Abstract
Of the 57 human cytochrome P450 (P450) enzymes, seven are mitochondrial: 11A1, 11B1, 11B2, 24A1, 27A1, 27B1, and 27C1. Mitochondrial P450s utilize an electron transport system with adrenodoxin (Adx) and NADPH-adrenodoxin reductase (AdR). AdR reduces Adx, which then transfers electrons to the P450. The interactions between proteins in the mitochondrial P450 system are largely driven by electrostatic interactions, though the specifics vary depending on the P450. Unlike other mitochondrial P450s, the interaction between P450 27C1, a retinoid 3,4-desaturase expressed in the skin, and Adx remains largely uncharacterized. In this work, we utilized an Alexa Fluor 488 C5 maleimide-labeled Adx to measure binding affinities between Adx and P450 27C1 or AdR. Both proteins bound Adx tightly, with Kd values < 100 nM, and binding affinities decreased with increasing ionic strength, supporting the role of electrostatic interactions in mediating these interactions. Cross-linking mass spectrometry and computational modeling were performed to identify interactions between P450 27C1 and Adx. While the residues of Adx identified in interactions were consistent with studies of other mitochondrial P450s, the binding interface of P450 27C1 was quite large and supported multiple Adx binding positions, including ones outside of the canonical Adx binding site. Additionally, Adx did not appear to be an allosteric effector of P450 27C1 substrate binding, in contrast to some other mitochondrial P450s. Overall, we conclude that P450-Adx interactions are P450-specific.