Heme binding to cold shock protein D, CspD, from Vibrio cholerae.

Cold shock protein D (CspD) is one of the homologous proteins of cold shock protein A (CspA), inhibiting DNA replication by binding to single-stranded DNA. We found that CspD from Vibrio cholerae (VcCspD) possesses one heme regulatory motif (HRM) sequence and specifically binds heme with a stoichiometry of 1:1. The binding of a synthetic single-stranded DNA oligomer (ssDNA) was followed by fluorescence quenching of Trp. The fluorescence quenching associated with the addition of ssDNA was suppressed in the presence of heme, indicating that heme binding to VcCspD inhibited the formation of the VcCspD-ssDNA complex. Such heme-induced inhibition was not observed for the VcCspD mutant with replacement of Cys22 in the HRM with alanine (C22A). Heme binding at Cys22 is, therefore, essential for the inhibition of ssDNA binding for VcCspD. The growth of Escherichia coli at 37 °C was slowed when VcCspD was overexpressed, indicating that VcCspD hampers the growth of E. coli. When the production of heme in cells was promoted by the addition of a heme precursor, δ-aminolevulinic acid, the growth of E. coli expressing VcCspD was decelerated, but the growth of E. coli expressing the C22A mutant was not decelerated. These observations allow us to conclude that heme specifically binds to the HRM region in VcCspD and inhibits the binding of target ssDNA, which suggests that heme functions as a regulatory molecule for DNA replication.

Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr).

The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr-ICE complex. The addition of EDTA to the Irr-ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr-ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29-mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29.