Tài liệu Báo cáo Y học: Role of electrostatics in the interaction between plastocyanin and

Role of electrostatics in the interaction between plastocyanin

and photosystem I of the cyanobacterium Phormidium laminosum

Beatrix G. Schlarb-Ridley1

, Jose´ A. Navarro2

, Matthew Spencer1

, Derek S. Bendall1

, Manuel Herva´ s

2

,

Christopher J. Howe1 and Miguel A. De la Rosa2

1

Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, UK; 2

Instituto de

Bioquı´mica Vegetal y Fotosı´ntesis, Centro de Investigaciones Cientı´ficas Isla de la Cartuja, Universidad de Sevilla y CSIC, Spain

The interactions between photosystem I and five charge

mutants of plastocyanin from the cyanobacterium Phormi￾dium laminosum were investigated in vitro. The dependence

of the overall rate constant of reaction, k2, on ionic strength

was investigated using laser flash photolysis. The rate con￾stant of the wild-type reaction increased with ionic strength,

indicating repulsion between the reaction partners. Remov￾ing a negative charge on plastocyanin (D44A) accelerated the

reaction and made it independent of ionic strength; removing

a positive charge adjacent to D44 (K53A) had little effect.

Neutralizing and inverting the charge on R93 slowed the

reaction down and increased the repulsion. Specific effects of

MgCl2 were observed for mutants K53A, R93Q and R93E.

Thermodynamic analysis of the transition state revealed

positive activation entropies, suggesting partial desolvation

of the interface in the transition state. In comparison with

plants, plastocyanin and photosystem I of Phormidium

laminosumreact slowly at low ionic strength, whereas the two

systems have similar rates in the range of physiological salt

concentrations. We conclude that in P. laminosum, in con￾trast with plants in vitro, hydrophobic interactions are more

important than electrostatics for the reactions of plastocya￾nin, both with photosystem I (this paper) and with cyto￾chrome f [Schlarb-Ridley, B.G., Bendall, D.S. & Howe, C.J.

(2002) Biochemistry 41, 3279–3285]. We discuss the impli￾cations of this conclusion for the divergent evolution of

cyanobacterial and plant plastocyanins.

Keywords: cyanobacteria; electron transfer; photosystem I;

plastocyanin; weak interaction.

Electron-transfer chains like that of oxygenic photosyn￾thesis impose special restraints on the proteins involved.

Reactions must be fast to allow rapid turnover of the

chain. Binding between the reaction partners must be

transient, while at the same time sufficient specificity needs

to be retained. Surface properties of proteinaceous reac￾tion partners play a crucial role in meeting these criteria.

The aim of our research was to increase our understand￾ing of how one property of the protein surface, the charge

pattern, influences the rate constant of the overall reaction

and how it may have evolved. Our model protein is

plastocyanin, a soluble photosynthetic redox protein

which accepts an electron from cytochrome f in the

cytochrome bf complex and passes it on to P700

+ in

photosystem I. In a previous study [1], we mutated

negatively and positively charged residues on the proposed

interaction site of plastocyanin with cytochrome f and

analysed the reaction of these mutants with the soluble

redox-active domain of cytochrome f (Cyt f) in vitro. This

paper presents results on the interaction in vitro between a

representative subset of these charge mutants with the

physiological electron acceptor of plastocyanin, photosys￾tem I. Hence, we can compare two sets of protein–protein

interactive surfaces operating in the same compartment

with similar functional selection pressures, with the aim of

identifying common features.

The organism from which plastocyanin and both its

reaction partners, Cyt f [1] and photosystem I (this paper),

were taken is a moderately thermophilic cyanobacterium,

Phormidium laminosum. Studying these photosynthetic

electron-transfer reactions of cyanobacteria is of evolu￾tionary interest: whereas the overall three-dimensional

structure of plastocyanin is highly conserved among plants

and cyanobacteria, the surface charge pattern varies

greatly [1]. Comparing cyanobacterial data with the wealth

of information available for the higher plant reaction [2–5]

reveals which functional aspects are variable. Further￾more, the type I copper protein plastocyanin can be

replaced by cytochrome c6, a redox protein of similar size

but entirely different folding, in a number of eukaryotic

algae and cyanobacteria including P. laminosum [6,7].

Hence two more sets of protein–protein interactive

surfaces with the same function as Cyt f – plastocyanin

and plastocyanin–photosystem I – are available for identi￾fication of features common to interprotein electron￾transfer reactions [4,7]. To our knowledge, this is the first

Correspondence to B. G. Schlarb-Ridley, Department of Biochemistry,

University of Cambridge, Building O, The Downing Site,

Cambridge CB2 1QW, UK.

Fax: + 44 1223 333345, Tel.: + 44 1223 333684,

E-mail: [email protected]

Abbreviations: Cyt f, soluble redox-active domain of cytochrome f;

kobs, observed first-order rate constant; kon, rate constant of protein

association; koff, rate constant of complex dissociation before electron

transfer has taken place; ket, rate constant of intracomplex electron

transfer; k2, bimolecular rate constant of the overall reaction; k¥, k2 at

infinite ionic strength.

(Received 10 June 2002, revised 5 September 2002,

accepted 15 October 2002)

Eur. J. Biochem. 269, 5893–5902 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03314.x