Tài liệu Báo cáo khoa học: Cytochrome b559 content in isolated photosystem II reaction center

Cytochrome b559 content in isolated photosystem II

reaction center preparations

Inmaculada Yruela1

, Francisca Miota1

, Elena Torrado1

, Michael Seibert2 and Rafael Picorel1

1

Estacio´n Experimental de Aula Dei (CSIC), Zaragoza, Spain; 2

National Renewable Energy Laboratory, Basic Sciences Center,

Golden, CO, USA

The cytochrome b559 content was examined in five types

of isolated photosystem II D1-D2-cytochrome b559 reaction

center preparations containing either five or six chlorophylls

per reaction center. The reaction center complexes were

obtained following isolation procedures that differed in

chromatographic column material, washing buffer compo￾sition and detergent concentration. Two different types of

cytochrome b559 assays were performed. The absolute heme

content in each preparation was obtained using the oxidized￾minus-reduced difference extinction coefficient of cyto￾chrome b559 at 559 nm. The relative amount of D1 and

cytochrome b559 a-subunit polypeptide was also calculated

for each preparation from immunoblots obtained using

antibodies raised against the two polypeptides. The results

indicate that the cytochrome b559 heme content in photo￾system II reaction center complexes can vary with the

isolation procedure, but the variation of the cytochrome b559

a-subunit/D1 polypeptide ratio was even greater. This

variation was not found in the PSII-enriched membrane

fragments used as the RC-isolation starting material, as

different batches of membranes obtained from spinach

harvested at different seasons of the year or those from sugar

beets grown in a chamber under controlled environmental

conditions lack variation in their a-subunit/D1 polypeptide

ratio. A precise determination of the ratio using an

RC1-control sample calibration curve gave a ratio of 1.25

cytochrome b559 a-subunit per 1.0 D1 polypeptide in photo￾system II membranes. We conclude that the variations

found in the reaction center preparations were due to the

different procedures used to isolate and purify the different

reaction center complexes.

Keywords: chromatography; cytochrome b559; detergent;

immunoblot; photosystem II.

Cytochrome (Cyt) b559 is a hemoprotein component of the

photosystem II (PSII) reaction center (RC) complex [1], and

it is an integral component of the minimal isolated RC

complex still capable of performing primary charge separ￾ation. It is composed of two small polypeptides, the a

(9 kDa) and b (4.5 kDa) subunits, encoded by the psbE and

psbF genes, respectively. Each polypeptide has a single

transmembrane a-helical domain [2,3]. The heme iron is

bound to a single histidine residue on each subunit [4], and it

is located close to the stromal surface of the membrane

[2,3,5–7]. However, a location for Cyt b559 heme on the

lumenal side of the PSII membrane has also been proposed,

suggesting that two hemes and two copies each of the two

subunits are present in the thylakoid membrane [8,9].

Despite numerous studies [8,10,11], the exact function of

Cyt b559 is still unclear but the following are possibilities: (a)

involvement in the electron transfer reactions on the

oxidizing side of PSII [12,13]; (b) participation in the

assembly of the water-splitting system [14]; and (c) protec￾tion of PSII against photoinhibition [15–19]. It is well

known that Cyt b559 can exist in a number of different redox

forms. At pH 6.0–6.5, PSII complexes, surrounded by their

natural membrane environments, as in chloroplasts, thyla￾koids and PSII membrane fragments, Cyt b559 exhibits

midpoint redox potentials (E¢m) of +400 mV [the high

potential (HP) form], +200–150 mV [the intermediate

potential (IP) form], and +70–60 mV [the low potential

(LP) form] [1,20–22]. The HP form dominates in thyla￾koids and PSII membranes with an intact water-oxidizing

complex.

A longstanding issue has been the number of Cyt b559 per

PSII complex. Shuvalov and coworkers argued that PSII

core complex from spinach with high O2-evolution activity

contains two Cyt b559 per PSII [8]. However, the currently

accepted value in isolated PSII RCs, based mainly on

absorption spectroscopy techniques [1,23–26], is one heme

per RC. Recent data based on the crystal structure of the

PSII core from Synecochoccus elongatus [2] and Thermo￾synechococcus vulcanus [3] are in agreement with this

proposal. But a second cytochrome might have been lost

during the preparation of the core material. Thus the

question of one or two Cyt b559 per PSII RC remains

unresolved because the stoichiometry might depend on the

isolation procedure used, the type of PSII preparation and/

or the organism examined.

Correspondence to R. Picorel, Estacio´n Experimental de Aula Dei

(CSIC), Ctra. Montan˜ana 1005, Zaragoza E-50080, Spain.

Fax: + 34 976 716145; Tel.: + 34 976 716053;

E-mail: [email protected]

Abbreviations: Cyt, cytochrome; D1/D2 HD, heterodimer made by

crosslinking of D1 and D2 polypeptides; DM, n-dodecyl b-D-malto￾side; HP, high potential; IMAC, immobilized metal affinity chroma￾tography; IP, intermidiate potential; LP, low potential; Mes,

2-(N-morpholino) ethane-sulfonic acid; PS, photosystem;

RC, reaction center.

Enzymes: glucose oxidase (EC 1.1.3.4); catalase (EC 1.11.1.6).

(Received 23 January 2003, revised 21 March 2003,

accepted 26 March 2003)

Eur. J. Biochem. 270, 2268–2273 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03597.x