Mn site. This type of mechanism could
then account for the observed decrease in the 18 O
exchange rates.
7.2. S1 to S2 transition
In contrast to the S0 to S1 transition, the S1 to S2
transition is commonly held to be coupled to the
oxidation of an MnIII ion to an MnIV ion in an
antiferromagnetically coupled di-W-oxo bridged Mn
dimer ([2], although see [13]). But surprisingly, the
slow phase of 18 O exchange increases by a factor of
V100, from V0.02 s31 to V2.0 s31 (Table 1), during this transition. Clearly, this ¢nding shows that
the site binding this substrate water molecule is not
the Mn site undergoing the oxidation increase (which
is presumably the same site that is oxidized from
MnII to MnIII on the S0 to S1 transition [78^82]).
To account for the unexpected increase in the exchange rate during the S1 to S2 transition, we note
that enhancements in the exchange rate can occur
upon deprotonation of neighboring ligands in the
complex (Tables 2 and 3). An attractive hypothesis
could be the deprotonation of a W-hydroxo bridge in
an Mn4 cluster. Current EXAFS measurements,
however, do not support a corresponding structural
change in the OEC during the S1 to S2 transition
[12,38]. Alternatively, the deprotonation may occur
at another site in the OEC, but close enough to in£uence the coordination sphere of the Mn which
binds the substrate.
7.3. S2 to S3 transition
Interestingly, the magnitude of the slow phase of
exchange in the S2 state is identical to its magnitude
in the S3 state (Table 1). This observation shows that
the a¤nity of the binding site for this substrate water
molecule stays the same during the S2 to S3 transition, despite the further accumulation of an oxidizing
equivalent in the OEC. However, the fast phase of
exchange, which is unresolvable in the S2 and earlier
205
S states, slows down by at least a factor of 5, to yield
a rate constant of V37 s31 in the S3 state (Table 1).
The decrease in the fast phase of exchange during
this transition may again be consistent with an oxidation increase at an Mn binding site or with a deprotonation of the bound substrate water molecule.
Unfortunately, the Mn oxidation state assignments
for the S2 and S3 states remain controversial as to
whether or not a metal-centered oxidation takes
place during this transition [12,79,80]. Irrespective
of whether Mn or a ligand is oxidized, it is important
to note that only fast phase of exchange is a¡ected
during the S2 to S3 transition and not the slow phase
of exchange. This observation provides strong evidence that the two binding sites are chemically separate in the S3 state. It is also interesting to point out
that the rate of the slow phase of exchange in the S2
and S3 states is very similar to what it is in the S0
state. This observation indicates that this binding site
is unlikely to di¡er by metal-centered oxidations.
8. Mechanism of substrate activation and O^O bond
formation
The overall results from the 18 O exchange measurements can be illustrated by the scheme shown
in Fig. 3. Here, the Mn ions of the OEC are shown
to be organized into two types: (1) those which are
involved in the direct accumulation of oxidizing
equivalents (designated by the brackets) and (2)
those which are involved in substrate binding. In
the scheme an Mn ion is proposed to bind a substrate water molecule beginning in the S0 state and
accounts for the slow phase in the 18 O exchange
kinetics. This Mn site is assumed not to undergo
any formal metal-centered oxidations up to the S3
state, though, as indicated, it could undergo a transient oxidation increase in the S4 state. Based on the
magnitudes of the exchange rates measured, this site
is likely to be MnIII , either in 5-coordinate geometry
or having a non-Jahn^Teller lengthened bond with
the substrate water. We consider MnII or MnIV to
be unlikely for the reasons given earlier in the discussion (i.e. the rate of water exchange at an MnII
site would most likely be too fast, while water bound
to an MnIV site would tend to form a slow exchanging oxo ligand). Likewise, we consider CaII to be
BBABIO 44978 1-12-00
206
W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
Fig. 3. A proposed scheme for the O2 evolving complex (OEC)
in photosystem II to explain the S-state dependence in the 18 O
exchange kinetics. The four Mn ions are organized such that
only two are associated with the accumulation of oxidizing
equivalents during the S-state cycle while the other two Mn
ions are involved in the binding of the substrate water. The
changes in the 18 O exchange rates are ascribed to deprotonation
events, either at the substrate water itself or at adjacent ligands,
which result from successive changes in the pK of the OEC
upon S-state turnover. Proton rearrangements are only implied
at the catalytic site and do not necessarily re£ect direct proton
release into the bulk. The second substrate water molecule is
proposed to only enter into the reaction sequence after the formation of the S3 state when the binding site becomes `open' to
the solvent water. See text for further discussion. From reference [11].
unlikely as well because of the peculiar S-state dependent changes in the slow phase kinetics.
