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Hemoglobin binding and catalytic heme extraction by IsdB NEAT domains Bowden, Catherine F. M.; Verstraete, Meghan M.; Eltis, Lindsay; Murphy, Michael E. P.
Abstract
The Isd (Iron-regulated surface determinant) system is a multi-protein transporter that enables bacterium Staphylococcus aureus to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall-anchored surface protein that contains 2 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 which included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (KD = 0.42 ± 0.05 µM) and to extract heme from Hb. Moreover, IsdB only extracted heme 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 heme transfer from metHb to IsdA with a Km for metHb of 0.75 ± 0.07 µN and a kcat of 0.22 ± 0.01 s-¹. The latter is consistent with the transfer of heme from metHb to IsdB as 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 carried out, representing all known Hb-heme uptake functions of the full-length IsdB protein.
Item Metadata
Title |
Hemoglobin binding and catalytic heme extraction by IsdB NEAT domains
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Creator | |
Contributor | |
Publisher |
Biochemistry
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Date Issued |
2014-03-20
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Description |
The Isd (Iron-regulated surface determinant) system is a multi-protein transporter that enables
bacterium Staphylococcus aureus to take up iron from hemoglobin (Hb) during human infection.
In this system, IsdB is a cell wall-anchored surface protein that contains 2 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 which included at least one
NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity
(KD = 0.42 ± 0.05 µM) and to extract heme from Hb. Moreover, IsdB only extracted heme 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 heme transfer from metHb to IsdA with a
Km for metHb of 0.75 ± 0.07 µN and a kcat of 0.22 ± 0.01 s-¹. The latter is consistent with the
transfer of heme from metHb to IsdB as 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 carried out, representing all known Hb-heme uptake functions
of the full-length IsdB protein.
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Subject | |
Genre | |
Type | |
Language |
eng
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Date Available |
2021-04-07
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Provider |
Vancouver : University of British Columbia Library
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Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
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DOI |
10.14288/1.0396561
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URI | |
Affiliation | |
Citation |
Bowden, C.M.F., Verstraete, M.M., Eltis, L.D. & Murphy, M.E.P. (2014) The IsdB NEAT domain 1 of Hemoglobin binding and catalytic heme extraction by IsdB NEAT domains. Biochemistry. 53, 2286-94.
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Publisher DOI |
10.1021/bi500230f
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Peer Review Status |
Reviewed
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Scholarly Level |
Faculty; Graduate
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Copyright Holder |
American Chemical Society
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Rights URI | |
Aggregated Source Repository |
DSpace
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Item Citations and Data
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International