Mutational analysis of residues forming hydrogen bonds in the Rieske [2Fe-2S] cluster of the cytochrome bc1 complex in Paracoccus denitrificans.
Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe-2S] cluster stability, the enzyme activity and the redox potential of the [2Fe-2S] cluster. In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 O gamma-S-1 and Tyr165 O eta-Cys139 S gamma [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567-579]. They are conserved in all known Rieske iron-sulfur proteins from bc1 complexes including that from P. denitrificans. Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain. The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc1 complex isolated from the parental strain was 145 s(-1), the turnover numbers for the mutants S157A and Y159F were 10 s(-1) and 52 s(-1), respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential Em of the Rieske cluster at pH 6 and 5 degrees C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.
Schröter
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Molekulare Genetik, Institut für Biochemie, Biozentrum, J.W. Goethe-Universität Frankfurt/Main, Germany.
Hatzfeld
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OM
Gemeinhardt
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S
Korn
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Friedrich
T
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Ludwig
B
B
Link
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TA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008929:Mitochondria, Heart; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000737:chemistry* / Q000235:genetics; D011199:Potentiometry; D013045:Species Specificity
D007506:Iron-Sulfur Proteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III