Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.

Cytochrome P450 (CYP) enzymes are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics, and more recently to pharmacogenomics. The human CYP2C9 is involved in the metabolism of over 15% of clinical drugs that include warfarin, losartan, tolbutamide, etc. More than eighty genetic variations have been identified in CYP2C9, and many of these have demonstrated significantly reduced activity compared with the wild‐type (WT) enzyme. The CYP2C9*8 allele, prevalent among African‐American population with a frequency of around 0.06, is associated with altered clearance of several drug substrates of CYP2C9. The *8 represents an amino acid variation from arginine to histidine at position 150 (R150H). The R150H variant was generated using CYP2C9 WT construct by site‐directed mutagenesis, and the enzyme was expressed in E. coli followed by protein purification and crystallization. The CYP2C9*8 was crystallized in the presence of the drug substrate losartan and the structure was determined using X‐ray crystallography at 2.3 Å resolution. The R150H, found on the surface of the protein on D‐helix that is distal from the active site, illustrates minimal effect on the overall conformation of the protein compared to the WT. Despite subtle changes in the structure itself, there were clear differences in the binding of losartan compared to the previously solved CYP2C9 WT complex. One molecule of losartan was bound in the active site and one on the surface, consistent to that observed in the WT complex. However, unlike the WT complex, the losartan in the access channel was not observed in the *8 complex. The region near the access channel was more compact than the WT enzyme. Furthermore, the losartan turn‐over rates measured using enzymatic assays differed significantly, with the variant demonstrating marked reduction in activity than the WT enzyme. Together, our findings from multiple techniques suggest an alternate mechanism may be involved in reduced activity of this variant located on the surface that leads to differences in binding of losartan near the active site. The results yield insights into the role of simultaneous binding of multiple substrate molecules, orientation of important amino acid side chains and altered hydroxylation profile of losartan with this variant. Support or Funding Information The X‐ray data collection at the Stanford Synchrotron Radiation Lightsource (SSRL) was done remotely. Use of the SSRL, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE‐AC02076SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (P41GM103393). The research was funded by the start‐up funds from the Albany College of Pharmacy and Health Sciences.