Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link

• Published in: The Journal of biological chemistry Vol. 267; no. 3; pp. 1438 - 1442
• Main Authors: Pikuleva, I A, Lapko, A G, Chashchin, V L
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.01.1992; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Apoenzymes - metabolism; Binding Sites; Biological and medical sciences; Carbon Monoxide - pharmacology; Cholesterol Side-Chain Cleavage Enzyme - metabolism; Chromatography, Gel; Cross-Linking Reagents - pharmacology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Heme - metabolism; Hemeproteins - metabolism; Isoxazoles - pharmacology; Kinetics; Molecular Sequence Data; Oxidation-Reduction; Oxidoreductases; Peptide Fragments - isolation & purification; Spectrophotometry; Hemoprotein; Cholesterol monooxygenase(side-chain cleaving); Hemin; Mother; Structure activity relation; Enzyme; Cytochrome P450; Crosslink agent; Ultraviolet visible spectrometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)45964-9

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.