The Effect Of Fe²⁺ On The Interaction Of Rhodococcus Sp.R04 CYP125A18 With Azole Drugs

Cytochrome P450 monooxygenase(cytochrome P450)is a class of monooxygenase superfamily which contains heme iron.It widely distributes in bacteria,fungi and higher organisms,such as bacteria,fungi and higher organisms.Its function includes transforming endogenous substances(such as cholesterol,steroids,fat,etc.)and catalyzing exogenous substances(such as drugs,environmental agents).The genome of Rhodococcus sp.R04 has 15 kinds of cytochrome P450 monooxygenases,of which CYP125A18 shares higher homology with CYP125 in Mycobacterium tuberculosis and Rhodococcus equi.In this study,we cloned and expressed the gene of CYP125A18,and obtained CYP125A18-Fe with foreign iron by adding Fe2+ in purification process.We analyzed the two groups of proteins,including its spectroscopic properties and the effect of interactions with azoles drug by using UY-Vis spectrophotometry.We also determined their circular dichroism spectra and the changes of dichroism spectra after them binding azole.Experimental results show that heme iron of the oxidation state CYP125A18 is mostly low spin state and a few is high spin state,while almost all of the heme iron of the oxidation state CYP125 A 18-Fe is high spin state.The difference-spectrum of oxidation state CYP125A18 and reduced state CYP125A18 combined with CO showed the typical CYP450 spectral characteristics.The CYP 125A 18-Fe protein has similar results,but Its absorption in 450nm is lower than CYP125A18.When CYP125A18 combined with the 4-cholesten-3-one,all the heme iron translate into high-spin state.Type II spectral happened after titration with azole drugs.Obvious changes happened in the ultraviolet spectrum after CYP125A18-Fe combined with the 4-cholesten-3-one and part of the heme iron recovery low spin state,which also have the same result after titration with azole drugs.The circular dichroism spectra of the CYP 125A18 and CYP 125A18-Fe show that there were no significant differences,declaring that Fe2+ had no effect on the secondary structure of proteins.The circular dichroism spectra of two proteins after binding with 4-cholesten-3-one and the number of secondary structure according to the circular dichroism spectra are consistent,indicating that its secondary structure amostly don't change after CYP125A18 combined with substrate and Fe2+ basically does not affect the combination of CYP125A18 with the substrate.The secondary structure calculating from the circular dichroism spectra of two proteins combined with azole drugs changes to ? helix and ? fold,showing that azole drugs changes the secondary structure of two groups of protein and triazole drugs have more dramatic effect on protein secondary structure.The difference of circular dichroism spectra of two proteins is not obvious,which further illustrate the Fe2+ has no effect on the combination of protein and azole drugs.The dissociation constants of CYP125A18 protein combined with azole drugs indicate that the affinity of CYP125A18 with seven azole drugs from strong to weak is ketoconazole,econazole,4-phenyl imidazole,fluconazole,4-methyl-2-phenyl imidazole,clotrimazole and metronidazole.The spectral changes of CYP125A18-Fe protein combined with azole drug significantly different from CYP125A18 protein,showing the absorption were increased at about 370 nm and 415 nm and the spectrum variation it has small difference with the CYP125A18 protein.The constant combined with seven azole drugs show that the affinity from strong to weak is same as the CYP125A18,Illustrating the Fe2+ has no effect on the combination of CYP125A18 protein and azole drugs.These findings take a significant role for the research on CYP125 metabolic of cholesterol,and provide valuable data and theoretical support for the resistance research of disease and selection of drug.