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J Biol Chem, Vol. 274, Issue 51, 36097-36106, December 17, 1999

The FMN to Heme Electron Transfer in Cytochrome P450BM-3
EFFECT OF CHEMICAL MODIFICATION OF CYSTEINES ENGINEERED AT THE FMN-HEME DOMAIN INTERACTION SITE^*

Irina F. Sevrioukova, James T. Hazzard^§, Gordon Tollin^§, and Thomas L. Poulos^¶

From the  Department of Molecular Biology and Biochemistry and the Program in Macromolecular Structure, University of California, Irvine, California 92697-3900 and the ^§ Department of Biochemistry, University of Arizona, Tucson, Arizona 85721

The crystal structure of the complex between the heme and FMN-containing domains of Bacillus megaterium cytochrome P450BM-3 (Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., and Poulos, T. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1863-1868) indicates that the proximal side of the heme domain molecule is the docking site for the FMN domain and that the Pro³⁸²Gln³⁸⁷ peptide may provide an electron transfer (ET) path from the FMN to the heme iron. In order to evaluate whether ET complexes formed in solution by the heme and FMN domains are structurally relevant to that seen in the crystal structure, we utilized site-directed mutagenesis to introduce Cys residues at positions 104 and 387, which are sites of close contact between the domains in the crystal structure and at position 372 as a control. Cys residues were modified with a bulky sulfhydryl reagent, 1-dimethylaminonaphthalene-5-sulfonate-L-cystine (dansylcystine (DC)), to prevent the FMN domain from binding at the site seen in the crystal structure. The DC modification of Cys³⁷² and Cys³⁸⁷ resulted in a 2-fold decrease in the rates of interdomain ET in the reconstituted system consisting of the separate heme and FMN domains and had no effect on heme reduction in the intact heme/FMN-binding fragment of P450BM-3. DC modification of Cys¹⁰⁴ caused a 10-20-fold decrease in the interdomain ET reaction rate in both the reconstituted system and the intact heme/FMN domain. This indicates that the proximal side of the heme domain molecule represents the FMN domain binding site in both the crystallized and solution complexes, with the area around residue 104 being the most critical for the redox partner docking.

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