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Isolation and characterization of the membrane-bound cytochrome c-554 from the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus

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Abstract

The membrane-bound photooxidizable cytochrome c-554 from Chloroflexus aurantiacus has been purified. The purified protein runs as a single heme staining band on SDS-PAGE with an apparent molecular mass of 43 000 daltons. An extinction coefficient of 28 ± 1 mM−1 cm−1 per heme at 554 nm was found for the dithionite-reduced protein. The potentiometric titration of the hemes takes place over an extended range, showing clearly that the protein does not contain a single heme in a well-defined site. The titration can be fit to a Nernst curve with midpoint potentials at 0, +120, +220 and +300 mV vs the standard hydrogen electrode. Pyridine hemochrome analysis combined with a Lowry protein assay and the SDS-PAGE molecular weight indicates that there are a minimum of three, and probably four hemes per peptide. Amino acid analysis shows 5 histidine residues and 29% hydrophobic residues in the protein. This cytochrome appears to be functionally similar to the bound cytochrome from Rhodopseudomonas viridis. Both cytochrome c-554 from C. aurantiacus and the four-heme cytochrome c-558-553 from R. viridis appear to act as direct electron donors to the special bacteriochlorophyll pair of the photosynthetic reaction center. They have a similar content of hydrophobic amino acids, but differ in isoelectric point, thermodynamic characteristics, spectral properties, and in their ability to be photooxidized at low temperature.

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Abbreviations

LDAO:

lauryl dimethyl amine-N-oxide

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

mV:

millivolt

Em.8 :

midpoint potential at pH 8.0

ODV:

optical density x volume in ml

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  1. John C. Freeman

    Present address: Department of Chemistry, The Pennsylvania State University, 16802, University Park, PA, USA

Authors and Affiliations

  1. Department of Chemistry, Arizona State University, 85287-1604, Tempe, AZ, USA

    John C. Freeman & Robert E. Blankenship

  2. Center for Study of Early Events in Photosynthesis, 85287-1604, Tempe, AZ, USA

    John C. Freeman & Robert E. Blankenship

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Freeman, J.C., Blankenship, R.E. Isolation and characterization of the membrane-bound cytochrome c-554 from the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus . Photosynth Res 23, 29–38 (1990). https://doi.org/10.1007/BF00030060

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