Interaction of tryptophan residues of cytochrome P450scc with a highly specific fluorescence quencher, a substrate analogue, compared to acrylamide and iodide.
The cytochrome P450scc tryptophan fluorescence was studied by the use of the three quenchers acrylamide, 25-doxyl-27-nor-cholesterol (CNO) and potassium iodide (KI). All the nine tryptophan residues were accessible to acrylamide. Whereas a strong interaction (static quenching) between acrylamide and tryptophan in the active site had been found previously for cytochrome P450c21 [Narasimhulu, S. (1988) Biochemistry 27, 1147-1153], in the case of P450scc the temperature dependence of the slope of the linear Stern-Volmer plots indicated a dynamic quenching mechanism. This mechanism was confirmed by fluorescence lifetime measurements. Of the three observed life-times tau 1 = 3.1 +/- 0.5 ns, tau 2 = 0.7 +/- 0.25 ns and tau 3 = 20 +/- 10 ps, tau 1 decreased noticeably as a function of the acrylamide concentration. CNO, a spin-labeled substrate which is known to bind tightly to the substrate-binding site of P450scc, quenched 15.5% of the total fluorescence. The Lehrer plot of this compound indicated a static quenching process with a reciprocal quenching constant of 1/Ks = 4 microM, a value which is in accord with the dissociation constant. Our data indicate that CNO quenches selectively one or two tryptophan residue(s) in the active site. The fluorescence spectrum of the residue(s) accessible to CNO was characterized by a red-shifted emission maximum (from 332 nm to 336 nm). The same residue(s) appeared to be quenched by potassium iodide, although much less effectively (1/Ks = 0.12 M). The most probable candidate for a complex formation with CNO is Trp417, which is rather close to Cys422 (the fifth heme ligand). Four arginine residues (Arg411, Arg420, Arg425 and Arg426) in the heme peptide may constitute the iodide-binding site.
Lange
R
R
Institut National de la Santé et de la Recherche Médicale, Unité 128, Montpellier, France.
Anzenbacher
P
P
Müller
S
S
Maurin
L
L
Balny
C
C
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D011193:Potassium Iodide / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D000178:Acrylamides; D005456:Fluorescent Dyes; C053027:25-doxylcholesterol; D011193:Potassium Iodide; D020106:Acrylamide; D014364:Tryptophan; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme