1

PHOTOSYSTEM II: THE WATER/PLASTOQUINONE OXIDO-

REDUCTASE IN PHOTOSYNTHESIS

T. WYDRZYNSKI AND K. SATOH, EDITORS

CHAPTER 25

PHOTO-ASSEMBLY OF THE CATALYTIC MANGANESE CLUSTER

G. Charles Dismukes*

Department of Chemistry and Princeton Environmental Institute

Princeton University

Princeton, NJ, USA

Gennady M. Ananyev

Department of Environmental Biophysics & Molecular Ecology

Rutgers University, E. Brunswick, NJ, USA

Richard Watt

Department of Chemistry

University of New Mexico, Albuqurque, NM, USA

*correspondence at dismukes@princeton.edu, FAX: 609-258-1980

2

Table of Contents

Abbreviations ............................................................................................................... 3

I. Introduction .......................................................................................................... 4

II. Function of PSII Subunits in Water Splitting and Photo-assembly ........................ 4

A. The PSII Core Complex. ................................................................................... 5

B. Extrinsic Proteins .............................................................................................. 7

1. The Manganese Stabilizing Protein. The MSP ................................................ 7

2. Other Extrinsic Proteins. ................................................................................ 10

C. Small Subunits ................................................................................................ 10

D. [Mn]4 Core ....................................................................................................... 10

E. The Calcium Effector Site ................................................................................ 11

III. Biogenesis of PSII-WOC ..................................................................................... 12

A. Subunit Assembly ............................................................................................. 12

1. Prokaryotes .................................................................................................... 12

2. Chloroplasts ................................................................................................... 14

B. Mn2+

Uptake Into the Cell ................................................................................. 15

C. Inorganic Core Assembly Involves no Protein Chaperones .............................. 16

IV. Roles of the Inorganic Cofactors from Photo-assembly ...................................... 18

A. Assembly of the Inorganic Core ....................................................................... 18

1. Instrumentation. ............................................................................................. 18

2. Kinetics and Mechanism. ............................................................................... 19

3. Proton Evolution. ........................................................................................... 20

4. Oxo/Hydroxo/Bicarbonate Sites. ................................................................... 21

5. Calcium. ......................................................................................................... 22

6. Protein Subunits Required for Photoactivation .............................................. 24

B. “Inorganic Mutants” of the Manganese and Calcium Sites .............................. 25

1. Manganese Mutants. ...................................................................................... 25

2. Calcium Mutants ............................................................................................ 26

3. Other Metal Ion Sites. .................................................................................... 28

V. Concluding Remarks ..................................................................................... 28

Acknowledgements .................................................................................................... 29

References .................................................................................................................. 38

3

Summary

The biogenesis of the photosystem II membrane protein complex is reviewed with emphasis on

the sequence and location of assembly of the subunits within the internal membrane systems of

prokaryotes and chloroplasts. The uptake and distribution of manganese in cyanobacterial cells is

discussed. The role of individual subunits in the isolated photosystem II in light-induced

assembly of the inorganic core of the water splitting complex is discussed (photoactivation

process). The roles of the inorganic cofactors (Mn2+

, Ca2+

, Cl- and bicarbonate) in water splitting

are revealed by examining the effects of their reconstitution with non-native cofactors. Novel

instrumentation for the measurement of O2 concentration and the kinetics and mechanism of

photoactivation are described.

Abbreviations

CA carbonic anhydrase; Chl chlorophyll; EPR electron paramagnetic resonance; MSP

manganese-stabilizing protein; RC reaction center; WOC, water oxidizing complex; XAS x-ray

absorption spectroscopy; XRD x-ray diffraction;.

4

I. Introduction

The photosynthetic water splitting enzyme (WOC) located within the Photosystem II reaction

center is responsible for nearly all biological O2 production on Earth. It is remarkable in that,

despite enormous diversification of the classes of oxygenic photoautotrophs, there is but one type

of enzymatic core that performs this chemistry (Dismukes et al. this series). The active site is a

unique mangano-calcium cluster comprised of five constituents plus water,

Mn4Ca1Clx(OHn)y(HCO3)z, that is assembled de novo from elementary ions, water and

bicarbonate by a light-driven photoassembly process within the cofactor-free apo-WOC-PSII

complex. This process is called photoactivation, after the defining works of George Cheniae and

coworkers.(Frasch et al. 2001)

This review covers recent advances dealing with the uptake, distribution, photoassembly and

function of the inorganic cofactors comprising the PSII-WOC from cyanobacteria and higher

plants. It covers aspects of Mn transport, localization and photoassembly, both in vivo during

biogenesis/repair and in vitro during reconstitution of isolated PSII-WOCs. This material

examines processes from the cellular level down to the atomic level.

II. Function of PSII Subunits in Water Splitting and Photo-assembly

A summary of the subunit positioning derived from X-ray diffraction (XRD) analysis can be

found in other chapters. Information derived from XRD is not considered in this review. We

begin with an overview of the composition of the intact PSII protein complex and the associated

inorganic core of the WOC.

5

A. The PSII Core Complex

The detergent-solubilized isolated PSII protein core complex from the thermophillic

cyanobacterium Thermosynechococcus elongatus is composed of 19 polypeptide subunits (Zouni

et al. 2001; Ferreira et al. 2004), while 19 or more subunits are present in higher plants like

spinach.(Hankamer et al. 2001). However, these numbers may be underestimates of the

composition in the native membrane based on more sensitive studies using mass spectrometry

and N-terminal amino acid sequencing methods that have identified 31 unique subunits within an

isolated PSII core complex from the cyanobacterium Synechocystis 6803.(Kashino et al. 2002)

This large increase in the number of subunits within the PSII core complex may reflect both

species differences and improvements in purification afforded by polyhistidine tagging of the

CP47 subunit. The native PSII complex exists as a dimer with two-fold symmetry comprised of

two sets of reaction centers and antenna complexes that operate more or less independently in

terms of redox chemistry, but do exchange excitation energy. No reports have indicated any

difference in the properties of the WOC in monomers and dimers, although PSII heterogeneity in

the thermal activation parameters associated with water splitting is known.(Burda et al. 2001)

The D1 (psbA gene) and D2 (psbD gene) proteins are homologous and form a hetero-dimeric

reaction center within each monomeric unit (RC). However, the smallest isolated RC complex

that performs primary charge separation also contains the two subunits of cytochrome b559,

(psbE gene) and (psbF gene) (Nanba et al. 1987; Seibert 1993). The D1, D2, CP47, CP43 and

cytochrome b559 subunits comprise what is called the PSII core, as no complex without these five

subunits had been isolated that evolved oxygen( a number of small subunits that lack cofactors

are also generally present in cores). A stable CP47RC subcore complex assembles within the

thylakoid membrane of cyanobacteria in the deletion mutant lacking the psbC gene for

6

CP43.(Rögner et al. 1991) These complexes can be isolated and are active in electron transport

but inactive in O2 evolution, presumably due to an incomplete Mn cluster. A stable CP47RC

subcore complex has been prepared from spinach by removal of CP43 with detergents, called the

CP47RC subcore. Although the CP47RC subcore is devoid of Mn and inactive in O2 evolution, it

is capable of full activity after reconstitution of the Mn cluster by photoactivation in the presence

of the free inorganic cofactors.(Buchel et al. 1999) The successful reconstitution of the CP47RC

subcore together with the subunit positioning data and XRD of the Mn binding site (below)

suggest that a RC subcore lacking CP47, might also be capable of O2 evolution. However, to

date no stable subcore complex lacking CP47 has been isolated to test this hypothesis.

The Mn-cluster is surrounded primarily by the lumenal surface of the D1 trans-membrane

protein of the PSII reaction center, as predicted by previous mutational studies[Diner, 1998

#79;[Debus, 2001 #2216] and model building(Svensson et al. 1996). Nixon and Diner showed

that Asp170 plays a critical role in the binding and oxidation of the first Mn2+

required for the

formation of the Mn cluster.(Nixon et al. 1992) Further spectroscopic analysis confirmed that

deletion of Asp170 significantly alters the EPR signal of the first photooxidized Mn3+

bound to

PSII. (Campbell et al. 2000) Chemical modification studies also confirmed that Asp170 is

required for the binding of Mn2+

at the high affinity site and that D1-His337 may play a role in

ligating the Mn cluster.(Ghirardi et al. 1998)

The YD radical has been found to advance the lowest S-state of the Mn cluster from S0 S1 in

the dark, while in its reduced form it shortens the lifetime of the S2 and S3 states. (Babcock et al.

1975; Styring et al. 1987; Vass et al. 1991) On this basis the YD radical was proposed to function

in vivo to stabilize the Mn cluster against over-reduction and thus possible loss of reduced

Mn2+

.(Rova et al. 1998)

7

B. Extrinsic Proteins

In plants three water-soluble extrinsic proteins are attached non-covalently to the lumenal

surface of the PSII complex. Their associated genes and molecular masses are used to identify

these proteins as the 33 kDa (psbO gene), 23 kDa (psbP gene) and 19 kDa (psbQ gene).[Debus,

1992 #683;Debus, 2000 #1766] The 33 kDa protein binds first at the interface of the D1/D2

dimer and the 23 kDa protein binding adjacent to the 33 kDa protein near to the CP43 protein

with the 19 kDa protein binding last.(Nield et al. 2002) The PsbO protein is found universally in

all oxygenic photoautotrophs. The PsbP and PsbQ proteins were until recently not believed to be

present in PSII core complexes from cyanobacteria and the role of these proteins appears to be

substituted by the cytochrome c550 (psbV gene) and to a lesser extent a 12 kDa protein. (psbU

gene).(Shen et al. 1995; Shen et al. 1995; Shen et al. 1998; Ohta et al. 1999; Katoh et al. 2001)

However, recently a smaller protein of 11 kDa with high homology to the psbQ gene product

was found in a PSII core complex isolated from Synechocystis 6803, suggesting a wider

distribution than previously thought.(Kashino et al. 2002)

1. The Manganese Stabilizing Protein. The MSP is so called because removal of this protein

exposes the Mn cluster to exogenous reductants and competition for replacement of manganese

by other metal ions. In vitro photoassembly of spinach apo-WOC-PSII is slowed by the binding

of this protein. This protein is unfolded in solution and only assumes a folded structure when

bound to the intrinsic proteins of PSII.(Lydakis-Simantiris et al. 1999) The isolated spinach MSP

was found not to bind Mn2+

or Ca2+

ions in solution based on low affinity (dissociation constant

KD ~3 mM) and low stoichiometry (0.14 Mn/MSP).(Hunziker et al. 1987) MSP binds to PSII

somewhat more tightly when the Mn cluster is present(Miyao et al. 1989; Kavelaki et al. 1991;

Leuschner et al. 1996) Changes in the oxidation state of the Mn cluster in the PSII core complex

8

alter the structure of the MSP making it susceptible to proteolysis.(Hong et al. 2001) PSII does

not require the MSP for oxygen evolution in vitro(Bricker 1992) nor in vivo (Burnap et al. 1991),

but in its absence, the steady-state oxygen evolution rates are decreased.(Miyao et al. 1987;

Burnap et al. 1992) This decrease is attributed to deactivation of the Mn-cluster by endogenous

reductants in the lumen. A deletion mutant of the MSP reveals that it acts as a diffusion barrier in

vivo by suppressing electron donation from Mn2+

and decreasing the quantum yield of

photoactivation in whole cells.(Chu et al. 1994; Burnap et al. 1996) When reductants are added

to PSII membranes in the presence of Ca2+

with the MSP, the Mn2+

ions are trapped and do not

diffuse away, indicating they are trapped in a compartment for later photooxidation and

reassembly of the Mn cluster.(Mei et al. 1992; Riggs-Gelasco et al. 1996) The removal of the

MSP results in the release of two Mn2+

ions from PSII membranes unless high concentrations of

Cl- and Ca

2+ are used.(Miyao et al. 1984; Ono et al. 1984) In the absence of the MSP: the S3

(S4) S0 transition is slowed 5-fold (Miyao et al. 1987; Vass et al. 1987; Burnap et al. 1992),

the S2 state is abnormally stable against dark decay to S1, the reduction kinetics of YZ are

altered(Razeghifard et al. 1997), the substrate water exchange rates are slightly decreased(Hillier

et al. 2001) and the susceptibility to photo-oxidative damage increases.(Mayes et al. 1991;

Philbrick et al. 1991)

PSII appears to possess its own mechanism and biochemical machinery for regulating the

bicarbonate concentration within the lumenal space near the WOC. A low level of carbonic

anhydrase (CA) activity has been found to be intrinsic to oxygen-evolving PSII membranes(Lu

et al. 2002). Careful analysis showed that after washing the membranes with 1 M CaCl2 to

remove the extrinsic polypeptides, the CA activity could be found in both fractions, an intrinsic

CA associated with the membranes that functions in the hydration direction and an extrinsic CA

in the soluble fraction that functions in the dehydration direction. The soluble CA activity was

9

found associated with a 33 kDa protein that comigrated with the 33-MSP extrinsic protein of

PSII(PsbO). The authors proposed, and later confirmed that these are the same protein and have

demonstrated that the 33 kDa protein of PSII possesses very low level CA activity (~ 60 s-1

vs

105 s

-1 of typical Zn

2+-CA) that is dependent on the concentration of Mn

2+ and Cl

-(personal

communication A. J. Stemler). The latter results are at variance with earlier work showing that

neither Mn2+

nor Ca2+

have specific binding sites on the spinach 33-MSP when studied as a free

protein.(Hunziker et al. 1987) The discrepancy may be resolved if the Mn-dependent CA activity

represents non-specifically associated Mn2+

arising from electrostatic attraction. These results are

provocative and should be tested further to judge whether this CA activity is found in vivo and

how it may be linked, if at all, to the possible function of bicarbonate as an alternate substrate for

O2 evolution.

In cyanobacterial cells MSP binding to the PSII complex is weakened by genetic mutations to

the lumenal e-loop of CP47 which introduce anionic residues(carboxylates) for positive

residues(arginines). These same mutants also support the highest quantum yields for

photoactivation of O2 evolution. The data point to two consequences of the lumenal surface

charges on the e-loop of CP47. In wild type, two arginine groups of the e-loop of CP47 help to

maintain tight MSP binding which blocks achieving a high quantum yield of photoactivation

(presumably under Mn-limiting conditions?), while the anionic mutants bind less MSP and

exhibit the highest yields of photoactivation. The latter result is consistent with photoactivation

studies showing that absorption of anionic molecules (tetraphenylboron) but not cationic

molecules (tetraphenylphosphonium) into isolated PSII membranes accelerates the rate and

increases the quantum yield of photoactivation under conditions of low Mn2+

concentrations.(Ananyev et al. 1996; Ananyev et al. 2001) Thus, surface-bound anionic groups

in PSII, both native and exogenous in origin, are able to attract/steer Mn2+

ions from solution

10

closer to the PSII complex. This role of surface anionic residues at the PSII protein/aqueous

interface is analogous to that played by the surface carboxylates in cation transporter

proteins.(Sansom et al. 2000) This function should not be confused with catalytic residues

involved in the direct coordination of Mn in the native complex.(Ishikawa et al. 2002)

2. Other Extrinsic Proteins. The 23 kDa protein (psbP gene) has been implicated in increasing

the affinity for Ca2+

to the WOC, while the 19 kDa protein (psbQ gene) enhances the Cl- binding

affinity. (Yocum 1991) As with the 33 kDa protein, when all 3 extrinsic proteins are bound, they

form a diffusion barrier that keeps the intrinsic Mn2+

, Ca2+

and Cl- ions sequestered so that the

manganese cluster can be reassembled rapidly upon illumination.(Adelroth et al. 1995;

Wincencjusz et al. 1998) In the absence of the 23- and 19-kDa proteins, oxygen evolution

requires higher concentrations of Ca2+

and Cl- ions, indicating these proteins mediate tighter

binding of these ions.

C. Small Subunits

There are several other intrinsic proteins found in the PSII protein complex, however, their role

in the photo-activation process is not yet clear. PsbI (psbI) 4.2 kDa, PsbTc (psbTc) 3.8 kDa,

PsbW (psbW) 5.9 kDa are intimately associated with the D1/D2 dimer (Debus 2000). The PsbL

(psbL), 4.3 kDa, and PsbK (psbK), 4.3 kDa, proteins are also found with D1/D2. PsbL appears to

aid in electron transfer (YzP680+ Yz

+P680)(Hoshida et al. 1997; Hoshida et al. 1997), while

both have been implicated in plastoquinone binding and are hypothesized to be positioned to

play a role in forming dimeric core units composed of two D1/D2 dimers.(Zheleva et al. 1998)

D. [Mn]4 Core

11

The composition and structure of the inorganic core of the intact PSII-WOC has been examined

by a number of techniques in higher plants and cyanobacteria (primarily Synechocystis 6803 and

Thermosynechococcus). There is emerging consensus that the stoichiometry is Mn4Ca1(OHn)xCly

with as few as 1-2 high affinity chlorides in spinach.(Debus 1992; Ananyev et al. 2001; Carrell et

al. 2002) (Vrettos et al. 2001) The number of tightly bound non-exchangeable O2-

/OH-/OH2

molecules has not been directly established and the proton ionization state depends on oxidation

state.

E. The Calcium Effector Site

Calcium is essential for water oxidation in the native enzyme and can be functionally replaced by

Sr2+

.(Boussac et al. 1988) EPR reveals that removal of Ca2+

from the holoenzyme changes the

distribution of unpaired spin density among the four Mn ions. Inhibition by lanthanides reveals

that the affinity for the Ca site in the holoenzyme increases with charge density.(Ono 2000;

Vrettos et al. 2001). However, this finding differs from photoactivation studies showing that Sr2+

,

a lower charge density ion, binds 8-fold tighter to the Ca effector site in PSII membranes

depleted of the 3 extrinsic subunits (Ananyev et al. 2001). EPR reveals that Ca2+

binding to apo-

WOC-PSII selectively templates the folding of the protein complex into a form that exhibits

higher affinity for Mn2+

and binds two Mn2+

ions as a dimanganese(II,II) binuclear site.(Ananyev

et al. 1997) Strontium XAS of Ca-depleted/Sr-reconstituted PSII indicates a close positioning

(~3.5 Å) between Sr and Mn.(Cinco et al. 1998) Calcium XAS of Ca-sufficient PSII also

suggests a heavy atom scatter in the second shell.(Cinco et al. 2002) These and other data

indicate that Ca2+

directly participates in the catalysis of water splitting as an integral cofactor.

The reader is referred to other chapters on the roles of Cl-, Ca

2+ and bicarbonate in the

holoenzyme.

12

III. Biogenesis of PSII-WOC

A. Subunit Assembly

1. Prokaryotes. The site of oxygenic photosynthesis in cyanobacteria, green algae and

chloroplasts is a specialized inner membrane called the thylakoid system. Photoactivation of the

inorganic cofactors of the WOC occurs within PSII complexes bound to the thylakoid

membrane.(Cheniae et al. 1971) Biochemical and electron micrographic studies of green algae

and chloroplasts indicates that the chloroplast envelope is a major biogenic structure, from which

thylakoid membranes emerge (Hoober et al. 1994). This function also holds true for

cyanobacteria. Analogous to other gram-negative bacteria, cyanobacteria have an envelope

barrier consisting of an outer membrane, a peptidoglycan layer and an inner (plasma) membrane,

which are physically and functionally distinct from the thylakoid membrane.(Gantt 1994) The

two membrane systems exchange components via vesicle transport. The localization of

photosystem proteins in cyanobacterial cells and chloroplasts has been studied by fractionation

methods. Thylakoid and plasma membranes have been purified from Anacystis nidulans.(Smith

et al. 1993) A number of polypeptides from both photosystems were found in both membranes.

In particular, the D1 and extrinsic 33 kDa proteins were localized in both membranes, but the

latter formed functional complexes only in the thylakoid membrane. Both CP43 and CP47 were

confined to the thylakoid membrane. Two-dimensional fractionation methods combined with

immunoblotting analysis has demonstrated that the purified plasma membrane from

Synechocystis 6803 contains a number of protein components closely associated with the

reaction centers of both photosystems.(Zak et al. 2001) Localization of a functional PSI reaction

center capable of primary charge separation was found, but not a functional PSII reaction center.

