Insertion of Heme b into the Structure of the Cys34‐Carbamidomethylated Human Lipocalin α 1 ‐Microglobulin: Formation of a [(Heme) 2 (α 1 ‐Microglobulin)] 3 Complex

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 .