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Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b 559 from higher plants

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Abstract

A study of the in vitro reconstitution of sugar beet cytochrome b 559 of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b 559 form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b 559. Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b 559 subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b 559-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b 559-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b 559. A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.

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Abbreviations

Abs:

Absorbance

BCA:

Bicinchroninic acid

Cyt:

Cytochrome

β-DM:

N-dodecyl-β-d-maltoside

DEAE:

Diethyl aminoethyl cellulose

E h :

Ambient redox potential

E m :

Midpoint redox potential

EPR:

Electron paramagnetic resonance

ε:

Extinction coefficient

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HP:

High potential

HS:

High spin

IP:

Intermediate potential

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

KDS:

Potassium dodecyl sulfate

LHCP:

Light-harvesting chlorophyll protein

LP:

Low potential

LS:

Low spin

MBP:

Maltose-binding protein

MES:

2-(N-Morpholino)ethanesulfonic acid

OD:

Optical density

PAGE:

Polyacrylamide gel electrophoresis

PMSF:

Phenylmethanesulfonylfluoride

PS:

Photosystem

SDS:

Sodium dodecyl sulfate

TRIS:

Tris(hydroxymethyl)aminomethane

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Acknowledgments

This work was supported by Grants AGL2008-00377, MAT2008-03461, and BFU2007-68107-C02-01 from the Spanish Ministry of Science and Innovation (MICINN), PADI CVI-261 from the Andalusia Regional Government, and DGA-GC E33 and DGA-GE B18 from Aragon Regional Government. All these Grants were partially financed by the EU FEDER Program. M. A. Luján would like to thank the FPI Fellowship Program of the MICINN for financial support.

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Authors and Affiliations

  1. Estación Experimental de Aula Dei (EEAD), Consejo Superior de Investigaciones Científicas (CSIC), Carretera Montañana 1005, 50059, Zaragoza, Spain

    María A. Luján, Inmaculada Yruela & Rafael Picorel

  2. Instituto Ciencia de Materiales de Aragón (CSIC-Universidad de Zaragoza), C/Pedro Cerbuna 12, 50009, Zaragoza, Spain

    Jesús I. Martínez & Pablo J. Alonso

  3. Instituto de Bioquímica Vegetal y Fotosíntesis (CSIC-Universidad de Sevilla), C/Américo Vespucio 49, 41092, Sevilla, Spain

    Fernando Guerrero, Mercedes Roncel & José M. Ortega

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  1. María A. Luján
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  2. Jesús I. Martínez
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  3. Pablo J. Alonso
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  4. Fernando Guerrero
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  5. Mercedes Roncel
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  6. José M. Ortega
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  7. Inmaculada Yruela
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  8. Rafael Picorel
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Correspondence to Rafael Picorel.

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Luján, M.A., Martínez, J.I., Alonso, P.J. et al. Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b 559 from higher plants. Photosynth Res 112, 193–204 (2012). https://doi.org/10.1007/s11120-012-9772-3

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