Domain-domain interaction in cytochrome P450BM-3.
The influence of ionic strength on the interactions between individually expressed functional domains of cytochrome P450BM-3 and the domains in the holoenzyme has been analyzed by spectrophotometric and fluorometric techniques. High ionic strength facilitated electron transfer from NADPH to the FMN moiety of the reductase domain (BMR) of P450BM-3 and did not affect the first electron transfer from FMN to the heme in the holoenzyme. The cytochrome c reductase activity of the holoenzyme was higher than that of BMR within the range of ionic strength tested. Two electron reduced FMN, ie incapable of transferring electrons to the heme iron of P450BM-3, was found to be capable of reducing cytochrome c. Fluorometric studies of the domains of P450BM-3 revealed that: 1) fluorescence of FAD is completely quenched in the FAD-binding domain; 2) BMR gives the highest quantum yield which is 2.5 times higher than that of the FMN-binding domain alone; 3) the heme domain (BMP) quenches a half and three-fourths of the fluorescence of the FMN in the linked BMP/FMN-binding domain and in the holoenzyme, respectively; 4) maximal quenching of the flavin fluorescence in the mixtures containing different combinations of the functional domains of P450BM-3 was observed at high ionic strength. The results indicate that the flavins in P450BM-3 are not in close proximity. Moreover, the presence of the FAD domain causes structural changes in the FMN domain resulting in an increase in the polarity of the FMN environment in BMR and may promote the interaction between FMN- and heme-binding domains in P450BM-3. Such domain interaction may facilitate the delivery of electrons from the FMN semiquinone to the heme and prevent the formation of the inactive two electron reduced species of the FMN. Thus, the high turnover number of P450BM-3 and tight coupling of the monooxygenation reaction are provided not only by the mechanism of reduction of the heme by the reductase but also by domain-domain interaction.
Sevrioukova
I
I
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038, USA.
Peterson
J A
JA
D001426:Bacterial Proteins; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005415:Flavins; D005456:Fluorescent Dyes / Q000737:chemistry; D008861:Microsomes; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D009994:Osmolar Concentration
D001426:Bacterial Proteins; D005415:Flavins; D005456:Fluorescent Dyes; D005182:Flavin-Adenine Dinucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases