Structural differences between soluble and membrane bound cytochrome P450s

The superfamily of cytochrome P450s forms a large class of heme monooxygenases with more than 13,000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochrome P450s from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochrome P450s reveals the presence of two distinct structural classes of cytochrome P450s. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which may result in an increase of the volume of the substrate binding pocket and an opening of a potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochrome P450s, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochrome P450s can be rationalized through the presence of several amino acid inserts in the latter class which are involved in direct interactions with the membrane, namely the F′- and G′-helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B–C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B–C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochrome P450s with important functional implications induced by the lipid bilayer.

Soluble and membrane bound cytochromes P450 have different configuration of the predominant substrate access pathways. This difference is determined by positions of the beta-domain with respect to the heme plane and to the F-G helical buindle, as seen from comparison of soluble CYP101 and membrane bound CYP3A4 shown in the figure.Figure optionsDownload as PowerPoint slideHighlights
► Comparison of all available X-ray structures of cytochromes P450 reveals new features.
► Position of the beta-domain defines the direction of substrate access channel.
► Soluble and membrane bound cytochromes P450 form two structural classes.
► MD study of the membrane bound CYP3A4 shows stable opening directed into the bilayer.