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    Kinetic and spectroscopic studies of hemin acquisition in the hemophore HasAp from Pseudomonas aeruginosa

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    Yukl_ACS_2010.pdf (1.045Mb)
    Issue Date
    2010-08-10
    Author
    Yukl, Erik T.
    Jepkorir, Grace
    Alontaga, Aileen Yung
    Pautsch, Lawrence
    Rodriguez, Juan C.
    Rivera, Mario
    Moënne-Loccoz, Pierre
    Publisher
    ACS
    Type
    Article
    Article Version
    Scholarly/refereed, author accepted manuscript
    Rights
    Copyright © 2010 American Chemical Society
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    Abstract
    The extreme limitation of free iron has driven various pathogens to acquire iron from the host in the form of heme. Specifically, several Gram negative pathogens secrete a heme binding protein known as HasA to scavenge heme from the extracellular environment and to transfer it to the receptor protein HasR for import into the bacterial cell. Structures of heme-bound and apo-HasA homologues show that the heme iron(III) ligands, His32 and Tyr75, reside on loops extending from the core of the protein and that a significant conformational change must occur at the His32 loop upon heme binding. Here, we investigate the kinetics of heme acquisition by HasA from Pseudomonas aeruginosa (HasAp). The rate of heme acquisition from human met-hemoglobin (met-Hb) closely matched that of heme dissociation which suggests a passive mode of heme uptake from this source. The binding of free hemin is characterized by an initial rapid phase forming an intermediate before further conversion to the final complex. Analysis of this same reaction using an H32A variant lacking the His heme ligand shows only the rapid phase to form a heme-protein complex spectroscopically equivalent to that of the wild type intermediate. Further characterization of these reactions using EPR and resonance Raman spectroscopy of rapid freeze quench samples provided support for a model where heme is initially bound by the Tyr75 to form a high-spin heme-protein complex before slower coordination of the His32 ligand upon closing of the His loop over the heme. The slow rate of this loop closure implies that the induced-fit mechanism of heme uptake in HasAp is not based on a rapid sampling of the H32 loop between open and closed configurations, but rather, that the H32 loop motions are triggered by the formation of the high-spin heme-HasAp intermediate complex.
    URI
    http://hdl.handle.net/1808/23693
    Collections
    • Chemistry Scholarly Works [557]
    Citation
    Yukl, E. T., Jepkorir, G., Alontaga, A. Y., Pautsch, L., Rodriguez, J. C., Rivera, M., & Moënne-Loccoz, P. (2010). Kinetic and spectroscopic studies of hemin acquisition in the hemophore HasAp from Pseudomonas aeruginosa. Biochemistry, 49(31), 6646–6654. http://doi.org/10.1021/bi100692f

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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