Abstract
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 Pro(382)-Gln(387) 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 Cys(372) and Cys(387) 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 Cys(104) 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.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Reagents for bacterial growth were purchased from Difco. pProEXTM HTb vector, rTEV protease, and Pwo DNA polymerase were from Life Technologies, Inc
The crystal structure of the complex between the heme and FMN-containing domains of Bacillus megaterium cytochrome P450BM-3
In order to evaluate whether electron transfer (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
Summary
Materials—Reagents for bacterial growth were purchased from Difco. pProEXTM HTb vector, rTEV protease, and Pwo DNA polymerase were from Life Technologies, Inc. To build double mutants C156S/L104C, C156S/E372C, and C156S/Q387C of the heme domain of P450BM-3, the MunI/PstI fragment of pProEx-BMP(C156S) was exchanged with corresponding fragments of pProEx-BMP plasmids carrying single mutations. In order to introduce mutations into the holoenzyme of P450BM-3 and the heme/FMN-binding domain, the PinAI/PmeI fragments of pProEx-BM3 and pProEx-BMP/FMN were exchanged with corresponding fragments of pProEx-BMP carrying single and double mutants. Protein Purification—Transformation, cell growth, and purification of the 6-histidine tag fused wild type (WT) and mutants of BMP, BMP/FMN, and P450BM-3 was performed as described previously for the 1– 649 heme/FMN-containing fragment of P450BM-3 [27]. After reduction of 100 M solutions of the mutants of BMP or BMP/FMN in 20 mM sodium phosphate buffer, pH 7.4, 0.1 M NaCl, with 20 mM dithiothreitol (DTT) at 37 °C for 1.5 h, the excess of DTT was separated from the proteins by chromatography on Sephadex G-25 in the same buffer. Transient kinetic data were collected using a Tektronix TDS 410A digitizing oscilloscope and analyzed on a personal computer using KINFIT (OLIS Co, Jefferson, GA)
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