PhotosynthesisResearch34: 287-300, 1992. © 1992 KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

A three-dimensional model of the Photosystem II reaction centre of Pisum sativum Stuart V. Ruffle 1, Dan Donnelly2'3, Tom L. Blundell 2 & Jonathan H.A. Nugent 1'4

1Department of Biology, Darwin Building, University College, University of London, Gower Street, London, WC1 E 6BT, UK; 2ICRF Unit of Structural Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London, WC1E 7HX, UK; 3present address: Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK; 4for correspondence and reprints Received 30 March 1992; accepted in revised form 3 July 1992

Key words:

computer model, D1 protein, D2 protein, manganese cluster binding site

Abstract

A three-dimensional model of the core proteins D1 and D2, including the cofactors, that form the Photosystem II reaction centre of pea (Pisum sativum), has been generated. This model was built with a rule-based computer modelling system using the information from the crystal structures of the photosynthetic reaction centres of Rhodopseudomonas viridis and Rhodobacter sphaeroides. An alignment of the primary sequences of twenty three D1, nine D2, eight bacterial L and eight bacterial M subunits predicts strong similarity between bacterial and higher plant reaction centres, especially in the transmembrane region where the cofactors responsible for electron transport are located. The sequence to be modelled was aligned to the bacterial structures using environment-dependent substitution tables to construct matrices, improving the alignment procedure. The ancestral relationship between the bacteria and higher plant sequences allowed both the L and M subunits to be used as structural templates as they were equally related to the higher plant polypeptides. The regions with the highest predicted structural homology were used as a framework for the construction of the structurally conserved regions. The structurally conserved region of the model shows strong similarity to the bacterial reaction centre in the transmembrane helices. The stromal and lumenal loops show greater sequence variation and are therefore predicted to be the structurally variable regions in the model. The key sidechain assignments and residues that may interact with cofactors are discussed.

Abbreviations: D - Tyrl61 in the D2 polypeptide; PS II - Photosystem II;

Q A - primary plastoquinone acceptor of Photosystem II; QB -secondary plastoquinone acceptor of Photosystem II; Z - T y r 1 6 1 in the D1 polypeptide

Introduction

Photosystem II (PSII) is a membrane-bound protein complex that catalyses the transfer of electrons from water to plastoquinone. PS II has many constituent protein subunits located in and adjacent to the thylakoid membrane, although

the function of some of these polypeptides is, at present, unknown. At the heart of the complex is a heterodimer of the D1 and D2 proteins which have functional and partial sequence homology to the L and M subunits of the bacterial reaction centre (Deisenhofer et al. 1985), the protein core that binds the cofactors involved in

288 primary photochemistry. The relationship between the reaction centres of higher plants and bacteria has been investigated (Michel and Deisenhofer 1988, Deisenhofer and Michel 1989). It has been suggested that a single gene duplication led to the heterodimeric structures before evolutionary divergence (Deisenhofer and Michel 1989). A second proposal, using parsimony analysis of the six sequences from spinach, Rhodopseudomonas viridis (R. viridis) and Chloroflexus aurantiacus (Chl. aurantiacus) argues that the heterodimers of plants and bacteria arose independently after divergence (Beanland 1990). However, this analysis was based on an incorrect sequence alignment where, for example, the histidines required to bind the non-haem iron were misaligned. The structure of the purple non-sulphur bacterial reaction centre was solved by X-ray crystallographic techniques for R. viridis at 3 A resolution (Deisenhofer et al. 1984) and later refined to 2.3 ,K resolution. The reaction centre from a second purple bacterium, Rhodobacter sphaeroides R-26 (Rb. sphaeroides) has been crystallized and its structure solved by two groups (Chang et al. 1986, 1991, Allen et al. 1986, 1987a, b). The architecture of the R. viridis and the Rb. sphaeroides structures has been reviewed and compared (Allen et al. 1986, EIKabbani et al. 1991). In higher plants the reaction centre was found to be located on the D1 and D2 polypeptides following detergent digestion of PS II membranes to produce and active complex of D1/D2/ Cytochrome b559 (Cyt b599) (Nanba and Satoh 1987). Modelling the structure of the PS II reaction centre on L and M has been attempted at the secondary structure level by many researchers with mixed results. Early studies suggested that the core proteins spanned the bilayer seven times (Rao et al. 1983). This was caused by the incorrect assignment of the amphipathic helices between the helices, now termed, 1 and 2 and between 4 and 5, as transmembrane helices. The secondary structure of the D1 and D2 polypeptides has now been predicted to contain five membrane spanning regions, (Deisenhofer et al. 1985, Trebst 1986) and this has been verified experimentally by immunological studies (Sayre et al. 1986). These spans are predicted to adopt

an a-helical conformation. A complex including D 1 / D 2 / C y t b559 and psb I gene product is known to contain an a-helical content of 67% as measured by Fourier transform infrared spectroscopy (He et al. 1991). The cofactors involved in electron transfer are, by analogy with the bacterial reaction centre, located between helices 4 and 5 of D1 and D2. Stoichiometric analysis of plant and bacterial reaction centres has produced variable results depending on preparation methods used. The consensus of these reports is that there are six chlorophyll and two pheophytin molecules associated with D1/D2/Cyt b559 complex. However, centres that have four chlorophyll and two pheophytin molecules, the situation in bacterial reaction centres, have been shown to carry out charge separation (Shuvalov et al. 1989). These pigments, along with a non-haem iron and the plastoquinone electron acceptors QA and QB are the essential cofactors of fully functional PS II. Many of the ligands for the bacterial cofactors are conserved in higher plant sequences but several are missing including the histidine ligands to the accessory bacteriochlorophylls (Deisenhofer and Michel 1989). No X-ray crystal structure of the PS II reaction centre has been reported. There have been previous reports of partial three-dimensional models of the D1 and D2 proteins, two of the QB binding region (Bowyer et al. 1990, Tietjen et al. 1991) and one of the redox active tyrosines (Svensson et al. 1990). Svensson et al. (1990), modelled regions of the lumenal side of the complex by superimposing the residues in the putative transmembrane helices, using previously suggested membrane boundary assignments (Trebst 1986, Michel and Deisenhofer 1988) onto the coordinates from the bacterial membrane spanning regions. The loop regions between the helices were fitted in using the F R O D O computer program (Jones 1985). The two models of the QB binding loop were constructed to investigate the herbicide binding site of the D1 protein (Bowyer et al. 1990, Tietjen et al. 1991). These models were generated using the SYBYL suite of programs (Tripos Associates, St. Louis. Missouri, USA), before the COMPOSER module was included in this package (Blundell et al. 1987, 1988, Sutcliffe et al. 1987a,b).

289 Bowyer et al. (1990) derived their coordinates by digitizing a stereoimage of the R. viridis reaction centre. Their model was generated by superimposing the D1 residues following an alignment of the amino acid sequences of D1 and the R. viridis L subunit. Fourteen residues were omitted as they had no aligned equivalents in the bacterial sequence. The model of Tietjen et al. (1991) was based on the crystallographic coordinates of the L subunit from R. viridis. The present study has produced a model of the Pisum sativum (P. sativum) D1 and D2 polypeptides including cofactors, using the rule-based computer modelling system COMPOSER (Blundell et al. 1987, 1988, Sutcliffe et al. 1987a,b). COMPOSER builds a framework of protein structure generated from multiple homologous X-ray crystallographic derived structures. The loop regions are built by matching fragments from solved structures and then adding them to the framework. This model may allow some interpretation of the location of the manganese cluster and the nature and behaviour of the key residues around the cofactors. It will be useful as a tool for suggesting molecular biological and biochemical experiments that will enhance the understanding of the mechanism of PS II.

Materials and methods

The structures of the L and M subunits from both R. viridis and Rb. sphaeroides were aligned using the computer program COMPARER (Sali and Blundell 1990). This program includes local structural information in the alignment procedure. In addition, the sequences of seventeen D1 and nine D2 subunits were aligned using the computer program MALIGN (Johnson and Overington, submitted). The structural alignment of the four bacterial subunits was then aligned with the alignment of the D1 and D2 sequences using the novel method of Johnson and Overington (submitted). This approach uses environment-dependent substitution tables (for details see Overington et al. 1990, 1992) to construct distance matrices for dynamic programming, ensuring that structural

information is used in the alignment procedure. The resulting alignment was used to calculate evolutionary distances between all pairs of sequences based upon percentage residue identities. Using the computer program KITSCH (Felsenstein 1985), a phyletic tree was computed based upon these proportional distances and their proposed branching order. Structurally conserved regions (SCRs) in the alignment of the four bacterial subunits were first defined using the program MNYFIT (Sutcliffe et al. 1987a). These regions were refined by consulting the larger alignment with the more distant D1 and D2 sequences so that the SCRs only included the regions for which a good alignment was possible. These SCRs were used to build both the D1 and D2 subunits of the P. sativum sequence using the rule-based modelling program COMPOSER (Blundell et al. 1987, 1988, Sutcliffe et al. 1987a,b). Certain regions outside the SCRs were modelled by 'collar building' which refers to the construction of a region of the model from a part of the bacterial structure that is only partially conserved in the structural alignment. The structurally variable regions (SVRs) were built by selecting fragments from the Brookhaven Protein Databank (Bernstein et al. 1977) and then adding them onto the framework by a least-squares process. Certain SVRs (D1 between Ala81 and Glyll0 and Met293 and Ile314 and in D2 between Ala78 and Cysl06) were not built since no suitable fragments could be found. The N- and C-terminal regions were also excluded. The cofactors in the PS II of P. sativum were constructed by modifying those of Rb. sphaeroides using interactive graphics facilities. The complete PS II complex was then built by least squares fitting the appropriate substructures onto their analogous counterparts in the structure of Rb. sphaeroides. Certain sidechain conformations were altered manually during the analysis of the model. The model was finally minimized using the default energy minimization options within the molecular graphic package SYBYL (Tripos Associates, St. Louis. Missouri USA). During this process, the cofactors were constrained so that only the protein could move. Computations were performed on a VAX 11/750, microvax or a Silicon Graphics Iris

290 workstation. Copies of the coordinates are available from J.H.A. Nugent.

eugdl cypadl - livdl riced1 ryedl bard1 soydl 'tobdl peadl alfdl

Results and discussion

Sequence alignment The alignment of L, M, D1 and D2 sequences (data not shown) identifies the conserved residues, some of which are likely to be those with key structural and functional roles. Obvious examples of this are the histidine residues that co-ordinate the non-haem iron and the special-pair chlorophyll molecules. Those residues that have a specific function in PS II, such as those binding the cofactors involved in water oxidation, are likely to be conserved in PS II sequences only. The family tree (Fig. 1, see also Table 1 for a key to the codes) was derived from sequence similarities (see Materials and methods). Figure 1 shows that the L and M subunits may have been derived from a common ancestral homodimer. The alignment between the sequences of oxygenic organisms and the purple bacteria reveals that D1 is related as closely to L, as it is M and the same applies to D2. This observation allowed each subunit of the model to be constructed using four known structures (2 L and 2 M subunits from R. viridis and Rb. sphaeroides), thus increasing the structural data available. The differences between the sequences in the oxygen evolving organisms was significant enough to allow differentiation between monocotyledonous and dicotyledonous plants within the family tree. It is interesting to note the position of Prochlorothrix hollandica (Pr. hollandica) (prcldl), which has been suggested as the modern counterpart of the ancestor of the chloroplast. It is a prokaryote, contains no phycobilins but has both chlorophyll a and b (Meyer et al. 1986). Pr. hollandica aligns with the cyanobacteria; diverging from them after the branch away from higher plants and algae. This suggests that chlorophyll b synthesis arose either independently in Pr. hollandica, or all cyanobacteria lost this capacity after the divergence. The flagellate protist Cyanophora paradoxa (C. paradoxa) contains chlorophyll a and phycobiliproteins. This organism is thought to be an evolutionary step between cyanobacteria and

E

~

chmodl

chredl prcldl caldl syndl synTdl syTaxiI syn7d2 chred2 -- livd2

spind2 peazl2 tobd2 bard2 riced2

ryed2 sm vm sl vl

Fig. 1. Phyletic tree of plant, cyanobacterial and selected purple non-sulphur bacterial reaction centre proteins derived from sequence similarity scores using the program KITSCH (Felsenstein 1985). For codes see Table 1.

chloroplast containing organisms. It is believed to be a result of an endosymbiosis event between a eukaryote and a cyanobacterium (Hall and Claus 1963). The C. paradoxa sequence (cypadl) is grouped with the algae and plants but is an outlier, at the extreme of that branch. The unusual organism, Euglena gracilis (E. gracilis), which has the higher plant complement of pigments, is an outlier in the D1 family tree (eugdl).

Structural template. The template generated for the model, which is shown in Fig. 2, shows the

291 Table 1. Codes used in this paper. Codes used in Fig. 1 are cross-referenced with those found in the OWL database (Bleasby and Wootton 1990) and the organism and gene product names Code in Fig. 1

Code in OWL database

Organism and gene product

alfdl bardl bard2 caldl chmodl chredl chred2 cypadl eugdl livdl livd2 peadl pead2 prcldl riced1 riced2 ryedl ryed2 soydl spind2 sl sm sy7adl syn7dl syn7d2 syndl tobdl tobd2 vl

PSBA~ ;MEDSA PSBA', ;HORVU PSBD: ;HORVU PSBA: ;FREDI PSBAI ;CHLMO PSBA: ;CHLRE PSBD: ;CHLRE PSBA'. ;CYAPA PSBA; ;EUGGR FMLV32 F2LVD2 PSBA$PEA F2PMD2 PSBASPROHO FMRZ32 F2RZD2 PSBA$SECCE PSBD$SECCE FMSY32 F2SPD2 WNRFLS WNFRMS PSB2$SYNP7 PSBI$SYNP7 PSBD$SYNP7 PSBA$SYNY4 FMSP32 F2NTD2 RCELSRHOVI RCEM$RHOVI

