Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link.
In order to evaluate structure-function relationships of heme moiety in cytochrome P-450scc, we carried out the reconstitution of apoprotein with Fe-protoporphyrin IX, one carboxyl group of which was converted to reactive enol ester by Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). Woodward's reagent K can be used as a cross-linking reagent, since amino groups can apparently react with the enol ester. Treatment of cytochrome P-450scc with H2O2 was used to obtain the apoprotein. Functional reconstitution of the hemin derivative with apocytochrome P-450scc was achieved. The reconstituted hemeprotein was purified, and the resulting preparation contained no P-420 form and had the same cholesterol-hydroxylating activity as a control preparation. 30% of the reconstituted hemin was covalently bound to protein. Heme-linked peptide (Gly177-Phe194) was isolated. Its possible role in the active site formation of cytochrome P-450scc is discussed.
Pikuleva
I A
IA
Institute of Bioorganic Chemistry, Byelorussian Republic Academy of Sciences, Minsk, Union of Soviet Socialist Republics.
Lapko
A G
AG
Chashchin
V L
VL
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D002248:Carbon Monoxide / Q000494:pharmacology; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000378:metabolism*; D007555:Isoxazoles / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D013053:Spectrophotometry
D001051:Apoenzymes; D003432:Cross-Linking Reagents; D006420:Hemeproteins; D007555:Isoxazoles; D010446:Peptide Fragments; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D006418:Heme; D002248:Carbon Monoxide; D002786:Cholesterol Side-Chain Cleavage Enzyme