Steps toward constructing a cytochrome b6 f complex in the purple bacterium Rhodobacter sphaeroides: an example of the structural plasticity of a membrane cytochrome.
We have modified the cytochrome b subunit of the cytochrome bc1 complex from the purple bacterium Rhodobacter sphaeroides to introduce two distinctive features of cytochrome b6 f complexes. In the first one, we have split cyt b into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f complexes. In the second, an extra residue was added between His198 and Phe199, thus extending the span between the histidine ligands for the two b-hemes in helix D. The properties of the mutant strains were determined using thermodynamic and kinetic analysis. The two mutant enzymes were assembled and functioned so as to allow the photosynthetic growth of the mutant strains. For the split enzyme, we show that two independently translated fragments of cyt b are inserted in the membrane. Our results indicate a decrease in the stability of the semiquinone formed at the quinone reduction (Qi) site in this mutant. This property, characteristic for b6 f complexes, indicates the functional importance of the connecting span between helices D and E. The presence of the inserted threonine in helix D modified the spectrum and redox potential of the bL-heme, shifting the potential difference between the two b-hemes from 140 mV in the wild-type to 55 mV in the mutant strain. This change in the driving force of electron transfer through the membrane was reflected in an inability of the mutant strain to accumulate a large transmembrane electrical potential on successive flashes.
Kuras
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R
Department of Microbiology, Center for Biophysics and Computational Biology, University of Illinois, Urbana 61801, USA.
Guergova-Kuras
M
M
Crofts
A R
AR
D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D045346:Cytochrome b6f Complex; D004579:Electron Transport / Q000235:genetics; D008564:Membrane Potentials / Q000235:genetics; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010782:Photolysis; D010788:Photosynthesis / Q000235:genetics; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D018717:Purple Membrane / Q000201:enzymology*; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013053:Spectrophotometry
D003573:Cytochrome b Group; D008565:Membrane Proteins; D045346:Cytochrome b6f Complex