Alfalfa psbA Barley psbA Barley psbD Calothrix psbA

vm

alignment of the D1 and D2 sequences of pea against the bacterial structural alignment. Regions of the four structures with the highest structural homology are designated as core (underscored with = symbols). Part o f . t h e designated core includes the transmembrane helices, shown in Table 2. For the D1 subunit this places helix 1 between Trp32 and Phe51. Previous studies have placed this helix between Met37 and Pro56 (Trebst 1986) or Ala44 and I1e63 (Michel and Deisenhofer 1988). The I helix described in Rb sphaeroides by Allen et al. (1987b) was designated as core in the structural alignment, indicating conservation of this structural feature in the PS II reaction centre. These amphipathic helices are found at the ends of the transmembrane helices and in the loop regions associated with the lumen.

Chlamydornonas moewusiipsbA Chlamydomonas reinhardtiipsbA Chlamydomonas reinhardtiipsbD Cyanophora paradoxa psbA Euglena gracilispsbA Liverwort psbA Liverwort psbD Pea psbA Pea psbD Prochlorothrix hollandica psbA Rice psbA Rice psbD Rye psbA Rye psbD Soybean psbA Spinach psbD Rhodobacter sphaeroides pulL Rhodobacter sphaeroides pufM Synechococcus PCC 7942 psbA II Synechococcus PCC 7942 psbA I Synechococcus PCC 7942 psbD Synechocystis PCC 6714 psbA Tobacco psbA Tobacco psbD Rhodopseudomonas viridispufL Rhodopseudomonas viridispufM

Table 2. Helix boundaries assigned using a Kabsch and Sander (1983) analysis of the model coordinates (Helices 1 to 5 transmembrane, I - interrupted helix) Helix

Position in D1

Position in D2

1 2 3 4 5 I

Trp32 - Ile53 Glyl06 - Leu136 Trp142 - Gln164 Pro196 - Set221 Ser268 - Ile289 Va167 - Gly73 I1e176 - His190 Asn315 - Ile319

Trp32 - Phe55 Glyl09 - Vail37 Tyr142- Gln165 Pro196 - Asn220 Lys265 - Arg295 Ala65 - Gly70 Ala178 - Hisl90 Glu307 - Thr316

A low/3-sheet content is seen in the model in agreement with the data from Fourier transform infra-red spectroscopy data (He et al. 1991). The model predicts that residues Gly79 and Leu173

292 vm

ady~£iyi~i~ai---gphi

sm

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IGkigdA~

fs~v~v~---gpadlgm~edvnl-aRrsgvgpfst

sl

al 1 s f~rkyivp---gg~l

vl

al Is f~rkyivf---ggt

llGwfgnA~

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...............

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...............

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9asGiaAfafg~ai

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sl

--pfYvGffgva~fffaalgii

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....

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fi~

....

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rd

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a a a a ~ a ~ a a a ~ a a a a a a a a a a

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pead2

---VFVGWSGLLLFPCAYFAVGGWFTGTTFVTS

vm

ffwLGly--pPka~yg&gi-P

..........

p-- lhdGgwWlmAgl

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Ifff~Le--PPap£yglsfaA

. . . . . . . . . .

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sl

-~1 isVy--pPaleyglg-gA

..........

p-- lakGGLWqiiti~atg

vl

. faiii~.-pPdlkyglg-aA

..........

p-- 1 leGgfWqal~vsalg

peadl

NNIISGAI IPTSAAIGLHFYPIW--EA-ASVDEWLYNGGPYELIVLHFLL

pead2

CNFLTAAVSTPANSLAHSLLLLWGPEAQGDLTRWCQLGGLWTFVALHGAF

vm

llgiw~i~vysLa1AlglgthiA@fifaaai

8m

av@i~wgr

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afvswal~vEi~rklgigyhipfafafai

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c .

.

.

.

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.

? 118

ffvl~igci~Pklvgi~i~G

tylraqAIgmgkhtAwafliaI~lw~VIGfiRPil~gi~A

•

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GVACYMGREWEL

pead2

GL IGFMLRQFELARSVQLRPYNA

126

.... WYTHGLASSYLEG

130

S FRLGMRPWI

•

•

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f~Pv~mga~g~A

•

llg!~GhA

ee

•

IYP IGQGS

F SDG

L I YP LGQSGWFFA

161

165

170

Fig. 2. T h e alignment between the model sequences and the consensus secondary structure of the bacterial reaction centre

proteins. T h e amino acid code is the standard one letter code formatted using the following convention to denote the local structural environment: U P P E R C A S E , inaccessible; lowercase, accessible; italic, positive phi angle; breve ( ' ) , cis-peptide bond; bold, hydrogen bond with mainchain amide proton; underline, hydrogen bond to mainchain carbonyl; tilde (~), hydrogen bond with other sidechain. The consensus secondary structure is shown immediately below the alignment: a, alpha helix; /3, beta strand; 3, 31° (three residues per turn) helix; e, conserved inaccessible residue. The topologically equivalent positions in the structures used as a framework for the model building are indicated by; = , designated core region and c, collar built region. T h e alignment of the pea D1 and D2 sequences to the structural alignment are shown below the framework. The n u m b e r i n g shown refers to some of the key residues in the higher plant sequences referred to in the text.

