The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻⁶ M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻⁸ M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.
Dlia issledovaniia roli belkov-partnerov v obrazovanii funktsional'nykh kompleksov v sistemakh tsitokhroma R450 s pomoshch'iu opticheskogo biosensora opredeleny kineticheskie konstanty i ravnovesnye konstanty dissotsiatsii kompleksov tsitokhroma CYP11A1 (P450scc) s adrenodoksinom dikogo tipa Adx WT i mutantnymi formami adrenodoksina R106D i D109R. Adrenodoksin dikogo tipa Adx WT (Kd =1,23±0,09)⋅10 M) i mutantnaia forma D109R (Kd = (2,37±0,09)⋅10 M) obrazovyvali kompleksy s tsitokhromom R450scc. Dlia mutantnoĭ formy R106D sviazyvanie ne bylo zaregistrirovano. Dlia analiza vozmozhnogo uchastiia adrenodoksinov i ikh mutantnykh variantov v protsesse perenosa élektronov v mitokhondrial'nykh tsitokhrom R450 sistemakh byli issledovany élektrokhimicheskie svoĭstva étikh zhelezosernykh belkov. Adrenodoksin dikogo tipa Adx WT i mutantnye formy adrenodoksinov R106D i D109R imeiut okislitel'no-vosstanovitel'nye potentsialy E1/2 v katodnoĭ otritsatel'noĭ oblasti potentsialov, prichem znachitel'no bolee otritsatel'nye, chem tsitokhromy R450 (-579±10 mV, -590±15 mV, i -528±10 mV sootvetstvenno). Takie rezul'taty ukazyvaiut na vozmozhnost' uchastiia étikh belkov v kachestve donorov élektronov.
Keywords: adrenodoxine; cytochrome P450; electroanalysis; mutant forms; redox potential; surface plasmon resonance (SPR).