In the scheme, the fast exchanging substrate site in
the S3 state is also predicted to be an MnIII ion,
mainly because its rate constant is not substantially
di¡erent from the rate constant for slow exchanging
site (di¡ering by only a factor of V20). For this
reason we may expect the fast phase kinetics to be
slow enough in the earlier S states to be resolvable by
our current methods. Since they are not, we invoke
the possibility that the second substrate water molecule may only enter the catalytic site after the formation of the S3 state. The entrance of the second
substrate molecule later in the reaction sequence may
serve to optimize the positioning of the reactants
[52]. Alternatively, the second substrate molecule
may indeed reside at a fast exchanging site in the
earlier S states, such as a Jahn^Teller lengthened
bond at another MnIII ion or a CaII ion, in which
the exchange slows down considerably upon the formation of the S3 state (see above). In either situation,
the O^O bond would still have to form during the
¢nal S3 to S4 to S0 transition, possibly through a
nucleophilic attack mechanism [11], in order to account for the separate kinetic behavior of the two
sites during the S2 to S3 transition.
The suggestion that MnIII ions in the OEC constitute the substrate binding sites throughout the Sstate cycle is at odds with the interpretations based
on the XANES [79,80,84] and kL £uorescence (J.
Messinger, personal communication) measurements.
However, we note that the Mn edge positions
strongly depend on the O/N ratio of the surrounding
ligands [13,85] and on the coordination geometry of
the complex [86,87]. Thus, the Mn oxidation states
predicted from the X-ray techniques are not necessarily unique, at least until more information becomes
available on the structure of the OEC. Therefore, to
avoid the generation of extreme Mn oxidation levels
in the higher S states, we propose in the scheme of
Fig. 3 that in the S1 state all four Mn ions exist in the
+3 oxidation state. During the S1 to S2 and S2 to S3
transitions there could then be either metal- or ligand-centered oxidations to account for the various
XANES and EPR results (for a discussion, see
[12,13]). These latter possibilities are merely indicated
in Fig. 3 by the + symbols next to the Mn pair which
accumulates the oxidizing equivalents. The variations
BBABIO 44978 1-12-00
W. Hillier, T. Wydrzynski / Biochimica et Biophysica Acta 1503 (2001) 197^209
in the 18 O exchange rates with the S states are then
explained in terms of protonation reactions of the
OEC, involving either the substrate water itself or
adjacent ligands, as discussed above.
The separation of the accumulation of oxidizing
equivalents from substrate binding and O^O bond
formation is an extension of an earlier hypothesis,
in which a special `water binding component' in the
OEC was invoked [88]. Regardless of the details,
however, our results are largely inconsistent with current models of O2 evolution, either because the observed changes in the 18 O exchange during the early
S-state transitions are incompatible with the expected
binding a¤nities in these models [4,8] or the models
do not account for the independent kinetic behavior
of the two binding sites upon the S2 to S3 transition
[5^7,9,10,13].
9. Conclusions
Comparisons of the 18 O exchange measurements
of the O2 evolving complex of photosystem II as a
function of the S states lead to the following conclusions: (1) One substrate water molecule is bound to
the catalytic site at the beginning of the S-state cycle.
(2) The second substrate water molecule is bound at
least in the S3 state, at a site which is separate from
the ¢rst substrate water site but one which is chemically similar. (2) If Mn ions form the binding sites for
the substrate water, then at least two of the Mn ions
must function independently of each other within the
catalytic cluster and are likely to be in the +3 oxidation state through the S-state cycle. (3) The O^O
bond forming step must occur during the ¢nal S3
to S4 to S0 transition. (4) Although the variations
in the 18 O exchange during the S-state cycle are not
straightforward, they probably re£ect deprotonation
reactions and dynamic structural changes of the
OEC rather than direct metal-centered oxidations
at the substrate binding sites. We feel that our ¢ndings provide some surprising insights into the photosynthetic O2 evolution and will need to be addressed
in future chemical mechanisms.
207
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