The 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(-1) 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 
heme-binding.