ABSTRACT Cytochrome P450 enzymes (CYPs) are membrane-associated heme proteins that mediate catalysis for a range of reactions. Seven of the 57 CYP enzymes in humans function in the inner mitochondrial membrane, where they perform important endogenous reactions in steroid metabolism and vitamin-D activation and inactivation. A primary focus of our research program is to understand how these CYPs are regulated on a structural level, particularly for catalytic steps requiring binding of substrate and molecular recognition of Adrenodoxin (Adx), the soluble ferredoxin required for electron delivery. This is the objective of a recently activated NIGMS award (R35 GM133375) in which our projects focus on two vitamin-D metabolizing enzymes (CYP24A1 and CYP27B1) and one vitamin-A metabolizing enzyme (CYP27C1). A central question is how substrate binding and Adx recognition events are structurally related, via conformational changes in the CYP, and how such allostery impacts CYP function. However, due to limitations inherent in our biochemical assays and our current instrumentation, we are not able to accurately quantify molecular interactions between CYP and ligand and between CYP and Adx. Our laboratory, along with the laboratory of Dr. Mark Sutton, who is also applying for this supplementation award (R01 GM130761), were recently able to observe an in-lab demonstration of the OctetRed96e bio-layer interferometry instrument (ForteBio). We have concluded that the design features of this instrument will allow us to quantify these interactions in a rapid, label-free way. This capability will allow our program to expand and pose two new questions: i) what are CYP-ligand affinities for interactions that occur independent of a change in heme spin state? … and ii) How do substrates and inhibitors alter the specific affinity of the CYP-Adx interaction? Answers to these questions have the potential to expand our current research program far beyond our current objectives.