293

vm

vpfgiwPhidwl

8m

vP~Gi

taFsiiYgmfyyCpw~gf~igfaygKgl

fshld~tnnfslvHGal

fy~pf~gl~iaflygsal

sl

fp~giw£hld&vsnTgy~yG~fhy~pahmiAis

vl

fp~gl

1 §hldwvn~fgyq~lKwhy~pghm~EvEf

peadl

MPLGI

SGTFNFMIVFQAEH-NI

pead2

PSFGVAAI

FRFI

I faahga~i

I

I famhga~i

I

f fft~alala[hgalvl I fvnama]glhggl

i l

LMHPFHMLGVAGVFGGSLFSAMHGSLVT

LFFQGFH-NWTLNPFHMMGVAGVLGAALLCAIHGATVE

T

1"

$

q"

"r

"r

1"

179

182

186

190

198

212

215

tAv~raal

vm

ava~fg---Gdr~ieqitdig

8m

aver

81

saanpe

....

kgk

vl

~vaapg

....

dgd .....

peadl

SSLI--RETTENESANEGYRFG--QEEETYNIVAAHGYFGRLIFQYASFN

pead2

NTLF

........

fg---G~r~leqiad~g .....

f@~w-~igf-fi-a

........

tAa~raal

f~rw-tmgf-n-a

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f fr~-

kvk

ta-~hefi~yf~-vvgy-~-

........

lvgy-~-

i i

¢ c c c ¢ ¢

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=

EDGDGANTFRAFNPTQAEETYSMVTANRFWSQ-

IFGVA-FS

T

$

231(DI) 228(D2)

2~

t i~ivhiwg~f

f~lmvmv!a~vGII

1~ ..... I IS .....

G-~f

....

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tmegihrwaiwmavlv~lTgGigi

sl

gtlgihr

lgl

ll~l~Avf

vl

gal!i~r

lgl

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pesdl

NSRSLHFFLAAWPVVGIWFTALGISTMAFNLNGFNF-NQSVVDSQGRVIN

pead2

NKRWLHFFMLFVPVTGLWMSALGVVGLALNLRAYDFVSQEIRAAEDPEFE

fsalEmiiT fl~gafg£ia~

g- Jv ....

v ....

.....

G-t

i ....

~ ....

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.....

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....

~ ....

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==

T 272(D1) 269(D2) vm

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8m

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.....

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vl

~w~gwwldi--p

peadl

TWADIINRANLGMEV-MH

.......

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pead2

TFYTKNILLNEGIRAWMA

.......

TQDQPHENLIFP

l--p

dypaylpa~pdpas

lpgapk

...................

wwafii

...................

f~e

332(D1)

Fig. 2 (continued).

342(D!)

294 in D1, and Ala76 and Phe173 in D2 are in /3-strand conformation.

Cofactor binding. This study has generated a D1/D2 complex that is 73% complete (Fig. 3). The N and C-termini (16% of the missing protein) were excluded and loops could not be fitted in D1 between Ala81 and Glyll0 and Met293 and I1e314 and in D2 between Ala78 and Cysl06. The model includes the PS II cofactors placed using the coordinates of the bacterial equivalents. Both the cofactors and electron transport" rates in PS II are similar to those in purple bacteria (Rutherford 1989, Diner et al. 1991). This implies similar relative orientations and separation between cofactors. Therefore, the PS II cofactors were located in the same orientation relative to each other as in the bacterial reaction centre. The special pair. The proposed ligands to the special pair of reaction centre chlorophylls are His198 in D1 and D2, since these residues are totally conserved in the alignment between the bacterial structures and the pea sequences (Fig. 2). The sigma-nitrogen atoms of His198 in both D1 and D2 are minimized to be approximately 2.2/~ away from the coordinating magnesium ions in the model.

Fe

Qb 2 1---

"

Qa 5' !: 4 ,~' Pheo

!

:

5"

:

', P680

l'""

'3

1

2

Fig. 3. Ribbon diagram generated by the program MOLSCRIPT (Kraulis 1991) of the model of the PS II reaction centre proteins D1 (shaded) and D2 (unshaded). The cofactors are represented in stick form.

