Conformational Changes near the Cytochrome P450 Active Site upon Binding of Two Different Ligands

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K. N. Myasoedova^1* and K. N. Timofeev²

¹Semenov Institute of Chemical Physics, Russian Academy of Sciences, ul. Kosygina 4, 119991 Moscow, Russia; E-mail: KseniaMyasoedova@gmail.com

²Biological Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received December 30, 2009

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It is shown that a stable nitroxyl radical, 4-cyano-2,2,6,6-tetramethylpiperidine-1-oxyl, forms a complex with cytochrome P4502B4 by analogy with the second type substrates by joining directly to pentacoordinate heme iron. The bound radical is inaccessible to water-soluble paramagnetic ions, which confirms its localization in a hydrophobic pocket near the heme. Benzphetamine and N,N-dimethylaniline, the first-type nonpolar substrates, induce conformational changes of the spin-labeled hemoprotein which are evidently accompanied by an increase in the volume of the pocket resulting in emergence of contact with aqueous phase, and the heme-bound spin label becomes accessible to water-soluble paramagnetics. In this case potassium ferricyanide broadens the spin-labeled cytochrome signal and, as a result, lowers the amplitudes of the spectral components. Similar changes were registered at non-micellar concentrations of nonionic detergent Emulgen 913, whose activating effect on hydroxylation reactions is associated, as we showed previously, with its presence in the CYP2B4 active site simultaneously with substrates.

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KEY WORDS: cytochrome P4502B4 (CYP2B4), ligands, spin label, EPR spectra, substrates, conformation

DOI: 10.1134/S0006297910070126

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