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Purification and characterization of cytochrome c 6 from Acaryochloris marina

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Abstract

Cytochrome c 6 , (cyt c 6) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.

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Abbreviations

cyt c 6 :

Cytochrome c 6

FPLC:

Fast protein liquid chromatography

IEF:

Isoelectrofocusing

CD:

Circular dichroism

E m :

Midpoint potential

NHE:

Normal hydrogen electrode

A. marina :

Acaryochloris marina

PSI:

Photosystem I

PC:

Plastocyanin

IEC:

Ion exchange chromatography

MALDI-TOF:

Matrix assisted laser desorption ionization-time of flight

LC-MS/MS:

Liquid chromatography tandem mass spectrometry

χ :

Fraction reduced

A :

Absorbance

A o :

Absorbance of fully oxidized species

A r :

Absorbance of fully reduced species

E :

potential

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Acknowledgments

We would like to thank the Washington University Mass Spectrometry Resource Center supported by NIH NCRR Grant No. 2P41RR000954 for their assistance. In addition, we would like to thank Jianzhong Wen for his help with the mass spectrometry and Aaron Collins for his help with the CD spectroscopy. This research was supported by a grant from the Exobiology program from NASA.

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Authors and Affiliations

  1. Department of Chemistry, Washington University in St. Louis, Campus Box 1137, St. Louis, MO, 63130-4899, USA

    Patrick D. Bell, Yueyong **n & Robert E. Blankenship

  2. Department of Biology, Washington University in St. Louis, Campus Box 1137, St. Louis, MO, 63130-4899, USA

    Robert E. Blankenship

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  1. Patrick D. Bell
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  2. Yueyong **n
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Correspondence to Robert E. Blankenship.

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Bell, P.D., **n, Y. & Blankenship, R.E. Purification and characterization of cytochrome c 6 from Acaryochloris marina . Photosynth Res 102, 43–51 (2009). https://doi.org/10.1007/s11120-009-9482-7

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