Selective chemical modification of Cys²⁶⁴ with diiodofluorescein iodacetamide as a tool to study the membrane topology of cytochrome P450scc (CYP11A1)

Abstract

CYS ZM of cytochrome P45Oscc (CYPllAl) was selectively labelled with diiodofluorescein iodacetamide in solution and in proteoliposomes. The lahelling affected the interaction of P45Oscc with adrenodoxin and significantly inhibited the side-chain cleavage activity of the soluble and membrane-bound hemeprotein in the reconstituted system. In proteoliposomes both the labelled and unlabelled hemepoteins were susceptible to trypsin and split into Fl and F2, two fragments corresponding to the two main domains of P45Oscc. These results suggest that the hinge connecting the two domains in the region Arg?4&' 1s exposed to the surface of the membrane and involved in the interaction of P45Oscc with adrenodoxin.

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc

“…Recently, numerous crosslinking and chemical modification experiments have provided evidence for specific residues and peptides at the Adx–P450scc interface [9,14–17,25,27–29]. Because no three‐dimensional structure of P450scc is currently available, we used a computer model of the protein as a basis for discussing the interaction of P450scc with Adx.…”

“…Several interaction sites deduced from chemical modification, site‐directed mutagenesis and crosslinking experiments cannot be explained by this model. For example, the peptides Pro97 to Trp108, Leu368 to Arg384, Ser390 to Pro397 [16], Arg250 to Asn257 [28], and Lys73, 109, 110, 145, 148, 154 and 270 of P450scc [15], as well as the Adx peptide Ile7 to Arg14 [17] are far away from the area proposed to be buried between Adx and P450scc. In support of our model, parts of these peptides, as well as Lys73 and Lys145 of P450scc, have been identified using mass spectrometric sequencing (Fig.…”

Complex Formation in Vesicle-Reconstituted Mitochondrial Cytochrome P450 Systems (CYP11A1 and CYP11B1) As Evidenced by Rotational Diffusion Experiments Using EPR and ST-EPR

“…Prior labeling of P450SCC with diiodofluorescin iodoacetamide (DIFIA), a fluorophore known to bind selectively to Cys264 (35), effectively prevented labeling with MSL(6)** (Figure 2, bottom spectrum). By double integration of the spectra, the level of residual labeling after blocking Cys264 was determined to about 15%.…”

Structure-Guided Activity Restoration of the Silkworm Glutathione Transferase Omega GSTO3-3

“…Incubation was carried out on ice for 2 h. After incubation, the mixture was applied to a Sephadex G-25F column equilibrated with 100 mM potassium phosphate buffer, pH 6.5, to remove the extra iodoacetamide. 36 Intrinsic fluorescence spectroscopy…”