title: Non-heme-binding domains and segments of the Staphylococcus aureus IsdB protein critically contribute to the kinetics and equilibrium of heme acquisition from methemoglobin.
abstract: The hemoglobin receptor IsdB rapidly acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. IsdB consists of N-terminal segment (NS), NEAT1 (N1), middle (MD), and heme binding NEAT2 (N2) domains, and C-terminal segment (CS). This study aims to elucidate the roles of these domains or segments in the metHb/IsdB reaction. Deletion of CS does not alter the kinetics and equilibrium of the reaction. Sequential deletions of NS and N1 in NS-N1-MD-N2 progressively reduce heme transfer rates and change the kinetic pattern from one to two phases, but have no effect on the equilibrium of the heme transfer reaction, whereas further deletion of MD reduces the percentage of transferred metHb heme. MD-N2 has higher affinity for heme than N2. MD in trans reduces rates of heme dissociation from holo-N2 and increases the percentage of metHb heme captured by N2 by 4.5 fold. NS-N1-MD and N2, but not NS-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction. NS-N1-MD-NIsdC, a fusion protein of NS-N1-MD and the NEAT domain of IsdC, slowly acquires heme from metHb by itself but together with N2 results in rapid heme loss from metHb. Thus, NS-N1 and MD domains specifically and critically contribute to the kinetics and equilibrium of the metHb/IsdB reaction, respectively. These findings support a mechanism of direct heme acquisition by IsdB in which MD enhances the affinity of N2 for heme to thermodynamically drive heme transfer from metHb to IsdB and in which NS is required for the rapid and single phase kinetics of the metHb/IsdB reaction.
authors: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, United States of America; Department of Physiology, Harbin Medical University, Harbin, People's Republic of China.
authors: Hui
authors: H
authors: Zhu
authors: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, United States of America.
authors: Dengfeng
authors: D
authors: Li
authors: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, United States of America.
authors: Mengyao
authors: M
authors: Liu
authors: Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, United States of America.
authors: Valerie
authors: V
authors: Copié
authors: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, United States of America.
authors: Benfang
authors: B
authors: Lei
mesh_terms: D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008706:Methemoglobin / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; C510010:IsdB protein, Staphylococcus aureus; D011994:Recombinant Proteins; D006418:Heme; D008706:Methemoglobin