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Tapping mode atomic force microscopy was applied for a study of the molecular organization and aggregation states of purified bovine adrenocortical cytochrome P450scc present in thin solid films. The films of pure cytochrome P450scc or cytochrome P450scc mixed with lipids were transferred onto substrates of highly oriented pyrolytic graphite or mica. Images of the cytochrome P450scc films show that the topology of the proteins is drastically dependent on surface pressure. An effect of hemeprotein on the structural organization of lipid was observed. Images of the hemeprotein molecules and/or their aggregates provide a new insight for the further understanding of the nature of hemeprotein-hemeprotein and hemeprotein−lipid interactions as well as the membrane topology of cytochrome P450scc.

Monolayers of cytochrome P450scc and its complex with adrenodoxin were formed by Langmuir techniques and covalently immobilized on the solid substrates. The orientation of hemeprotein molecules was studied using polyclonal antibodies specific to the intact cytochrome P450scc molecule and its tryptic fragments F1 and F2, representing N- and C-terminal parts of the hemeprotein molecule. Specific interactions of the Langmuir films of cytochrome P450scc with adrenodoxin were investigated, and the position of the ferredoxin binding site at the hemeprotein molecule was identified. It was shown that the molecular orientation of the P450scc-AD (adrenodoxin) complex at the water-air interface is dependent on the surface density of the monolayer. The P450scc molecules do not denature upon spreading on the water surface. The formed monolayer can be transferred from the air-water interface to the surface modified with siloxane polymer and covalently immobilized without damage to the structure. A model considering the mode of orientation of cytochrome P450scc molecules on the air-water interface in dependence on the surface pressure is discussed.