Langmuir--Blodgett Films of Cytochrome P450scc and Its Complex with Adrenodoxin

O. L. Guryev,¹ T. B. Dubrovsky,² A. A. Chernogolov,¹ S. B. Dubrovskaya,³ C. Nicolini,³ and S. A. Usanov^1,4

¹Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, ul. Zhodinskaya 5/2, Minsk, 220141 Belarus; fax: (0172) 63-72-74; E-mail: usanov@eco2.iasnet.com

²Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia.

³Institute of Biophysics, University of Genoa, via Giotto 2, Genoa Sestri Ponente 16153, Italy.

⁴To whom correspondence should be addressed.

Submitted December 24, 1996; revision submitted March 5, 1997.

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Langmuir--Blodgett films prepared from cytochrome P450scc and its complex with adrenodoxin have been prepared and studied. Adrenodoxin was preliminarily selectively modified with fluorescein isothiocyanate and the effect of this modification on the interaction with cytochrome P450scc was studied. Using selectively modified adrenodoxin the ratio of the proteins in the film was found to be 1 mole of adrenodoxin per 2 moles of cytochrome P450scc. Langmuir--Blodgett films were also prepared from adrenodoxin-reductase and it was shown that this flavoprotein is transferred to the substrate as an apo-protein. It is also shown that the adrenodoxin-binding region of cytochrome P450scc is exposed to the subphase under all pressures in the interval studied. The relationship between the orientation of cytochrome P450scc--adrenodoxin complex in monolayers on the water--air interface and the pressure produced at the interface at the moment of monolayer formation was found. Our data are in excellent accordance with ideas on the molecular organization of cytochrome P450scc in the inner adrenocortical membrane and allows the use of this approach to model membrane structures containing cytochrome P450.

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KEY WORDS: cytochrome P450scc, adrenodoxin, adrenodoxin-reductase, Langmuir--Blodgett films.

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