Kinetic and spectroscopic studies of hemin acquisition in the hemophore hasap from pseudomonas aeruginosa

Erik T. Yukl, Grace Jepkorir, Aileen Y. Alontaga, Lawrence Pautsch, Juan C. Rodriguez, Mario Rivera, Pierre Moënne-Loccoz

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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 matches 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 electron paramagnetic resonance and resonance Raman spectroscopy of rapid freeze quench samples provides support for a model in which 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.

Original languageEnglish (US)
Pages (from-to)6646-6654
Number of pages9
JournalBiochemistry
Volume49
Issue number31
DOIs
StatePublished - Aug 10 2010

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ASJC Scopus subject areas

  • Biochemistry

Cite this

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. https://doi.org/10.1021/bi100692f