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Electrostatic Attraction between Cytochrome bc1 and Cytochrome c Affects Kinetics of Cytochrome c Reduction

V. Dadák1* and M. Holík2

1Institute of Biochemistry, Faculty of Science, Masaryk University, CZ-62500 Brno, Czech Republic; fax: +420-549492690; E-mail: dadak@chemi.muni.cz

2Institute of Chemistry, Faculty of Science, Masaryk University, CZ-62500 Brno, Czech Republic; E-mail: holik@chemi.muni.cz

* To whom correspondence should be addressed.

Received November 22, 2007; Revision received January 31, 2008
The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc1 complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c1 of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent kcat values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.
KEY WORDS: cytochrome c, cytochrome bc1, electrostatic attraction, QCR kinetics, ubiquinol-cytochrome c oxidoreductase, Paracoccus denitrificans

DOI: 10.1134/S000629790808004X