Hemoglobin Binding and Catalytic Heme Extraction by IsdB Near Iron Transporter
Domains
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Abstract
The
Isd (iron-regulated surface determinant) system is a multiprotein
transporter that allows bacterium <i>Staphylococcus aureus</i> to take up iron from hemoglobin (Hb) during human infection. In
this system, IsdB is a cell wall-anchored surface protein that contains
two near iron transporter (NEAT) domains, one of which binds heme.
IsdB rapidly extracts heme from Hb and transfers it to IsdA for relay
into the bacterial cell. Using a series of recombinant IsdB constructs
that included at least one NEAT domain, we demonstrated that both
domains are required to bind Hb with high affinity (<i>K</i><sub>D</sub> = 0.42 ± 0.05 μM) and to extract heme from
Hb. Moreover, IsdB extracted heme only from oxidized metHb, although
it also bound oxyHb and the Hb–CO complex. In a reconstituted
model of the biological heme relay pathway, IsdB catalyzed the transfer
of heme from metHb to IsdA with a <i>K</i><sub>m</sub> for
metHb of 0.75 ± 0.07 μN and a <i>k</i><sub>cat</sub> of 0.22 ± 0.01 s<sup>–1</sup>. The latter is consistent
with the transfer of heme from metHb to IsdB being the rate-limiting
step. With both NEAT domains and the linker region present in a single
contiguous polypeptide, high-affinity Hb binding was achieved, rapid
heme uptake was observed, and multiple turnovers of heme extraction
from metHb and transfer to IsdA were conducted, representing all known
Hb–heme uptake functions of the full-length IsdB protein