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.
Highlights
Iron is essential for growth and survival of most bacteria
We have found that: 1) the domain between N1 and N2 enhances the affinity of N2 for heme by slowing down heme dissociation from N2 and favorably driving the metHb/IsdB heme transfer reaction; 2) IsdB N-terminal segment is required for the rapid and single phase kinetics of the metHb/IsdB heme transfer reaction; and 3) the C-terminal segment does not contribute to the kinetics and equilibrium of the metHb/IsdB reaction
The mature IsdB protein is comprised of two NEAT domains (N1 and N2) that segment the protein into five distinct regions or domains: an Nterminal segment (NS); N1; a middle domain (MD); N2; and a C-terminal segment (CS) (Figure 1A) [14]
Summary
Iron is essential for growth and survival of most bacteria. There is little free iron to support bacterial growth in mammalian hosts, which is due to the extremely low solubility of ferric iron in water at physiological pH and the presence of proteins that avidly bind iron. Heme is a preferred iron source for some bacteria associated with mammals, including medically relevant Gram-positive pathogens S. aureus and Streptococcus pyogenes [2,3]. The S. aureus heme uptake system is made of the surface proteins, IsdA, IsdB, and IsdC, IsdH, and the membrane transporter IsdDEF, where IsdE is the lipoprotein component [4,5]. The heme acquisition machinery of S. pyogenes consists of the surface proteins, Shr and Shp, and the membrane transporter HtsABC [6,7,8,9,10]. Rapid heme transfer occurs from one protein to another among methemoglobin (metHb) and the heme acquisition proteins of S. aureus and S. pyogenes [15,16,17,18,19], supporting heme acquisition pathway schemes of metHb R IsdB R IsdA R IsdC R IsdDEF in S. aureus [17] and metHb R Shr R Shp R HtsABC in S. pyogenes [19]
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