Bach1 is a transcriptional repressor of heme oxygenase-1 and β-globin genes, both of which are known to be transcriptionally induced by heme. To test the hypothesis that heme regulates the activity of Bach1, we expressed wild type and mutated versions of Bach1 together with or without its heterodimer partner MafK in human 293T and GM02063 cells and examined their subcellular localization. Inhibition of heme synthesis enhanced the nuclear accumulation of Bach1, whereas treating cells with hemin resulted in nuclear exclusion of Bach1. While the cadmium-inducible nuclear export signal (NES) of Bach1 was dispensable for the heme response, a region containing two of the heme-binding motifs was found to be critical for the heme-induced nuclear exclusion. This region functioned as a heme-regulated NES dependent on the exporter Crm1. These results extend the regulatory roles for heme in protein sorting, and suggest that Bach1 transduces metabolic activity into gene expression

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

No abstract

Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H 2 , but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (Gln3 Arg), Asn (Gln3 Asn), and Val (Gln3 Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a sixcoordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.Prostaglandin endoperoxide H synthase (PGHS, 3 also known as cyclooxygenase or COX) is a membrane-bound heme-containing protein catalyzing the first committed step in prostanoid biosynthesis (1-3) including two sequential enzymatic reactions, bis-oxygenation of arachidonic acid to prostaglandin G 2 (PGG 2 ) (cyclooxygenase reaction) and the reduction of PGG 2 to prostaglandin H 2 (PGH 2 ) (peroxidase reaction). PGH 2 is, then, further metabolized to various kinds of prostaglandins, prostacyclins, and thromboxanes by the appropriate synthase (2). Two isoforms of PGHS have been discovered thus far, PGHS-1 and PGHS-2, both of which are composed of ϳ600 amino acid residues, sharing more than 60% sequence identity (4, 5), and their crystal structures are essentially superimposable (6 -8).Although both the PGHS isozymes function as a homodimer of ϳ70-kDa subunits and have similar catalytic properties, they have distinctly different biological functions. PGHS-1 is a "housekeeping" enzyme, expressed constitutively in most tissues, and produces prostaglandins to regulate cellular responses to hormonal stimulation and to regulate vascular homeostasis. PGHS-2, in contrast, is inducibly expressed i...