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Photosynthesis Research

, Volume 85, Issue 3, pp 359–372 | Cite as

Structure and Activity of the Photosystem II Manganese-Stabilizing Protein: Role of the Conserved Disulfide Bond

  • Aaron J. Wyman
  • Charles F. Yocum
Regular paper

Abstract

The 33-kDa manganese-stabilizing protein (MSP) of Photosystem II (PS II) maintains the functional stability of the Mn cluster in the enzyme’s active site. This protein has been shown to possess characteristics similar to those of the intrinsically disordered, or natively unfolded proteins [Lydakis-Simantiris et al. (1999b) Biochemistry 38: 404–414]. Alternately it was proposed that MSP should be classified as a molten globule, based in part on the hypothesis that its lone disulfide bridge is necessary for structural stability and function in solution [Shutova et al. (2000) FEBS Lett. 467: 137–140]. A site-directed mutant MSP (C28A,C51A) that eliminates the disulfide bond reconstitutes O2 evolution activity and binds to MSP-free PS II preparations at wild-type levels [Betts et al. (1996) Biochim. Biophys. Acta 1274: 135–142]. This mutant was further characterized by incubation at 90 °C to determine the effect of loss of the disulfide bridge on MSP thermostability and solution structure. After heating at 90 °C for 20 min, C28A,C51A MSP was still able to bind to PS II preparations at molar stoichiometries similar to those of WT MSP and reconstitute O2 evolution activity. A fraction of the protein aggregates upon heating, but after resolubilization, it regains the ability to bind to PS II and reconstitute O2 evolution activity. Characterization of the solution structure of C28A,C51A MSP, using CD spectroscopy, UV absorption spectroscopy, and gel filtration chromatography, revealed that the mutant has a more disordered solution structure than WT MSP. The disulfide bond is therefore unnecessary for MSP function and the intrinsically disordered characteristics of MSP are not dependent on its presence. However, the disulfide bond does play a role in the solution structure of MSP in vivo, as evidenced by the lability of a C20S MSP mutation in Synechocystis 6803 [Burnap et al. (1994) Biochemistry 33: 13712–13718].

Keywords

intrinsically disordered protein manganese-stabilizing protein oxygen evolution Photosystem II 

Abbreviations

CD

circular dichroism

Chl

chlorophyll

CP

chloroplast protein

DCBQ

2,6-dichloro-p-benzoquinone

EDC

1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide

EDTA

ethylenediamine tetraacetic acid

FT-IR

Fourier transform infrared

IPTG

isopropyl-β-D-thiogalactopyranoside

MES

2-(N-morpholino) ethanesulfonic acid

MSP

manganese-stabilizing protein

OEC

O2-evolving complex

PAGE

polyacrylamide gel electrophoresis

PS

photosystem

psbO

gene encoding precursor MSP

SDS

sodium dodecyl sulfate

SMN

buffer composed of sucrose (0.4 M), MES (50 mM, pH 6.0), NaCl (10 mM))

SW-PS II

Photosystem II preparation treated with 2 M NaCl to extract Ca2+ and the 23 and 17 kDa extrinsic polypeptides

USW-PS II

Photosystem II preparation treated with 2 M NaCl to extract Ca2+ and the 23 and 17 kDa extrinsic polypeptides, followed by incubation with 3.1 M urea and 240 mM NaCl to remove the manganese-stabilizing protein

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Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Department of Molecular, Cellular and Developmental BiologyUniversity of MichiganAnn ArborUSA
  2. 2.Department of ChemistryUniversity of MichiganAnn ArborUSA
  3. 3.Department of BiochemistryPurdue UniversityWest LafayetteUSA

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