However, evidence for localization of D1, D2 and cytb559 subunits in the plasma membrane was

13

established, but not CP43 or CP47. The absence of CP47 and CP43 indicates that assembly of a

functioning WOC is highly unlikely in the plasma membrane, based on preliminary observations

that PSII subcomplexes from which both CP43 and CP47 have been dissociated do not assemble

a functional inorganic core during photoactivation.(Buchel et al. 1999)

The newly synthesized D1 protein contains an extension of the carboxyl terminus that is cleaved

by a protease in the mature PSII. The D1 C-terminal extension was shown not to be required for

assembly of functional PSII complexes, nor for growth under optimal conditions in both

Synechocystis 6803(Nixon et al. 1992) and Chlamydomonas.(Lers et al. 1992) However, the

carboxyl extension must be cleaved beyond Ala344 by an soluble protease, called ctpA, prior to

assembly of an active inorganic core.(Nixon et al. 1992; Trost et al. 1997) In Synechocystis 6803

cultures which lack the carboxyl extension or have a genetically engineered two-fold longer

extension no difference in growth rates or O2 evolution rates versus wild type were

detected.(Ivleva et al. 2000) Thus, following cleavage, the carboxyl extension is not involved in

the assembly of an active WOC. Nonetheless, the C-extension in wild-type does provide a

selective advantage, albeit for reason that remain unclear. The timing and location of D1

processing in relation to the sequence of PSII biogenesis has been explored, although much

remains to be understood (Zhang et al. 2000). Translation elongation of newly synthesized D1 is

tightly regulated, as it occurs in vivo only if proper insertion into the thylakoid membrane pre-

exists and a trans-membrane potential is required. However, co- and post-translational assembly

steps of D1 into PSII reaction center and core complexes occur without requirement for

photosynthetic electron transfer or trans-thylakoid proton gradient.

In summary (Table 1), assembly of PSII subunits in prokaryotes begins in the plasma membrane

where the components of the reaction center and 33kDa extrinsic subunit are found in a non-

14

functional state. Final assembly, addition of the two inner antenna subunits of the PSII core

complex, uptake of the inorganic cofactors and binding of a functional extrinsic 33 kDa protein

all occur outside of the plasma membrane in the thylakoid system.

2. Chloroplasts. The thylakoid membrane system in chloroplasts has a more complex structure

than the undifferentiated thylakoid membrane of cyanobacteria. SDS-PAGE of the polypeptide

distribution(Wollenberger et al. 1994) and biophysical studies (Wollenberger et al. 1995;

Mamedov et al. 2000) of the light-harvesting antenna attachment and the localization and

specific O2 evolution activity of PSII-WOC complexes found within different regions of the

thylakoid membrane system in chloroplasts indicates that a gradient exists in the number of

subunits and complexity of functional activity. PSII size and functional complexity increases in

going from the individual stroma lamellae, to the periphery of the appressed multi-membrane

grana lamaellae (so-called margins) to the centers of the appressed grana lamaellae (core grana),

as summarized in Table 1. At least two sub-types of PSII, designated PSII-α and PSII-β, are

known that are distinguished by the number of light-harvesting antenna Chl a/b binding proteins

that funnel energy to the reaction center. PSII-α has more LHC II and a larger antenna size

compared to PSII-β.(Wollenberger et al. 1994) The Mn cluster is absent or inactive in the PSII

found in the stroma membrane; a majority fraction of the peripheral grana PSII-WOC are active

but have slow QA QB electron transfer kinetics; while greater than 80-90% of the core grana

PSII-WOC are fully active in O2 evolution and have normal acceptor side kinetics. (Mamedov et

al. 2000) The authors hypothesize that the “photoactivation” process (here meaning both subunit

assembly and inorganic core uptake) is initiated in the stroma lamellae, diffuses into the grana

periphery, and is completed within the appressed regions of the inner grana core.

15

In summary, studies of PSII-WOC biogenesis in both cyanobacteria and chloroplasts support an

“assembly line” model for the manufacturing of functional PSII core complexes. Beginning with

insertion into the plasma membrane of PSII reaction center subunits (D1, D2, cytb559,

association unknown) and a non-functional version of the 33-kDa MSP (likely pre-protease

processing?). This is followed by transport to stroma lamaellae where acquisition of inefficient

primary electron transport (slow QA QB) occurs. Some CP43 and CP47 is present,(Mamedov

et al. 2000) although gels reveal that CP43 is present at lower levels than the reaction center

subunits.(Wollenberger et al. 1994) Photoactivation of O2 evolution occurs in some centers (≤

40%), but the activity is unstable in the dark, suggesting that stable binding of the extrinsic 33

kDa protein has not been established in those centers that do photoactivate. PSII core assembly

develops further in the grana margins where gels reveal more prominent staining from CP47 and

CP43 and fluorescence reveals opening of QA- QB transfer. Lastly, diffusion to the core grana

lamaellae occurs where the most stable and active PSII core complex-WOC is found, also in

association with additional LHC II subunits that create the light-efficient PSII-β core complex

(Wollenberger et al. 1994; White et al. 1996). The high stability of the O2 evolution activity in

the latter complexes is associated with the binding of the extrinsic 33 kDa protein.(Burnap et al.

1996) Not all of the details of the assembly steps needed to support this biogenesis model have

been tested. However, we note that deletion of a single gene called VIPP1 (vesicle-inducing

protein in plastids 1) is deleterious to thylakoid membrane formation. VIPP1 is a hydrophilic

protein that is found in both the inner envelope and the thylakoid membranes and is a potential

candidate for facilitating protein transport between the two membrane systems.

B. Mn2+

Uptake Into the Cell

16

The Mn content of cyanobacterial cells is generally well in excess of that needed to supply all

PSII complexes, however intracellular distribution and transport are other determinants not as

well known. The capsular peptidoglycan comprising the cyanobacterial envelope plays a major

role in concentrating free Mn2+

from extracellular solution.(Mohamed 2001) Mature cells of

Synechocystis 6803 take up or exchange free Mn2+

at a rate of about 2 x 106 Mn

2+/cell in a period

of 30 minutes.(Ogawa et al. 2002) There are two pools of intracellular Mn2+

which have been

identified.(Keren et al. 2002) The largest pool, A~ 108 atoms/cell, is found closely associated

with the outer membrane possibly on either the outside or the inner leaflet (periplasm). This pool

can be removed by EDTA, is electrogenically generated under light and can be prevented by

electron transport inhibitors (DCMU) or abolished by proton uncouplers (CCCP). A tightly

associated second pool, B ~ 1.5x106

atoms/cell, is not affected by EDTA, light or proton

uncouplers and has traversed the inner membrane. Under Mn2+

starvation conditions pool B

contains about 105

Mn/cell which is essential for photosynthesis, of which the majority is

targeted to PSII. By comparison, the marine cyanobacterium Synechococcus sp. PCC7002

contains 1.5x106 Mn/cell in pool B, while the non-oxygenic purple bacterium Rhodobacter

capsulatus contains 1% of this amount. A two-component regulatory machinery for sensing of

extracellular Mn2+

concentrations and maintenance of intracellular Mn homeostasis in

cyanobacteria and non-photosynthetic prokaryotes has begun to emerge. (Ogawa et al. 2002)

C. Inorganic Core Assembly Involves no Protein Chaperones

Intracellular transport of transition metal ions used in the enzymatic machinery is typically under

tight control by proteins or small molecule chelators that ensure proper targeting of the metal to

its final destination and also protect the nascent system from unwanted reactions. These

“chaperone” proteins are widely distributed in the case of iron and copper regulator systems. The

17

thermodynamic potentials for aqueous Fe2+

/Fe3+

and Cu+/Cu

2+ metals indicate that these metals

are susceptible to redox changes within cells and thus will undergo major changes in speciation

if not coordinated during transport. Thus it may seem curious that no essential chaperone protein

has yet been identified for delivery of Mn2+

to the apo-WOC-PSII complex during biogenesis.

The explanation is likely due to the inaccessibility of the Mn3+

oxidation state in aqueous media

at neutral pH owing to the large thermodynamic barrier which prevents Mn2+

oxidation (standard

reduction potential 1.2 V at pH 7 vs NHE). Moreover, divalent Mn2+

with its spherical

electronic distribution and half-filled 3d5 electronic configuration (two sigma anti-bonding

electrons) forms relatively weak coordination complexes with all monodentate O, N, S

ligands.(Smith et al. 1976) Thus, free Mn2+

does not generally associate tightly with the internal

machinery of the cell unless there are preorganized chelation sites present with multiple ligand

donors. Generally, the latter sites are specific for ions of a particular size, charge and dehydration

energy so that biosynthetic discrimination is almost always ensured. Surface electrostatics plays

a major role in guiding metal ions to their functional binding sites and this mechanism is

important for steering Mn2+

to the apo-WOC-PSII site.(Ananyev et al. 1996) Binding of aquo-

Mn2+

to the high affinity site in apo-WOC-PSII is influenced by three factors: alkaline pH,

bicarbonate ions and Ca2+

are discussed in the next sections. Although no chaperone for Mn2+

insertion appears to exist, repair of the PSII protein complex caused by light absorption does

involve chaperone-like proteins.(Schroda et al. 2001)

A new and novel protein called Slr0286 was identified in Synechocystis 6803 which has no

homologues in other organisms and is not known to be associated with PSII.(Kufryk et al. 2001)

Deletion of slr0286 did not affect photoautotrophic capacity in wild type, but led to a marked

decrease in the population of Ca2+

bound to the WOC of PSII. A chaperone role for slr0286 in

18

photoactivation was postulated. However, an alternate role in elevating the Ca2+

supply within

the lumenal space of the thylakoid membrane would also be consistent with the data.

IV. Roles of the Inorganic Cofactors from Photo-assembly

A. Assembly of the Inorganic Core

1. Instrumentation. In 1995-96 we constructed a modified Clark-type electrochemical cell for

the detection of dissolved O2 gas in photosynthetic samples (Ananyev et al. 1995; Ananyev et al.

