Insertion of Heme b into the Structure of the Cys34‐Carbamidomethylated Human Lipocalin α₁‐Microglobulin: Formation of a [(Heme)₂(α₁‐Microglobulin)]₃ Complex

Abstract

α(1)-Microglobulin (α(1)m) is a 26 kDa plasma and tissue protein belonging to the lipocalin protein family. Previous investigations indicate that the protein interacts with heme and suggest that it has a function in heme metabolism. However, detailed characterizations of the α(1)m-heme interactions are lacking. Here, we report for the first time the preparation and analysis of a stable α(1)m-heme complex upon carbamidomethylation of the reactive Cys34 by using recombinantly expressed human α(1)m. Analytical size-exclusion chromatography coupled with a diode-array absorbance spectrophotometry demonstrates that at first an α(1)m-heme monomer is formed. Subsequently, a second heme triggers oligomerization that leads to trimerization. The resulting (α(1)m[heme](2))(3) complex was characterized by resonance Raman and EPR spectroscopy, which support the presence of two ferrihemes, thus indicating an unusual spin-state admixed ground state with S=(3)/(2), (5)/(2).

Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

“…A1M is a 26 kDA glycoprotein belonging to the Lipocalin family with immunoregulatory characteristics and is involved in heme catabolism [41]. Bikunin is a Kunitz-type serine protease inhibitor of the inter- α -inhibitor family and a structural component of the extracellular matrix [23, 42].…”

A1M, an extravascular tissue cleaning and housekeeping protein

“…It was shown that the cell cytosol and thiol groups of cytosolic proteins of the erythroid cell line K562 were reduced by A1M [39]. Furthermore, A1M reduced the number of carbonyl groups on oxidized collagen in a reaction which was entirely dependent on the presence of Cys34, [43]. A possible heme-binding site consisting of a Cys-Pro-dipeptide motif similar to those seen in the cytochrome P450 family is located at loop 1 (Cys34-Pro35) [8] (Figure 2).…”

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

“…The g Ќ value of 5.75 was too low for a pure high spin state. Based on the pH-independent g Ќ value of 5.75, the spin state is best described as a quantum spin admixture having 12% of an S ϭ 3/2 iron spin state with unresolved g 1 and g 2 (37,38).…”