The accessory chlorophylls. The residues that provide ligands to the bacteriochlorophyll monomers are not conserved in the alignment with the sequence to be modelled. In the purple bacterial reaction centre the magnesium ion of these cofactors is bound by His153 in L and Hisl80 in M (EI-Kabbani et al. 1991). In the higher plant sequence, these residues are replaced with Thr179 in D1 and I1e179 in D2. The model structure shows both D2 11e179 and D1 Thr179 are incapable of making ligands to their respective magnesium atoms. There may be a hydrogen bond to the mainchain oxygen atom of Met199 which is conserved in D1 and D2, from the keto oxygen of ring V of the chlorophyll ring complex. In the model these atoms are separated by 3.2 .~. The residue between the accessory bacteriochlorophyll and bacteriopheophytin on the active branch is Tyr210 in the M subunit, in Rh. sphaeroides, it has been implicated as a conduit for electron transport (Allen et al. 1987a) is replaced by Leu206 of D2 in the higher plant system. The amino acid in an equivalent position on the inactive branch is a phenylalanine in all cases. The pheophytins. The hydrogen bond to the pheophytin provided by D1 Glul30 is conserved in the sequence to be modelled and the L subunit in purple bacteria. Resonance Raman spectroscopy has previously inferred a hydrogen bond between the 9-keto carbonyl of pheophytin and Glul30 of D1 (M6enne-Loccoz et al. 1989). In some cyanobacteria however, the residue has mutated to glutamine and this is also seen in D2. These residues model close to the keto-carbonyl of pheophytin ring V. The sigma-carbonyl oxygen atom of Glul30 in D1 models to be 3.2/~ away from the A branch pheophytin while the sigma-nitrogen of Glnl30 in D2 models to be 2.7 ,~ from the corresponding atom of the B branch pigment. Tyr126 in D1 may form a hydrogen bond with the oxygen of the ester group on ring V of the A branch pheophytin as its sidechain phenol oxygen refines to 2.4A away. Significantly, this tyrosine residue is conserved in all D1 sequences. In bacteria Trp252 on the M subunit is between the primary acceptor and QA and is be-

295 lieved to be involved in the electron transport process (Allen et al. 1987a). The equivalently residue in the D2 subunit is Trp254 which is conserved. The general position of these residues in the bacterial and model structures are similar although distances and orientations to the cofactors differ slightly. The non-haem iron. The non-haem iron is liganded to the sigma-nitrogen atoms of residues His215 and His272 in D1 and His215 and His269 in D2, with modelled bond distances within 2.2~_. These ligands are predicated from the alignment since they are conserved. One turn of the helix before His215 in D2 is the conserved residue Cys212. This residue is predicted to interact with the iron binding histidine. In the purple non-sulphur bacteria two iron binding ligands are provided by Glu232 of the M subunit. It has been suggested that the structure around the iron in the higher plant protein is different because D2 does not have an equivalent residue to Glu232 in the appropriate region of the alignment. However, one or more of these ligands may be provided by Glu231 in D1, which is aligned in the template with Glu232 of the two M subunits and Lys204 in Rb. sphaeroides and Asp204 in R. viridis L subunits. The deltacarbonyl oxygen atom of D1 Glu231 models to be 2.3 ~ from the non-haem iron (Fig. 4). This may be the coordination in reaction centres depleted of bicarbonate. Michel and Deisenhofer (1988) have suggested that a bicarbonate anion in PS II can replace the bond/s formed by Glu232 of the M subunit in

photosynthetic bacteria. Bicarbonate may be anchored to the reaction centre by the formation of a iysine carbamate ion on residue Lys265 of D2, made possible by its neighbour D2 Arg266. When modelled into the space between the lysine sidechain and the iron, the bicarbonate carbonyl group can provide a ligand to the metal when bound to the lysine (data not shown). The quinones. Residues that interact with the quinone electron acceptors were found by calculating the sidechain accessibilities (Richmond and Richards 1978) with the plastoquinone molecules present and absent from the model. The buried residues, Leu211, Thr218, Trp254, Phe262 and Leu268 all become accessible when the quinone is removed from the QA binding loop of D2 (Fig. 5). Smaller changes in accessibility are recorded for His215 and Phe258. Residues identified by previous studies as being involved in forming the binding niche of the secondary quinone, QB include Phe211, Met214, His215, Leu218, Va1219, Tyr237, I1e248, Ala251, His252, Phe255, Gly256, Ala263, Ser264, Phe265, Asn266, Ser268 and Leu275 (Wolber et al. 1986, Trebst 1987, Dostani et al. 1988, Bowyer et al. 1990, Etienne et al. 1990, Tietjen et al. 1991). In the QB binding site of the model (Fig. 6), residues that change in their sidechain accessibility include Phe211, Met214, His215, Leu218, Va1219, Ala251, Phe255, I1e259, Tyr262, Ser264, Ser268, and Leu271. The residue associated with the binding of QB and some herbicides, Ser264, contributes to the quinone binding loop on the

DI Glu231

DI His272 DI Hi~15 I)2 His215

Fig. 4. Stereo image of the residues forming ligands to the non-haem iron (D1 His215, Glu231 and His272; D2 His215 and His269).