1996; Ananyev et al. 1996). It enabled measurements of O2 concentration with exceptional

sensitivity (50 femtomoles O2), micro-volume sample volume (5 μL) and 10-fold faster time-

resolution than the commercially available Clark electrodes. The electronic layout and novel

material innovations have been described and circuit diagrams are available upon request. It

features a platinum-iridium (Pt:Ir = 75:25) electrode which is 10 times harder than Pt and more

chemically resistant to oxidation, reducing the formation of PtO2 and the resulting photocurrents.

By using a thin (1 m) silicone membrane instead of Teflon, both rapid response (0.1 second rise

time) and substantial reduction in the dark current and loss of membrane fatigue were achieved.

The electrode assembly includes an integral preamplifier, further reducing susceptibility to

transients. This cell is integrated with a versatile LED light source for both red and blue

wavelength excitation, digital control of the pulse-shape and duration (0.5 ms to infinity), and

digital data acquisition system and programming environment (Labview). This instrument

permits measurement of the rate and yield of O2 production in samples containing 1 M PSII

centers and exhibiting as little as 0.5% activity and, by integration, as little as 0.05% of the

activity.

19

2. Kinetics and Mechanism. In the 1960s George Cheniae discovered the photoactivation

process, and together with his coworkers established many of the fundamental properties of the

WOC over the next three decades. This includes determination of the Mn stoichiometry (3-5

Mn/PSII), cooperative nature of the Mn association, and two-step sequence of intermediates

(light-dark-light) required for reconstitution of O2 evolution.(Frasch et al. 2001) The insights

provided by the pioneering results from his laboratory are an enduring legacy made more

remarkable by the fact that they were performed using simple tools: a Petri dish, a commercial

Clark electrode and a light bulb as the photoactivation reactor.

Application of the photoactivation instrument described above has enabled kinetic resolution of

the first two steps prior to the rate-limiting step in the assembly of the inorganic

cofactors.(Ananyev et al. 1996) The steps that follow the rate-limiting step are not individually

resolved, but their Mn stoichiometry needed to restore O2 evolution has been determined.

Photoactivation follows a two-step sequence, as originally discovered by Cheniae and Tamura on

the basis of steady-state O2 yield measurements(Tamura et al. 1987) and confirmed by Miller

and Brudvig.(Miller et al. 1989) Kinetic resolution of these steps revealed a reversible, light-

dependent lag phase where no O2 is produced, followed by a single exponential recovery

phase(Zaltsman et al. 1997). The kinetic resolution provides values of the three rate constants

shown in Figure 1 (k1, k-1 and k2). The sensitivity of the cell permits measurement of these rate

parameters using sub-stoichiometric concentrations and higher of Mn2+

. This permitted

exceptionally accurate titrations of the Mn stoichiometry required for O2 evolution, based on

measuring both an extensive variable (O2 flash yield)(Ananyev et al. 1996) and an intensive

variable (initial rate of recovery of O2 evolution capacity).(Ananyev et al. 2001) The latter

approach provided a particularly accurate assay of Mn stoichiometry because it does not require

determination of the number of apo-WOC centers. The photoactivation of PSII-WOC in whole

20

cells appears to occur similarly to the isolated PSII core complex. The ability to examine the role

of accessory proteins in assembly of the inorganic core under native conditions is possible.

(Burnap et al. 1996; Qian et al. 1997)

Two mild methods have been described for removal of the inorganic cofactors and extrinsic

proteins from PSII complexes (apo-WOC-PSII) that is essential for quantitative work and avoids

the denaturation created by the alkaline TRIS method (Ananyev et al. 1996; Ananyev et al. 1998;

Baranov et al. 2004).

Application of these methods has enabled measurement of the following characteristics of the

photoactivation process, primarily using spinach PSII membranes and PSII core

complexes(Ananyev et al. 1995; Ananyev et al. 1996; Ananyev et al. 1996; Ananyev et al. 1998;

Ananyev et al. 2001): (1) optimization of the total yield of reactivated holo-enzyme capable of

O2 production (70%-100%); (2) a molecularity of 1.0 Mn2+

/PSII on the first photooxidation step;

(3) linear light intensity dependence of the first step reflecting a single turnover that forms Mn3+

;

(4) determination of the proton stoichiometry of the first two steps (1H+ + 1H

+, as described in

the next section).; (5) determination of the rate constant of the subsequent rate-limiting dark step

that is independent of Mn2+

concentration and exhibits a molecularity of approximately 1

Ca2+

/PSII (step IM1 IM2*); (6) the Ca2+

affinity of this site (denoted the Ca2+

effector site)

increases following binding and photoxidation of the high affinity Mn2+

, reflecting mutual

cooperativity in binding; (7) subsequent rapid light and dark steps that are kinetic unresolved and

involve the uptake of 3.0 Mn2+

/PSII; (8) strong cooperativity in the uptake of the Mn ions is seen

in Mn2+

titration of the final O2 evolution flash (25, 50, 75 and > 90% with 1, 2, 3, and 4

Mn/PSII).

3. Proton Evolution. Alkaline pH accelerates the rate of Mn-dependent photoactivation by 10-

fold between pH 5.5 and 7.5, while the final yield of O2 per flash follows closely the known pH

21

dependence of the holo-enzyme. The data indicate that protons are released during the first two

steps of assembly (Ananyev et al. 1996; Ananyev et al. 2001). Direct measurements of proton

release during photoactivation have been described using a commercial ISFET (ion-selective

field-effect transistor) detector.(Ananyev et al. 2001) There are advantages to the ISFET detector

that can help resolve ambiguities obtained with glass electrodes or pH dyes as detectors.

Measurement of the proton concentration in bulk solution during photoactivation by flashes

reveals that a single proton is released upon photooxidation of the initial dark precursor,

attributed to MnII(OH)

+ based on studies of Cesium inhibition (see below). Thus, the first light-

induced assembly intermediate contains one fewer proton and is formulated as, MnIII

(OH)2+, in

which ionization of a water ligand is postulated.

4. Oxo/Hydroxo/Bicarbonate Sites. Bicarbonate ions were shown to efficiently replace alkaline

pH buffers in accelerating the rate of photoactivation, while increasing the yield of reactivated

centers, quite contrary to the reduction seen upon alkalinization. Bicarbonate binds to two sites

within apo-WOC complex as depicted in Figure 2 and described next. A high affinity site

operates at concentrations as low as 10 μM in the presence of stoichiometric Mn2+

/PSII

concentrations where it accelerates the first step of Mn2+

binding and/or photooxidation (Baranov

et al. 2000). The high affinity site is attributed to the ionization of carboxylate residues or

neutralization of cationic residues on the surface of the WOC protein complex, possibly the D1

protein, which electrostatically attract Mn2+

from solution close to the WOC (Baranov et al.

2004). The effect is much like that seen with lipid soluble anions like tetraphenylboron(Ananyev

et al. 1996). In more extensive studies covering 10 and 100 fold higher concentrations of Mn2+

and Ca2+

, respectively, a second bicarbonate binding site to apo-WOC-PSII having lower affinity

that depends upon the Mn2+

concentration has been identified (hence called the ternary site).

Bicarbonate produces a much larger 3-fold rate acceleration at the ternary site. The data suggest

22

that bicarbonate may serve to deliver of oxide/hydroxide needed for Mn-oxo/hydroxo assembly

or, possibly, as an integral cofactor within the WOC (Baranov et al. 2004). Since its conjugate

acid CO2 is highly soluble in the membrane phase, there will be higher concentration of

bicarbonate near the thylakoid membrane than in bulk solution at equilibrium in any system

which gets all of its inorganic carbon from CO2.

At higher (millimolar) concentrations of bicarbonate and Mn2+

the formation of free Mn2+

-

bicarbonate complexes occurs in solution. The formation of these species coincides with a

reduction in the O2 yield and slowing of the rate of photoactivation. These free complexes appear

to serve as a competitive electron donors to the functional site for photoassembly (ie., the ternary

site, Figure 2). Recent EPR studies of the role of calcium have extended the model in Figure 2

by establishing an electronic interaction between MnIII

and Ca2+

in the first photo-intermediate,

IM1 (Baranov et al. 2004).

5. Calcium. The kinetics of photoactivation reveal that Ca2+

binding affinity increases following

photooxidation of the high affinity Mn2+

. Ca2+

binding is coupled to a slow dark process that is

postulated to represent a protein conformational change. This process leads to more rapid uptake

and photooxidation of the remaining 3 Mn2+

.(Zaltsman et al. 1997) EPR spectroscopy reveals

that Ca2+

binding in the dark also induces binding of a dimanganese(II,II) center to apo-WOC-

PSII.(Ananyev et al. 1997) The yield of this Ca-induced dimanganese signal is optimal at a ratio

of 2 Mn/PSII, and saturates with increasing Ca2+

concentration. The calcium concentration for

induction of the Mn2(II,II) signal matches the value observed for both the activation of steady-

state O2 evolution and also the light-induced assembly of the functional WOC by

photoactivation. Formation of the Mn2(II,II) signal is specific for Ca2+

as it does not occur in the

presence of Mg2+

. The spectral features of this signal are indicative of a variety of weakly

23

interacting Mn2(II,II) pairs that have Mn2+

ions coupled by magnetic dipolar forces in the range

of inter-manganese separations of ca. 4.1 ± 0.4 Å.(Khangulov et al. 1995) This Ca-induced

Mn2(II,II) center is a more efficient electron donor to the photooxidized tyrosine radical, Yz ,

than is the mononuclear Mn2+

center present in the absence of Ca2+

, as measured by the Yz▪

radical yield. Formation of the Mn2(II,II) EPR signal by addition of Ca2+

correlates with

reduction of flash-induced catalase activity, indicating that calcium modulates the accessibility or

reactivity of the Mn2(II,II) core with added H2O2. These EPR studies indicates that Ca2+

organizes the binding site for higher affinity cooperative binding of the first two Mn2+

ion to the

apo-WOC protein.

