Influence of cholesterol on kinetic parameters for human aromatase (P450 19A1) in phospholipid nanodiscs.

Cholesterol, a significant constituent of the endoplasmic reticulum membrane, exerts a substantial effect on the membrane's biophysical and mechanical properties. Cholesterol, however, is often neglected in model systems used to study membrane-bound proteins. For example, the influence of cholesterol on the enzymatic functions of type 2 cytochromes P450, which require a phospholipid bilayer and the redox partner P450-oxidoreductase (POR) for activity, are rarely investigated. Human aromatase (P450 19A1) catalyzes three sequential oxygenations of 19­carbon steroids to estrogens and is widely expressed across various tissues, which are characterized by varying cholesterol compositions. Our study examined the impact of cholesterol on the functionality of the P450 19A1 complex with POR. Nanodiscs containing P450 19A1 with 20% cholesterol/80% phospholipid had similar rates and affinity of androstenedione binding as phospholipid-only P450 19A1 nanodiscs, and rates of product formation were indistinguishable among these conditions. In contrast, the rate of the first electron transfer from POR to P450 19A1 was 3-fold faster in cholesterol-containing nanodiscs than in phospholipid-only nanodiscs. These results suggest that cholesterol influences some aspects of POR interaction with P450 19A1 and might serve as an additional regulatory mechanism in this catalytic system.

The catalytic function of cytochrome P450 is entwined with its membrane-bound nature.

Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts-cytochrome P450-reductase and cytochrome b 5 -are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s) played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.