Characterization of the Redox Switches in Human Heme Oxygenase-2 and a Human Heme Oxygenase-2 and a Human-Responsive Potassium Channel.

Abstract

Heme oxygenase (HO) catalyzes heme catabolism, generating CO, biliverdin, and Fe2+. HO-2, the constitutively expressed isoform of HO, contains three Cys-Pro signatures (heme-regulatory-motifs, HRMs). In HO-2, we demonstrated that the C-terminal HRMs constitute a thiol/disulfide redox switch, regulating affinity of HO-2 for heme. HO-2 has lower affinity for Fe3+-heme (Kd=350 nM) when the C-terminal HRMs are in the dithiol state, but 10-fold higher affinity for Fe3+-heme (Kd=33 nM) when the C-terminal HRMs switch to the disulfide state. The three-dimensional structure of the core domain in HO-2 was determined in our research and found to be nearly identical to that of other HOs. Furthermore, our in vivo thiol trapping results demonstrated that the thiol/disulfide switch in human HO-2 is physiologically relevant, with its redox potential near the ambient intracellular redox potential (-200 mV). In human HEK293 cells, the C-terminal HRMs were found to be 60-70% reduced under normal growth conditions, while oxidative stress conditions convert most (86-89%) of the C-terminal HRMs to the disulfide state. Treatment with reductants converts the C-terminal HRMs largely (81-87%) to the dithiol state. Our research on the human large-conductance Ca2+ and voltage-activated K+ (BK) channel demonstrates a novel thiol/disulfide-mediated regulatory mechanism by which it can respond to cellular hypoxic/normoxic conditions. Our results demonstrate that heme, CO, and HO-2 bind to the 134-residue heme-binding domain (HBD) of human BK channel, which contains a characteristic CXXCH motif. The histidine residue in this motif serves as the axial heme ligand, with the CXXC forming a thiol/disulfide redox switch that regulates the HBD’s affinity for Fe3+-heme and CO. The dithiol state was demonstrated to bind Fe3+-heme (Kd=210 nM) 14-fold more tightly than the disulfide state. Furthermore, the HBD-Fe2+-heme complex demonstrated tight binding of CO (Kd=50 nM), with the CXXC motif regulating its affinity for CO. Fluorescence quenching experiments suggested that HBD is the key domain for BK channel’s interaction with HO-2. These combined findings indicate that thiol/disulfide redox switches in the C-terminal HRMs of HO-2 and the CXXCH motif of the BK channel establish a novel mechanism that allows hemoproteins to respond to variations in redox environments of cells.