RESUMO
The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.
Assuntos
Proteínas de Bactérias/metabolismo , Domínio Catalítico , Heme/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Catalase/metabolismo , Heme/química , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Modelos Moleculares , Mutação , Oxirredução , Ligação Proteica , Conformação Proteica , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxido Dismutase/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Familial lecithin: cholesterol acyltransferase (LCAT) deficiency is an autosomal recessive disorder characterized by corneal opacity, hemolytic anemia, proteinuria, and a low serum level of high-density lipoprotein cholesterol (HDL-C). Also, LCAT activity is remarkably decreased or absent. A 57-year-old Japanese man presented with corneal opacity, proteinuria, and a very low serum level of HDL-C. His LCAT activity was too low to measure. From clinical observations and results of examinations, we suspected LCAT deficiency. We performed a kidney biopsy and gene analysis. Light microscopy revealed the vacuolation of glomerular capillary tufts. Electron microscopy revealed small deposits in the glomerular basement membrane (GBM), extracellular matrix, and vascular endothelial cells. We identified a homozygous C to T point mutation at nucleotide 501 (g.501 C>T) of exon 4 at codon 140, resulting in an arginine (Arg) to cysteine (Cys) amino acid substitution (A140C) in the patient. These findings were characteristic of LCAT deficiency, which was confirmed to be due to a mutation that has only been reported in Japan.