296

-

Phe262

~{_/

[ 11e214 "7s~ /

ny,265

: )/

l.zl211

Fig. 5. Stereo image of the QA binding site (D2 Leu211, I1e214, His215, Thr218, Met247, Ala250, Trp254, Phe262, Asn264, Lys265, Leu268, part of the primary quinone (bold)).

His252 k.l251 A.m26'7~ i.eS~u2[i 8/~.,Vt ., l219 Ile259~ ~ ~ Met214 Tyr262 Fae211

~ ~

S

Fig. 6. Stereo image of the Q~ binding site (D1 Phe211, Met214, His215, Leu218, Va1219, Ala251, His252, Phe255, I1e259, Tyr262, Ser264, Asn266 (not labelled), Asn267, Ser268, Leu271, part of the secondary quinone (bold)).

opposite side of the quinone from the non-haem iron. The sidechain hydroxyl-oxygen of Ser264 models to be 3.1,~ from one of the quinone head group oxygen atoms and therefore would be able to form a hydrogen bond. This places the quinone head group between the delta-nitrogen of His215 and the hydroxyl group of Ser264 as previously assumed in other modelling studies (Bowyer et al. 1990, Tietjen et al. 1991). Metal ion binding sites and the environment around the redox active tyrosines. The sidechains of the redox active tyrosine residues are located in clefts in the protein between the lumenal ends of transmembrane helices 3 and 4. Both amino acids were found to be one turn from the lumenal end of their respective transmembrane helices, helix 3 in D1 and D2. The orientation of the phenol ring portion (C1 to O axis) of the sidechain of D2 Tyrl61 (D) minimized to be 86 ° to the plane of the membrane. This is in agreement

with the E P R orientation data available (O'Malley et al. 1984, Rutherford 1985). The nearest point of this residue to the special pair is 8.1 A. In D1, the phenolic sidechain of Tyrl61 (Z), is modelled at 30 ° to the plane of the membrane. The special pair is 7.6 A from the nearest point of the tyrosine sidechain. The protein environment surrounding the redox active residues differs between D1 and D2. Around the donor Z, the aromatic amino acids, D1 Phe182 and Phe186, are located between the tyrosine and the special pair (Fig. 7). They are both in van der Waals contact with the tyrosine sidechain. Other residues between Z and the special pair are D1 Val157 Val185 and Ile289. These residues are conserved in all D1 sequences except Val157, where a change to threonine occurs in Synechocystis PCC 6803. There are several polar residues between the donor Z and the lumen, including Ser169, Aspl70, Glu189 and Hisl90.

297

I1e290

~

Phe182

Gtn165

Fig. 7. Stereo image of the region around residue Tyr161 of D1 (D1 Tyrl61, Pro162, Gln165, Asp170, Gly171 (not labelled), Phe182, Phe186, Hisl90, I1e290 and the chlorin rings of the special pair chlorophylls (bold)).

The environment around D, Tyr161 in D2, is more hydrophobic. There are three phenylalanine residues in the close proximity of electron donor D, (D2 Phel70, Phe182 and Phe186) and Trp192 (Fig. 8). There are also large hydrophobic residues at the ends of helices 4 and 5 which may close the cleft making it inaccessible to solvent. The most striking difference in the environments around the redox active tyrosines in the different branches, is the residue opposite the phenolic sidechain in the cleft (position 171 in D1 and 170 in D2). The C-alpha atom of the conserved glycine residue in D1, models to only 3.1 A from the phenolic oxygen of Z. In D2 the equivalent position is the large hydrophobic residue Phel70, necessitating a rearrangement of sidechain orientations relative to D1, in order to

avoid sidechain clashes. These changes bring the sigma-nitrogen and oxygen atoms of D2 Gin165 to 3.0 and 3.1 ~ , respectively, from the phenolic oxygen of D, which are close enough to form hydrogen bonds. An alternate hydrogen bond is possible with the sidechain of His190 of D2. This has been proposed previously (Svensson et al. 1990). These differences between the environments of D and Z may partly explain the observed differences in the stability of EPR signals from the tyrosine radicals. The less hydrophobic, nonhydrogen bonded environment of Z is contrasted with that of D which is stabilized by a hydrogen bond and is located in a very hydrophobic pocket. The plant heterodimer binds the manganese cluster that is responsible for water oxidation on the lumenal side of PS II, using amine and car-

Glnl615 ~

"

Leu294 Phel70

Fig. 8. Stereo image of the region around residue Tyrl61 of D2 (D2 Tyrl61, Pro162, Gln165, Phel70, Phe182 (not labelled),

Phe186 (not labelled), Hisl90, Trp192, Leu294 and the chlorin rings of the special pair chlorophylls (bold)).

