Bach1, a heme‐dependent transcription factor, reveals presence of multiple heme binding sites with distinct coordination structure

Abstract

SummaryThe mammalian transcription factor Bach1 functions as a repressor of the enhancers of heme oxygenase-1 (HO-1) gene (Hmox-1) by forming heterodimers with the small Maf proteins such as MafK. The transcription of Hmox-1 is regulated by the substrate of HO-1, heme. Heme induces expression of Hmox-1 in part by inhibiting the binding of Bach1 to the enhancers and inducing the nuclear export of Bach1. A dipeptide motif of cysteine and proline (CP motif) in Bach1 is essential for the heme-mediated regulation. In this study, we show that five molecules of heme bind to Bach1 by the heme-titration assay. The Bach1-heme complex exhibits an absorption spectrum with a major Soret peak at 371 nm and Raman band at 343 cm 71 in high amounts of heme and a spectrum containing the major Soret peak at 423 nm at low heme concentrations. The spectroscopic characterization indicates that Bach1 has two kinds of heme-binding sites with different coordination structures. Mutagenesis studies have established that four molecules of heme bind to the cysteine residues of four CP motifs in the C terminus of Bach1. These results raise the possibility that two separated activities of Bach1, DNA-binding and nuclear export, are regulated by heme binding at the different CP motifs of Bach1 respectively, but not by cooperative hemebinding. IUBMB Life, 59: 542-551, 2007

Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding

“…The Soret absorption maximum of ferric PRX1 was 370 nm, and the visible maxima were 521 and 653 nm. The far blue-shifted Soret peak at ~370 nm is known as a signature of a five-coordinate high-spin heme with an axial thiol ligand [23,24], as shown in Table 1, indicating that PRX1 binds heme through Cys.…”

Wearing Red for Signaling: The Heme-Bach Axis in Heme Metabolism, Oxidative Stress Response and Iron Immunology

“…The 371-nm Soret peak becomes dominant at higher heme concentrations, which indicates the presence of another heme binding mode with affinity lower than that of the 423-nm six-coordinate mode. This binding appears to reflect a five-coordinate heme complex and involves the CP motifs (Hira et al 2007) (Fig. 6C).…”

“…2). Each of the four C-terminal CP motifs (CP3-CP6) is involved in one molecule of heme binding (Hira et al 2007). When heme binds to Bach1, heme decreases the DNA binding activity of Bach1 in vitro (Ogawa et al 2001;Sun et al 2002), reducing its half-life by promoting polyubiquitination and subsequent proteasome-mediated degradation in vivo (Zenke-Kawasaki et al 2007) (Fig.…”

“…Spectroscopic studies have been performed to elucidate the interactions between Bach1 and heme (Hira et al 2007). A recombinant fragment of Bach1 containing all of the CP motifs (176-739 a.a.) has been expressed in E. coli and purified.…”

Unique Heme Environmental Structures in Heme-regulated Proteins Using Heme as the Signaling Molecule

“…Based on the Raman spectra, the Soret peak at 423 nm can be assigned to sixcoordinated Cys-ligated heme, and another ligand trans to Cys is likely to be a histidine residue. 2 In contrast, the 371 nm absorption band becomes dominant at higher concentrations of heme, indicating the presence of another mode of heme binding to Bach1 with a lower affinity than heme binding for the 423 nm absorption band ( Figure 7B, solid line). The far blue-shifted Soret peaks at approximately 370 nm have been reported for five-coordinated Cys-ligated heme, 31,37,44 and the low-frequency region of the resonance Raman for truncated Bach1 showed a Raman line at 343 cm…”