The photoactivation and spectroscopic data noted above provide a self-consistent picture locating

the Ca effector site as an integral part of the Mn4 core, probably via shared bridging ligands to

Mn (aqua/hydroxo/carboxylato/chloro) (Carrell et al. 2002). Binding of the calcium cofactor to

Ca-depleted holoenzyme induces an increase in the energy of the Mn k-shell electron edge by

0.6-1.0 eV in three oxidation states (S1, S2 and S3 states) where Ca-depletion/reconstitution has

been measured by XANES.(reviewed in (Carrell, 2002 #2198). Therefore, a reasonable

interpretation of the role of Ca2+

is that it serves as an electrostatic promoter to raise the Mn

oxidation potential. This increase could be achieved if Ca2+

were to increase the extent of

electron transfer from shared (bridging) ligands (oxo, hydroxo, aquo?) to the empty 4p valence

orbitals on Mn. This interpretation suggests a possible direct role for Ca2+

in increasing the Mn-

ligand covalency and thus activating the Mn-water (substrate) molecule(s) for easier oxidation.

We have postulated that this role may involve Ca-induced ionization of protons from the

substrate water molecules that are bound to the Mn4 cluster(Carrell et al. 2002). This role is also

consistent with the observed correlation between equilibrium binding affinity for divalent metals

bound to the Ca-effector site and their pKa (Vrettos et al. 2001).

24

6. Protein Subunits Required for Photoactivation. Büchel and coworkers demonstrated that the

smallest PSII protein complex that is capable of catalyzing water oxidation is the CP47RC

subcore complex(Büchel et al. 1999). This complex is isolated from thylakoid membranes by

fractionation of a detergent-solubilized complex. It is comprised of only four major subunits (D1,

D2, Cytb559 and CP47) and a number of small subunits, but lacks CP43 and all of the inorganic

cofactors of the WOC. The isolated complex is inactive in O2 evolution, but does photoactivate

at slower rate (lower quantum efficiency) and produces centers having O2 flash yield of 30% vs

PSII membranes. These studies established that CP43 is not an essential subunit for water

splitting, but does provide a functional advantage for both assembly and O2 evolution activity.

YD is the symmetry related photoactive tyrosine in the D2 subunit(tyr-160) that is homologous to

the essential photoactive tyrosine-161 of the D1 subunit of PSII (YZ). It has been implicated in

stabilizing the higher S-states and in photoactivation, (Styring et al. 1987) but is not essential as

mutants lacking YD grow photoautotrophically, although at much slower rates. Diner and

coworkers investigated YD in both wild-type and a mutant strain (D2-Y160F) in which

phenylalanine replaces YD in the cyanobacterium Synechocystis sp. The quantum yield for O2 in

the intact holo-enzyme was found to be identical in the mutant and wild-type PSII cores using

long (saturating) pulses or continuous illumination, confirming earlier work. However, the O2

yield was shown to be appreciably reduced in the mutant using short non-saturating light pulses

(< 50 ms). Evidence was presented that the positive charge associated with the presence of a hole

on the YD-HisH+ radical center may promote the higher quantum yield for O2 production in wild-

type centers via increasing the oxidation potential of P680+, thereby accelerating the rate of

electron transfer from Mn4YZ to P680+. It was also found that the mutant can assemble a

functional WOC from the free inorganic cofactors, but at a 3-fold slower rate and with reduced

25

quantum efficiency vs wild type. The steps responsible for the slower photoactivation kinetics in

the mutant were identified. These features indicate that the YD▪ radical plays at least two

important roles during assembly of a functional WOC, including increasing the probability of

oxidation of Mn2+

in the dark and suppressing decay of the assembly intermediates by charge

recombination at low light flux. They contribute a competitive advantage to organisms that retain

the YD residue and therefore may account for its retention in the genome of all oxygenic

photoautotrophs.

B. “Inorganic Mutants” of the Manganese and Calcium Sites

Inhibitor binding at the high affinity Mn2+

site is characterized by slowing of the rate constant k1

and is competitive with Mn2+

concentrations. Acceleration of the decay of the first photooxidized

MnIII

intermediate is described by rate constant k-1. Inhibition at the Ca-effector site is

characterized by slowing of the rate constant k2 and is competitive with Ca2+

concentration.

1. Manganese Mutants. Mn appears to be unique in catalyzing O2 evolution in all oxygenic

photoautotrophs. No other metals have been found to date that can replace it. We have examined

several metal ions as potential surrogates for Mn2+

in the assembly process of spinach apo-WOC-

PSII.(Ananyev et al. 2001) These studies were conducted by replacing Mn2+

, while keeping all

other components fixed at the optimal concentrations for Mn-dependent photoactivation (Cl-,

Ca2+

and the electron acceptor ferricyanide). The following metal ions were found to inhibit Mn-

dependent photoactivation with varying affinities and none were found capable of supporting O2

evolution in the absence of Mn2+

(Cs+, Ba

2+, VO

2+, V

3+, Cr

3+, Fe

2+, Co

3+, Ni

2+, Cu

2+, Zn

2+,

MoO42-

,Ru3+

, Rh3+

, Re3+

, UO22+

). However, only a very limited range of conditions were studied:

26

fixed pH 6 and concentrations of Ca2+

, Cl- and electron acceptor. Other pH and concentrations

conditions ought to be examined before it is known for sure whether Mn can be replaced.

Cesium (Cs+) is the strongest, competitive, inhibitor of the high affinity Mn

2+ site, while the

smaller alkali metal ions bind with progressively weaker affinity (Cs+> Rb

+> K

+> Na

+> Li

+). The

equilibrium thermodynamic binding affinities were measured for all alkali metal ions and a clear

correlation with charge density was seen. Cs+ binding is highly selective for the Mn site, versus >

104

weaker binding to the Ca effector site. These data and indicate that Cs+ mimics the size and

charge of the functional Mn2+

species that binds to apo-WOC-PSII and initiates the assembly

process. Assignment of this dark precursor to Mn(OH)+ rather than Mn

2+ is implied by these

results. This conclusion is in full accord with the proton evolution results noted above and the

bicarbonate dependence. This proposal is further supported by the observation that metal-oxo

cations like UO22+

and VO2+

are among the strongest competitive inhibitors of the Mn site. These

results enabled the Mn and hydroxide composition of the first two assembly intermediates to be

determined (up to IM1, Figure 1).

2. Calcium Mutants Boussac discovered that Sr2+

can functionally exchange Ca2+

in the intact

holo-enzyme of PSII membranes by restoring O2 evolution activity.(Boussac et al. 1988) Later

we found that there is a kinetic advantage for uptake of Sr2+

versus Ca2+

at the Ca-effector site

during assembly of the apo-WOC enzyme and determined how each of the three rate constants

measured by photoactivation are affected (Table 2). (Ananyev et al. 2001) Sr2+

was shown to be

five times more effective than Ca2+

in accelerating the rate of the first two assembly steps (k1 and

k2) and two times better in retarding the deactivation step (k-1). Sr2+

competes with Ca2+

and thus

occupies the Ca-effector site of the photoassembled PSII. A 65% lower O2 evolution yield per

flash is obtained for the Sr-photoactivated enzyme, which is very close to the decreased steady-

27

state O2 evolution rate in the Sr-exchanged holo-enzyme. This decrease in O2 flash yield is

known to reflect a retardation of the rate of the final step, S3 S0 + O2.(Westphal et al. 2000)

Thus, the data taken together indicate that Ca2+

is clearly involved in accelerating the final step

in the S-state catalytic cycle. This step includes the O-O bond formation reaction. No other metal

ion other than Sr2+

has been found to functionally replace Ca2+

in water splitting.

Another interesting probe of the Ca2+

site is the vanadyl ion. VO2+

stimulates Mn-dependent

photoactivation by accelerating the first step (k1) and increasing the yield of photoactivated

centers when using stoichiometric Mn2+

concentrations or less (Table 2).(Ananyev et al. 2001)

The apparent Michaelis constant at this site is 15 M. By contrast, at higher concentrations

vanadyl slows the first step and reduces the overall yield of photoactivated centers (I50~200 M)

by serving as a competitive electron donor to Yz. The location of the VO2+

binding site appears

to be the Ca-effector site, since Ca2+

blocks the inhibition by VO2+

. Previous studies had

observed stimulation of Mn-dependent O2 evolution by VO2+

in the absence of Ca2+

.(Lockett et

al. 1990) However, by using the photoactivation method we were able to show that this result is

likely due to mobilization of residual Ca2+

by vanadyl ions. The vanadyl stimulation of the rate

constant k1 (Mn site) through binding at the Ca2+

site suggests a cooperative interaction between

the two sites, with binding of VO2+

at the Ca-effector site causing higher affinity binding of Mn2+

at the Mn site. This cooperativity suggests the possibility of either electrostatic or conformational

coupling of the two sites. This interpretation is consistent with equilibrium binding studies

showing that lanthanides of higher charge density bind more strongly to the Ca-depleted holo-

enzyme.(Ono 2000) This cooperativity between the Mn and Ca sites provides a possible

mechanism for the observed correlation in other physico-chemical properties, including XAS and

EPR properties, noted previously. Alternatively, tt is possible that the acceleration of the Mn-

dependent k1 step observed by the VO2+

ion is due to stimulation of proton evolution from MnII

28

or MnIII

, since vanadyl is a weak base (pK1 = 4.3, pK2 = 11.5). In previous studies we found that

organic bases stimulate the rate of the initial step of photoactivation.(Ananyev et al. 2001)

Evidence was presented that the mechanism involves stimulation of proton evolution from (or

hydroxide binding to) Mn (assembly of MnII(OH)

+ and Mn

III(OH)2

+). Distinction between these

alternative proposals for the vanadyl effect on k1 may be resolved by examination of the effect

on photoactivation of lanthanides binding to the Ca-effector site.