298 boxyl ligation (Tamura et al. 1989). The binding site may be exclusive to the D1 subunit (Virgin et al. 1988). A previous modelling study has used spectroscopically derived distance constraints to give a generalised area of binding. This placed the cluster at least 28 A but less than 43 A from Tyr-D and further than 10 .~ from Tyr-Z (Svensson et al. 1990). New spectroscopic data places the manganese cluster approximately 7 A from the radical formed from the S 2 to S 3 transition (Baumgarten et al. 1990). This radical has recently been suggested to involve Tyr-Z (Hallahan et al. 1992) and not a histidine radical as previously suggested (Boussac et al. 1990). This places the water oxidising complex within a sphere of approximately 10 A radius from Tyr-Z. It can be no closer to the special pair of chlorophylls than Z itself, to account for the route of electron donation. The region suggested by Svensson et al. (1990) to be the binding site of the cluster (the loop between helices 1 and 2) is between 10 and 3 0 A away from Tyr-Z and therefore will not form the major part of the site. Residues from D1 included in the model that are within 10 A of Tyr-Z and capable of forming ligands include, Gln165, Ser167, Aspl70, Glu189 and Hisl90. All of the sidechains of these residues, which are located in the loop between helices 3 and 4, point towards the lumen and appear to form the surface of one half of a space directly below Tyrl61, which is approximately 10 .~ in the diameter. This space and the residues surrounding it, generate a polar environment between the donor Z and the manganese cluster. Aspl70 has been proposed as a high affinity binding site for manganese by site directed mutagenesis experiments (Nixon and Diner 1992). It is proposed that the C-terminus portion of the D1 protein contributes to the manganese binding site (Diner et al. 1988), since site directed mutagenesis experiments have demonstrated an absolute requirement for residues His332 and Asp342 (Diner et al. 1991). Only after correct post translational processing will an active oxygen evolving complex assemble (Taylor et al. 1988). This suggests that the terminal residue Ala344 may also provide a ligand to the manganese cluster and so the C-terminus would be required to fold back under the reaction centre leaving Ala344 close to the base of helix 3 as previously proposed (Diner et al. 1991). Further modelling is

being carried out to fit the C-terminus of D1 to test this prediction. Other residues of interest. Other amino acids of interest include two conserved histidine residues that are located in helix 2 of D1 and D2 (Hisll8 in both). These have their sidechains directed out into the lipid bilayer. This situation suggests that the sidechains are interacting with either another protein or a cofactor, since it is unlikely that a charged residue would be conserved and facing the lipid. It is possible that these histidines may provide a site of interaction with the Cyt b559 or other core polypeptides which is found in reaction centre core complex preparations (Nanba and Satoh 1987). Alternately, they may bind additional chlorophyll molecules. Stoichiometric analyses of the reaction centre has given a variety of values for the number of chlorophylls associated with the D1/D2 complex. Three recent studies have assayed 6 chlorophyll a molecules per D1/D2/Cyt b559 complex (Gounaris et al. 1990, Kobayashi et al. 1990, Montoya et al. 1991)

Conclusions This work has generated a three-dimensional model of the PS 2 reaction centre core proteins D1 and D2 in P. sativum. The methods used are more extensive and rigorous than those in previous models. In earlier studies only parts of the complex were generated (Bowyer et al. 1990, Svensson et al. 1990, Tietjen et al. 1991). This model, generated by a rule-based computer program uses a framework derived from all of the available bacterial reaction centre structures. The loop regions between the SCRs, selected from the Brookhaven Database, were added to the framework using a least-squares method. The identification of amino acids that may be involved in the binding of cofactors provides useful targets for site directed mutagenesis studies.

Acknowledgements Professor G. Feher and his coworkers are acknowledged for providing the coordinates of the

299

Rb. sphaeroides reaction c e n t r e . We are also grateful to Prof J. Norris and coworkers for making available their coordinates for the Rb. sphaeroides reaction centre. Grateful thanks are also extended to Prof J. Thornton and her coworkers for the use of graphics facilities and the plotting programs P R E V I E W and M O L S C R I P T (Kraulis 1991). We are indebted to Dr M. Johnson for the use of the M A L I G N program. S.V.R. was supported by a S.E.R.C. studentship and D.D. was'in receipt of a S . E . R . C . / M e r c k , Sharp and D o h m e C.A.S.E. award.

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