Cytochrome P450-containing monooxygenase system participates in detoxication of mutagenic and cancinogenic substances and drug and functions by transfer of electrons from the protein partners cytochrome P450 reductase to active center of cytochrome P450. This process may be accompanied by formation of complexes between redox partners. The mechanism of formation of such complexes is not as yet fully clarified. In this study, the AFM method was used for the visualization of putidaredoxin reductase (PdR), putidaredoxin (Pd) and cytochrome P450 reductase cytochrome P450cam (P450cam) from the cytochrome P450cam - and cytochrome P450 reductase (d-Fp), cytochrome b5 (b5) and cytochrome P4502B4 (2B4) from the cytochrome P450 2B4 (2B4) containing monooxygenase system. Also visualized were the binary complexes between these molecules. Also, the visualization of the ternary PdR/Pd/P450cam in the mixture of appropriate proteins was reported.The work used of an atomic force “Solver P47H” microscope (“NT-MTD”, Russia) as well as cantilevers produced by “Micromash”. Obtained images were processed with “Smena” software (“NT-MTD”). As support for manufacturing of AFM nanochips of water-soluble proteins from the P450cam system - putidaredoxin (Pd), putidaredoxin-reductase (PdR) and P450cam - was used (negatively charged) mica. Visualization of membrane-bound protein from the P4502B4 system - NADPH-cytochrome P450 reductase (d-Fp), cytochromes b5 (d-b5) and 2B4 and protein complexes was accomplished using (a hydrophobic) highly-oriented pirolytic graphite nanochips. Typical heights of P450cam, Pd and PdR images were 2.6±0.3 nm, 2.0±0.3 nm è 2.8±0.3 nm, respectively. Pd/P450cam formed a group with a height of 3.9-5.7 nm. The heights of Pd/PdR and PdR/P450cam complexes constituted 4.9±0.3 nm and 5.1±0.3 nm, respectively. The 9.1-nm images correspond to ternary complexes, i.e. to aggregates containing molecules of PdR, Pd and P450cam. Typical heights of 2B4, Fp and b5 monomers were 2.2±0.3 nm, 2.3±0.3 nm and 1.8±0.3 nm, respectively. The height of 2B4/Fp complexes makes up 3.4 nm. The height of 2B4/b5 complexes is estimated to be 2.8 nm and 3.7 nm . No Fp/b5 complexes were registered.
This work was supported by RFBR grant N 01-04-48245, Scientific School RFBR 0015-97926