3. Other Metal Ion Sites. Recently, Cu2+

was shown to bind to PSII complexes from tobacco at

stoichiometric concentrations and produce stimulation of the electron transfer rate from

water.(Burda et al. 2002) The location of the Cu2+

binding site was not identified, but the authors

speculated a possible site in the WOC. This finding is novel as there is no precedent for an

endogenous functional Cu2+

site in any WOC. An alternative binding site for this novel Cu2+

may

be the acceptor side, based on data showing that a binding site for exogenously added Ni2+

or

Zn2+

has been identified near the QB acceptor in purple bacterial reaction centers and found to

slow the rate of proton transfer into reduced QB2-

.(Utschig et al. 1998; Keller et al. 2001)

V. Concluding Remarks

The universality of the inorganic core of the WOC among all oxygenic phototrophs, its high

efficiency of assembly and the absence of protein chaperones during assembly indicates that the

inorganic core can be assembled spontaneously from elementary ions, light and the apo-WOC

protein. Kinetically, the process is simple. It can be resolved into only two steps in which 1 Mn2+

and 1 Ca2+

are taken up in light and dark steps, respectively, followed by rapid light-dependent

uptake of 3 Mn2+

. This kinetic simplicity indicates minimal control by the protein and thus

implies the core structure may be intrinsically stable relative to other core topologies. Hence, we

can expect it to be among the known Mn4Ox core types previously prepared or accessible by self-

29

assembly (Mukhopadhyay et al. 2002). This sequence of uptake of the cofactors during

photoactivation, depicted in Figure 1, the strong cooperativity in binding of the high affinity

Mn3+

and Ca2+

ions, their coupling to proton release, and the rapid cooperative uptake of the

remaining 3 Mn2+

ions provide support for two of the eight possible Mn4Ox core types proposed

on the basis of EPR/EXAFS spectroscopies (Carrell et al. 2002). The two Mn4Ox core types

deduced by EPR/EXAFS that are consistent with the photoactivation data are depicted in Figure

3. The placement of a Ca2+

ion within each of these structures located along the pseudo three-

fold axis produces core types that are structurally in good agreement with the Mn4Ca1

heterocubane core type proposed on the basis of XRD of a cyanobacterial PSII core complex

(Ferreira et al. 2004). We may conclude that the Mn4Ca heterocubane core topology is

supported not only by the available spectroscopic data from various sources, including XRD,

EPR, and EXAFS, but also with the photoactivation data on assembly.

Acknowledgements

We thank Drs. M. Seibert, H. Pakrasi and J. Barber for preprints. GCD appreciates helpful

discussions with Drs. S. Baranov, A. Tyryshkin and A Stemler.

30

References

Adelroth P, Lindberg K and Andreasson L-E (1995) Studies of Ca2+

binding in spinach

photosystem II using 45

Ca2+

. Biochem 34: 9021--9027.

Ananyev GM and Dismukes GC, Eds. (1995) Photoassembly of the tetra-Mn site of

photosynthetic water oxidation: EPR evidence for dark ligation of a binuclear Mn

intermediate. Montpellier, Kluwer. Photosynthesis: From Light to Biosphere.

Montpellier, Kluwer.

Ananyev GM and Dismukes GC (1996) Assembly of the tetra-mn site of

photosynthetic water oxidation by photoactivation: Mn stoichiometry and detection of a

new intermediate. Biochem 35(13): 4102--4109.

Ananyev GM and Dismukes GC (1996) High-resolution kinetic studies of the

reassembly of the tetra- manganese cluster of photosynthetic water oxidation: Proton

equilibrium, cations, and electrostatics. Biochem 35(46): 14608--14617.

Ananyev GM and Dismukes GC (1997) Calcium induces binding and formation of a

spin-coupled dimanganese(II,II) center in the apo-water oxidation complex of

photosystem ii as precursor to the functional tetra-mn/ca cluster. Biochem 36(38):

11342--11350.

Ananyev GM, Zaltsman L, McInturff RA and Dismukes GC (1998). Assembly

intermediates and "inorganic mutants" of the psII oxygen evolving complex. In: G

Garob. II: 1347--1350. Dordrecht, Kluwer.

Ananyev GM, Zaltsman L, Vasko C and Dismukes GC (2001) The inorganic

biochemistry of photosynthetic oxygen evolution/water oxidation. BBA-Bioenergetics

1503(1-2): 52--68.

Babcock GT and Sauer K (1975) A rapid, light-induced transient in electron

paramagnetic resonance signal II activated upon inhibition of photosynthetic oxygen

evolution. Biochim Biophys Acta 376(2): 315--28.

Baranov S, Tyryshkin A, Katz D, Dismukes G, Ananyev G and Klimov V (2004)

Bicarbonate is a native cofactor for assembly of the manganese cluster of the

photosynthetic water oxidizing complex: II. Kinetics of reconstitution of O2 evolution

by photoactivation†. Biochem 43: 2070--2079.

Baranov SV, Ananyev GM, Klimov VV and Dismukes GC (2000) Bicarbonate

accelerates assembly of the inorganic core of the water-oxidizing complex in

manganese-depleted photosystem II: A proposed biogeochemical role for atmospheric

carbon dioxide in oxygenic photosynthesis. Biochem 39(20): 6060--6065.

Boussac A, F R, Carrier P, Verbavatz J-M, Gobin R, Kirilovsky DL, Rutherford AW

and Sugiura M (2004) Biosynthetic Ca2+

/Sr2+

exchange in the photosynthetic oxygen

evolving enzyme of thermosynechoccocus elongatus. J Biol Chem 279(22): 22809--19.

31

Boussac A and Rutherford AW (1988) The nature of the inhibition of the oxygen

evolving enzyme of psII which is induced by NaCl washing and reversed by the

addition of Ca2+ or Sr2+. Biochem 27: 3476--3483.

Bricker TM (1992) Oxygen evolution in the absence of the 33-kilodalton manganese-

stabilizing protein. Biochem 31(19): 4623--4628.

Büchel C, Barber J, Ananyev GM, Eshaghi S, Watt R and Dismukes GC (1999)

Photoassembly of the manganese cluster and oxygen evolution from monomeric and

dimeric CP47-reaction center photosystem II complexes. Proc Nat Acad Sci USA

96(25): 14288--14293.

Burda K, Kruk J, Stralka K and Schmid GH (2002) Stimulation of oxygen evolution ins

psii by copper(II) ions. Z Naturforsch 57 C: 853--857.

Burda K and Schmid GH (2001) Heterogeneity of the mechanism of water splitting in

photosystem II. BBA-Bioenergetics 1506(1): 47--54.

Burnap RL, Qian M and Pierce C (1996) The manganese-stabilizing protein of

photosystem II modifies in vivo deactivation and photoactivation kinetics of the H2O

oxidation complex in synechocystis sp. Pcc6803. Biochem 35: 874--882.

Burnap RL, Shen JR, Jursinic PA, Inoue Y and Sherman LA (1992) Oxygen yield and

thermoluminescence characteristics of a cyanobacterium lacking the manganese-

stabilizing protein of photosystem-II. Biochem 31(32): 7404--7410.

Burnap RL and Sherman LA (1991) Deletion mutagenesis in synechocystis sp pcc6803

indicates that the Mn-stabilizing protein of photosystem-II is not essential for O2

evolution. Biochem 30(2): 440--446.

Campbell KA, Force DA, Nixon PJ, Dole F, Diner BA and Britt RD (2000) Dual-mode

EPR detects the initial intermediate in the photoassembly of the PSII Mn cluster. J Am

Chem Soc 122: 3754--3761.

Carrell TG, Tyryshkin A and Dismukes GC (2002) An evaluation of structural model

for the photosynthetic water-oxidizing complex derived from spectroscopic and x-ray

diffraction signatures. J Biol Inorg Chem 7: 2--22.

Cheniae GM and Martin IF (1971) Photoactivation of manganese catalyst of O2

evolution .1. Biochemical and kinetic aspects. Biochim Biophys Acta 253(1): 167--181.

Chu H-A, Nguyen AP and Debus RJ (1994) Site-directed PSII mutants with perturbed

oxygen evolving properties. 1. Instability or inefficient assembly of the Mn cluster in

vivo. Biochem 33: 6137--6149.

Cinco RM, McFarlane Holman KL, Robblee JH, Yano J, Pizarro SA, Bellacchio E,

Sauer K and Yachandra VK (2002) Calcium exafs establishes the Mn-Ca cluster in the

oxygen-evolving complex of photosystem II. Biochem 41(43): 12928--12933.

32

Cinco RM, Robblee JH, Rompel A, Fernandez C, Yachandra VK, Sauer K and Klein

MP (1998) Strontium EXAFS reveals the proximity of calcium to the manganese

cluster of OEC-PSII. J Phys Chem B 102: 8248--8256.

Debus RJ (1992) The manganese and calcium ions of photosynthetic oxygen evolution.

Biochim Biophys Acta 1102: 269--352.

Debus RJ (2000). The polypeptides of photosystem ii and their influence on

manganotyrosyl-based oxygen evolution. In: Metal ions in biological systems:

Manganese and its role in biological processes. A Sigel, Sigel, H.37: 657--711. New

York, Marcel Dekker, Inc.

Dismukes GC and Blankenship RE (this series). The origin and evolution of

photosynthetic oxygen production. In: Photosystem II: The water/plastoquinone oxido-

reductase in photosynthesis. T Wydrzynski and K Satoh.Ch 32. Dordrecht, Kluwer.

Ferreira KN, Iverson TM, Maghlaoui K, Barber J and Iwata S (2004) Architecture of

the photosynthetic oxygen-evolving center. Science 303(19 March): 1831--38.

Frasch W and Sayre RT (2001) Remembering George Cheniae. Photosyn Res 70: 245--

247.

Gantt E (1994). In: The molecular biology of cyanobacteria. D Bryant: 119--138.

Dordrecht, Kluwer.

Ghirardi ML, Lutton TW and Seibert M (1998) Effects of carboxyl amino acid

modification on the properties of the high-affinity, manganese-binding site in

photosystem II. Biochem 37: 13559--13566.

Hankamer B, Morris E, Nield J, Gerle C and Barber J (2001) Three-dimensional

structure of the photosystem II core dimer of higher plants determined by electron

microscopy. J Struct Biol 135(3): 262--269.

Hillier W, Hendry G, Burnap RL and Wydrzynski T (2001) Substrate water exchange

in photosystem II depends on the peripheral proteins. J Biol Chem 276(50): 46917--24.

Hong SK, Pawlikowski SA, Vander Meulen KA and Yocum CF (2001) The oxidation

state of the photosystem ii manganese cluster influences the structure of manganese

stabilizing protein. Biochim Biophys Acta 1504(2-3): 262--74.

Hoober JK, White RA, Marks DB and Gabriel JL (1994) Biogenesis of thylakoid

membranes with emphasis on the process in chlamydomonas. Photosyn Res 39(1): 15--

31.

Hoshida H, Nakano Y and Toyoshima Y (1997) PSII-L is required in the electron

transfer at the donor side of P-680 to generate Y(z)(+)P(680)Pheo(-)state in PSII. Plant

Physiol 114(3): 1032--1032.

33

Hoshida H, Sugiyama R, Nakano Y, Shiina T and Toyoshima Y (1997) Electron

paramagnetic resonance and mutational analyses revealed the involvement of

photosystem II-L subunit in the oxidation step of Tyr-Z by P-680(+) to form the Tyr-

Z(+)P(680)Pheo(-) state in photosystem II. Biochem 36(40): 12053--12061.

Hunziker D, Abramowicz DA, Damoder R and Dismukes GC (1987). Manganese and

calcium binding properties of the extrinsic 33 kDa protein and of photosystem II

membranes. In: Prog Photosyn Res. J Biggins.I: 5997--5600. Dordrecht, Martinus-

Nijhoff.

Ishikawa Y, Yamamoto Y, Otsubo M, Theg SM and Tamura N (2002) Chemical

modification of amine groups on psII protein(s) retards photoassembly of the

photosynthetic water-oxidizing complex. Biochem 41(6): 1972--1980.

Ivleva NB, Shestakov SV and Pakrasi HB (2000) The carboxyl-terminal extension of

the precursor D1 protein of photosystem II is required for optimal photosynthetic

performance of the cyanobacterium synechocystis sp PCC 6803. Plant Physio 124(3):

1403--1411.

Kashino Y, Lauber WM, Carroll JA, Wang QJ, Whitmarsh J, Satoh K and Pakrasi HB

(2002) Proteomic analysis of a highly active photosystem II preparation from the

cyanobacterium synechocystis sp PCC 6803 reveals the presence of novel polypeptides.

Biochem 41(25): 8004--8012.

Katoh H, Itoh S, Shen JR and Ikeuchi M (2001) Functional analysis of psbV and a

novel c-type cytochrome gene psbV2 of the thermophilic cyanobacterium

thermosynechococcus elongatus strain BP-1. Plant Cell Physiol 42(6): 599--607.

Kavelaki K and Ghanotakis DF (1991) Effect of the manganese complex on the binding

of the extrinsic proteins (17-Kda ; 23-Kda and 33-Kda) of photosystem-II. Photosyn

Res 29(3): 149--155.

Keller S, Beatty JT, Paddock M, Breton J and Leibl W (2001) Effect of metal binding

on electrogenic proton transfer associated with reduction of the secondary electron

acceptor (Q(B)) in rhodobacter sphaeroides chromatophores. Biochem 40(2): 429--439.

Keren N, Kidd MJ, Penner-Hahn JE and Pakrasi HB (2002) A light-dependent

mechanism for massive accumulation of manganese in the photosynthetic bacterium

synechocystis sp. PCC 6803. Biochem 41: 15085--15092.

Khangulov SV, Pessiki PJ, Barynin VV, Ash DE and Dismukes GC (1995)

Determination of the metal-ion separation and energies of the 3 lowest electronic states

of dimanganese(Ii,Ii) complexes and enzymes - catalase and liver arginase. Biochem

34(6): 2015--2025.

Kufryk GI and Vermaas WF (2001) A novel protein involved in the functional

assembly of the oxygen-evolving complex of photosystem II in synechocystis sp. PCC

6803. Biochem 40(31): 9247--55.

34

Lers A, Heifetz PB, Boynton JE, Gillham NW and Osmond CB (1992) The carboxyl-

terminal extension of the d1 protein of photosystem-II is not required for optimal

photosynthetic performance under CO2-saturated and light-saturated growth-

conditions. J Biol Chem 267(25): 17494--17497.

Leuschner C and Bricker TM (1996) Interaction of the 33 kDa extrinsic protein with

photosystem II: Rebinding of the 33 kDa extrinsic protein to photosystem II

membranes which contain four ; two ; or zero manganese per photosystem II reaction

center. Biochem 35(14): 4551--4557.

Lockett CJ, Demeriou C, Bowden SJ and Nugent JA (1990) Studies on calcium

depletion of PSII by ph 8.3 treatment. Biochim Biophys Acta 1016: 213--218.

Lu YK and Stemler AJ (2002) Extrinsic photosystem II carbonic anhydrase in maize

mesophyll chloroplasts. Plant Physiol 128(2): 643--9.

Lydakis-Simantiris N, Hutchison RS, Betts SD, Barry BA and Yocum CF (1999)

Manganese stabilizing protein of photosystem II is a thermostable, natively unfolded

polypeptide. Biochem 38(1): 404--14.

Mamedov F, Stefansson H, Albertsson PA and Styring S (2000) Photosystem II in

different parts of the thylakoid membrane: A functional comparison between different

domains. Biochem 39(34): 10478--10486.

Mayes SR, Cook KM, Self SJ, Zhang ZH and Barber J (1991) Deletion of the gene

encoding the photosystem-II 33-kDa protein from synechocystis Sp Pcc-6803 does not

inactivate water-splitting but increases vulnerability to photoinhibition. Biochim

Biophys Acta 1060(1): 1--12.

Mei R and Yocum CF (1992) Comparative properties of hydroquinone and

hydroxylamine reduction of the Ca2+-stabilized O2-evolving complex of PSII:

Reductant-dependent Mn2+ formation and activity inhibition. Biochem 31: 8449--

8454.

Miller A-F and Brudvig G (1989) Manganese and calcium requirement for

reconstruction of oxygen-evolution activity in manganese-depleted photosystem II

membranes. Biochem 28: 8181--8190.

Miyao M and Murata N (1984) Role of the 33-Kda polypeptide in preserving Mn in the

photosynthetic oxygen-evolution system and its replacement by chloride-ions. Febs

Letters 170(2): 350--354.

Miyao M and Murata N (1989) The mode of binding of 3 extrinsic proteins of 33-Kda;

23-Kda and 18-Kda in the photosystem-II complex of spinach. Biochem Biophys Acta

977(3): 315--321.

Miyao M, Murata N, Lavorel J, Maisonpeteri B, Boussac A and Etienne AL (1987)

Effect of the 33-Kda protein on the S-state transitions in photosynthetic oxygen

evolution. Biochim Biophys Acta 890(2): 151--159.

35

Mohamed ZA (2001) Removal of cadmium and manganese by a non-toxic strain of the

freshwater cyanobacterium gloeothece magna. Water Research 35(18): 4405--4409.

Mukhopadhyay S, Staples R and Armstrong W (2002) Toward synthetic models for

high oxidation state forms of the photosystem II active site metal cluster: The first

tetranuclear manganese cluster containing a [Mn4(mu-O)5]6+ core. Chem Commun

8(April 21): 864--5.

Nanba O and Satoh K (1987) Isolation of a photosystem-II reaction center consisting of

D-1 and D-2 polypeptides and cytochrome-B-559. Proc Natl Acad Sci U S A 84(1):

109--112.

Nield J, Balsera M, De Las Rivas J and Barber J (2002) Three-dimensional electron

cryo-microscopy study of the extrinsic domains of the oxygen-evolving complex of

spinach : Assignment of the PsbO protein. J Biol Chem 277(17): 15006--15012.

Nixon P, Trost J and Diner B (1992) Role of the carboxy terminus of polypeptide D1 in

the assembly of a functional water-oxidizing manganese cluster in photosystem II of

the cyanobacterium synechocystis sp. PCC 6803: Assembly requires a free carboxyl

group at C-terminal position 344. Biochem 31: 10859–10871.

Nixon PJ and Diner BA (1992) Asp170 of the PSII reaction center polypeptide D1 is

involved in the assembly of the oxygen-evolving mn cluster. Biochem 31: 942--948.

Ogawa T, Bao D, Katoh H, Shibata M, Pakrasi H and Bhattacharyya-Pakrasi M (2002)

A two-component signal transduction pathway regulates manganese homeostasis in

synechocystis 6803, a photosynthetic organism. J Biol Chem 277(32): 28981--28986.

Ohta H, Okumura A, Okuyama S, Akiyama A, Iwai M, Yoshihara S, Shen JR, Kamo M

and Enami I (1999) Cloning, expression of the psbu gene, and functional studies of the

recombinant 12-kda protein of photosystem II from a red alga cyanidium caldarium.

Biochem Biophys Res Commun 260(1): 245--50.

Ono T (2000) Effects of lanthanide substitution at Ca2+-site on the properties of the

oxygen evolving center of photosystem II. J Bioinorg Chem 82(1-4): 85--91.

Ono T and Inoue Y (1984) Ca-2+-dependent restoration of O-2-evolving activity in

Cacl2-washed PS-II particles depleted of 33-Kda; 24-Kda and 16-Kda proteins. Febs

Letters 168(2): 281--286.

Philbrick JB, Diner BA and Zilinskas BA (1991) Construction and characterization of

cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem-

II. J Biol Chem 266(20): 13370--13376.

Qian M, Al-Khaldi SF, Putnam-Evans C, Bricker TM and Burnap RL (1997)

Photoassembly of the PSII Mn4 cluster in site-directed mutants impaired in the binding

of the Mn-stabilizing protein. Biochem 36: 15244--15252.

36

Razeghifard MR, Wydrzynski T, Pace RJ and Burnap RL (1997) Yz. Reduction

kinetics in the absence of the manganese-stabilizing protein of photosystem II.

Biochem 36(47): 14474--14478.

Riggs-Gelasco PJ, Mei R, Yocum CF and Penner-Hahn JE (1996) Reduced derivatives

of the Mn cluster in the oxygen-evolving complex of photosystem II: An EXAFS study.

J Am Chem Soc 118: 2387--2399.

Rögner M, Chisholm DA and Diner BA (1991) Site-directed mutagenesis of the psbC

gene of photosystem II: Isolation and functional characterization of CP43-less

photosystem II core complexes. Biochem 30: 5387--5395.

Rova M, Mamedov F, Magnuson A, Fredriksson PO and Styring S (1998) Coupled

activation of the donor and the acceptor side of photosystem II during photoactivation

of the oxygen evolving cluster. Biochem 37(31): 11039--11045.

