title: Aluminum-substituted heme domain of P450BM-3 (BMP): introducing a heme-derived fluorescent probe for studies of substrate binding and protein-protein interactions in cytochromes P450.
abstract: We describe a novel technique for heme removal and replacement in the heme domain of P450BM-3 (BMP). The method was applied to obtain the aluminum-protoporphyrin IX (Al-PP) substituted derivative of BMP (Al-BMP). The overall yield of the purified Al-BMP was about 15% as related to the initial amount of the hemeprotein. Al-BMP possesses extensive fluorescence in the 550-650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM-3 (palmitic, arachidonic, and cis-parinaric acids) with affinities similar to those of the native enzyme (3-6 µM). However, the substrate-induced changes in fluorescence of Al-PP reveal the existence of a second, low-affinity substrate-binding site, which cannot be detected by the spin shift in the native, heme-containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al-BMP forms a complex with the flavoprotein domain of P450BM-3 labeled with 7-ethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin maleimide, revealing the affinity similar to that of native BMP (Kd = 5 µM at 0.06 M ionic strength). Therefore, aluminum-substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.
authors.affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA. ddavydov@ucsd.edu
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
authors.forename: Gelii V
authors.initials: GV
authors.lastname: Ponomarev
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Bobrovnikova-Marjon
authors.forename: Donovan C
authors.initials: DC
authors.lastname: Haines
authors.forename: Julian A
authors.initials: JA
authors.lastname: Peterson
mesh_terms: D000535:Aluminum / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D000535:Aluminum; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases