C‐terminal membrane spanning region of human Heme Oxygenase‐1 mediates a time‐dependent complex formation with NADPH cytochrome P450 reductase

Abstract

Heme oxygenase‐1 (HO‐1) catalyzes the oxidative degradation of heme to biliverdin, carbon monoxide, and free iron in a reaction requiring the interaction of HO‐1 with NADPH‐cytochrome P450 reductase (CPR). Full‐length HO‐1 (flHO1) is bound to the endoplasmic reticulum by 23 C‐terminal amino acids, however a soluble HO‐1 (sHO‐1) lacking this membrane spanning region has been extensively studied. The goal of this project was to characterize the effect of the C‐terminal hydrophobic domain on formation of the flHO‐1/CPR complex. In previous experiments, we demonstrated a catalytic difference between flHO‐1 and sHO‐1; indicating that the C‐terminal region influences HO‐1 binding to CPR. Recent kinetic data proposed that the increased specific activity of flHO‐1 may be attributable to a time dependent formation of a high affinity flHO‐1/CPR complex that was not seen with sHO1. Gel filtration analysis confirmed the formation of multiple high molecular weight complexes in the presence and absence of the synthetic lipid dilauroylphosphatidylcholine (DLPC). However, the largest complex appeared following a two hour incubation of flHO‐1 and CPR in DLPC, suggesting that C‐terminal region was required for flHO‐1/CPR complex formation and membrane incorporation. These data displayed that the C‐terminal region influenced flHO‐1 complex formation and ultimately its affinity for CPR. (Supported by NIEHS ES004344)