Sansom M, Shrivastava I, Ranatunga K and Smith G (2000) Simulations of ion

channels –watching ions and water move. Trends Biochem. Sci. 25(August): 368--374.

Schroda M, Krpat J, Oster U, Rudiger W, Vallon O, Wollman F-A and Beck CF (2001)

Possible role for molecular chaperones in assembly and repair of PSII. Biochem Soc

Trans 29(4): 413--418.

Seibert M (1993). Biochemical, biophysical, and structural characterization of the

isolated PSII reaction center complex. In: The Photosynthetic Reaction Center Vol. I

:319--356, Academic.

Shen JR, Burnap RL and Inoue Y (1995) An independent role of cytochrome c-550 in

cyanobacterial photosystem ii as revealed by double-deletion mutagenesis of the psbO

and psbV genes in synechocystis sp. PCC 6803. Biochem 34(39): 12661--8.

Shen JR, Qian M, Inoue Y and Burnap RL (1998) Functional characterization of

synechocystis sp. PCC 6803 delta psbu and delta psbv mutants reveals important roles

of cytochrome c-550 in cyanobacterial oxygen evolution. Biochem 37(6): 1551--8.

Shen JR, Vermaas W and Inoue Y (1995) The role of cytochrome c-550 as studied

through reverse genetics and mutant characterization in synechocystis sp. PCC 6803. J

Biol Chem 270(12): 6901--7.

Smith D and Howe CJ (1993) The distribution of photosystem-I and photosystem-II

polypeptides between the cytoplasmic and thylakoid membranes of cyanobacteria.

Fems Microbiol Lett 110(3): 341--347.

Smith RM and Martell AE (1976) Critical stability constants. New York, Plenum.

Styring S and Rutherford AW (1987) In the oxygen-evolving complex of PSII the S0

state is oxidized to the S1 state by D+. Biochem 26(9): 2401--2405.

37

Svensson B, Etchebest C, Tuffery P, Van Kan P, Smith J and Styring S (1996) A model

for the photosystem II reaction center core including the structure of the primary donor

P680. Biochem 35: 14486--14502.

Tamura N and Cheniae GM (1987) Photoactivation of the water-oxidizing complex in

photosystem II membranes depleted of Mn and extrinsic proteins. I. Biochemical and

kinetic characterization. Biochim Biophys Acta 890: 179--194.

Trost JT, Chisholm DA, Jordan DB and Diner BA (1997) The D1 C-terminal

processing protease of photosystem II from scenedesmus obliquus - protein purification

and gene characterization in wild type and processing mutants. J Biol Chem 272(33):

20348--20356.

Utschig LM, Ohigashi Y, Thurnauer MC and Tiede DM (1998) A new metal-binding

site in photosynthetic bacterial reaction centers that modulates QA to QB electron

transfer. Biochem 37(23): 8278--8281.

Vass I, Ono T and Inoue Y (1987) Removal of 33 Kda extrinsic protein specifically

stabilizes the S2qa- charge pair in photosystem-II. Febs Letters 211(2): 215--220.

Vass I and Styring S (1991) Ph-dependent charge equilibria between tyrosine-d and the

s states in photosystem II. Estimation of relative midpoint redox potentials. Biochem

30(3): 830--9.

Vrettos JS, Reifler MJ, Kievit O, Lakshmi KV, de Paula JC and Brudvig GW (2001)

Factors that determine the unusually low reduction potential of cytochrome c(550) in

cyanobacterial photosystem II. J Biol Inorg Chem 6(7): 708--716.

Vrettos JS, Stone DA and Brudvig GW (2001) Quantifying the ion selectivity of the

Ca2+ site in photosystem II: Evidence for direct involvement of Ca2+ in O-2

formation. Biochem 40(26): 7937--7945.

Westphal KL, Lydakis-Simantiris N, Cukier RI and Babcock GT (2000) Effects of

Sr2+-substitution on the reduction rates of y- z(center dot) in PSII membranes -

evidence for concerted hydrogen-atom transfer in oxygen evolution. Biochem 39(51):

16220--16229.

White RA, Wolfe GR, Komine Y and Hoober JK (1996) Localization of light-

harvesting complex apoproteins in the chloroplast and cytoplasm during greening of

chlamydomonas reinhardtii at 38 degrees C. Photosyn Res 47(3): 267--280.

Wincencjusz H, Yocum CF and van Gorkom HJ (1998) S-state dependence of chloride

binding affinities and exchange dynamics in the intact and polypeptide depleted O2

evolving complex of PSII. Biochem 37: 8595--8604.

Wollenberger L, Stefansson H, Yu SG and Albertsson PA (1994) Isolation and

characterization of vesicles originating from the chloroplast grana margins. BBA-

Bioenergetics 1184(1): 93--102.

38

Wollenberger L, Weibull C and Albertsson PA (1995) Further characterization of the

chloroplast grana margins - the non-detergent preparation of granal photosystem-I

cannot reduce ferredoxin in the absence of Nadp(+)reduction. BBA- Bioenergetics

1230(1-2): 10--22.

Yocum CF (1991) Calcium activation of photosynthetic water oxidation. Biochim

Biophys Acta 1059: 1--15.

Zak E, Norling B, Maitra R, Huang F, Andersson B and Pakrasi HB (2001) The initial

steps of biogenesis of cyanobacterial photosystems occur in plasma membranes. Proc

Nat Acad Sci USA 98(23): 13443--13448.

Zaltsman L, Ananyev G, Bruntrager E and Dismukes GC (1997) A quantitative kinetic

model for photo-assembly of the photosynthetic water oxidase from its inorganic

constituents: Requirements for Mn and Ca in the kinetically resolved steps. Biochem

36: 8914--8922.

Zhang L, Virpi Paakkarinen V, van Wijk K and Aro E-M (2000) Biogenesis of the

chloroplast-encoded d1 protein: Regulation of translation elongation, insertion, and

assembly into photosystem II. The Plant Cell 12: 1769–1781.

Zheleva D, Sharma J, Panico M, Morris HR and Barber J (1998) Isolation and

characterization of monomeric and dimeric CP47-reaction center photosystem II

complexes. J Biol Chem 273: 16122--16127.

Zouni A, Kern J, Loll B, Fromme P, Witt H, Orth P, Krauß N, Saenger W and

Biesiadka J (2001). Biochemical characterization and crystal structure of water

oxidizing photosystem II from synechococcus elongatus. In: PS2001 Proc 12th Int

Cong Photosyn. Melbourne, CSIRO.

39

A B C Dk1 = 0.034

k-1 = 0.01

k2 = 0.0038

dark

k3 ~ 0.006

[MnIIOH]

+

Mn(III)

OH

OHMn

O

Mn

OH

apo-WOC

CahhMn

IM1*IM1

X X

Ca

IM2

Mn4Ca1Ox

HCO3-

nh

2Mn

*Mn

HCO3

light

MnIII

HO

Ca

OH

3+

MnIII

O Ca

3+H2O

Figure 1. The sequence of kinetic intermediates (top) formed during assembly of the inorganic

core of the WOC by photoactivation (A, B, C) (Ananyev et al. 2001). (bottom) proposed

structures of intermediates.

40

COO

XH

R+

HX

COO

X

X

HCO3

A Mn2+Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

D1 D1)R(CO2

3

COO

OH2

HX

Mn2+

COO

X

X

Mn2+

XH

HCO3

HCO3

B

Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

Mn2+

R+

D1 D1 Mn2+

)R(CO2

3

H2O

H+

Scheme 2 Baranov et al.

Figure 2. The two proposed sites for bicarbonate stimulation of Mn

2+ assembly during

the photoactivation reaction (Baranov et al. 2004). (A) The high-affinity bicarbonate

site: At low Mn2+

concentrations (< 8 Mn2+/PSII), bicarbonate anions electrostatically

steer Mn2+

ions into the apo-WOC-PSI complex from solution by partitioning or

ionization of protons or through ion-pairing with cationic side chains arginines, R+).

(B) The low-affinity bicarbonate site: At high Mn2+

concentrations ( > 30 Mn2+

/PSII),

bicarbonate binds to the photoactive high-affinity Mn2+

binding site.

41

O

MnMn

O

O

Mn

OMn

( 3-oxo)2( 2-oxo)2

Mn

O

O

Mn

O

Mn

Mn

O

HO OH

( 2-oxo)4( 2-hydroxo)2

Figure 3. Two of the manganese-oxo core types proposed for the PSII-WOC on the

basis of EPR and EXAFS data (Carrell et al. 2002).

42

Table 1. Sequence and location of assembly of PSII core complex protein subunits and inorganic

core cofactors in cyanobacteria and chloroplasts.

Membrane: Plasma Stroma Grana Margin Core Grana

PSII-WOC

Maturation

Unassembled

reaction center

PSII-β

Inner antenna insertion

labile inorganic core

PSII core-β

Completed

polypeptide core

labile inorganic core

PSII core-α

Assembled

polypeptide core

stable inorganic

core + LHC II

Major Subunits RCb,

33-MSP(nf)a RC+CP47 ~CP43 RC+CP43+CP47

All major su +

33-MSP(Boussac et al.)

Electron

Transport

none Slow QAQB

Slow QAQB

Normal PSII

Kinetics

WOC

Photoactivation

none ~40% photoactivatible

labile O2↑

Photoactivation

yield increases

Fully assembled

stable O2↑

a (nf) non-functional; (Boussac et al.) functional

b RC subunits D1, D2, cytb559

Table 2. Activator/inhibitors at the Ca-effector site of Mn-dependent photoactivation of

O2 evolution in spinach apo-WOC-PSII(Ananyev et al. 2001)

Metal Photoactivation

step affected

O2 yield*

At pH 6.5

Michaelis

Const. O2 ?

Ca2+

k1 no effect

k1 accelerated

k2 accelerated

70-100% 1.5mM

Sr2+

k1 no effect

k1 accelerated

k2 accelerated

30% ~30 mM

Mg2+

Weak non-specific

effect

<0.5% >0.1 M

(inhibition)

Ba2+

k1 slowed <0.5% Blocks Mn

Not Ca site

VO2+

k1 accelerated

k1 slowed

<0.5%

15 μM (k1)

200 μM