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J. Biol. Chem., Vol. 279, Issue 33, 34269-34276, August 13, 2004

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Stripping Down the Mitochondrial Cholesterol Hydroxylase System, a Kinetics Study^*

Burkhard Schiffler, Andy Zöllner, and Rita Bernhardt

From the Naturwissenschaftlich-Technische Fakultät III, FR 8.8-Biochemie, Universität des Saarlandes, P. O. Box 151150, D-66041 Saarbrücken, Germany

The origin of steroid hormones in mammals is cholesterol that is metabolized by the mitochondrial CYP11A1 system. The cytochrome P450 is fed with reduction equivalents via a small electron transfer chain consisting of NADPH, adrenodoxin reductase, and adrenodoxin. Though the redox behavior of the individual protein components has been studied previously, the kinetics of the system in its entirety has not yet been analyzed. In this study we combine surface plasmon resonance experiments to determine the binding constants for the different pairs of redox partners with measurements of the pre-steady-state kinetics of the different reaction steps of this system and steady-state kinetics. We could correlate the individual protein-protein interactions with the effect of distinct reduction-oxidation steps on the overall catalytic activity of the CYP11A1 system. For the first time, we were able to follow the reduction of each of the protein components of this system within one measurement when we mixed all oxidized protein components with NADPH. These measurements allowed the determination of the individual apparent rate constants for the reduction of all three proteins involved. In addition, variation of the ionic strength in these experiments revealed different optimum salt concentrations for the reduction of adrenodoxin reductase and adrenodoxin, respectively, and unraveled dramatically changing reduction rates of CYP11A1 by adrenodoxin.

Received for publication, March 12, 2004 , and in revised form, June 4, 2004.

^* This work was supported by a grant of the Deutsche Forschungsgesallschaft (Be1343/12/1-2), the Fonds der chemischen Industrie, and the Volkswagen Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-681-302-4241; Fax: 49-681-302-4739; E-mail: ritabern{at}mx.uni-saarland.de.

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