The structural and functional analysis of BhuR: The outer membrane hemin receptor of Bordetella bronchiseptica
Mocny, Jeffrey C.
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Hemin uptake by the Bordetellae is mediated by BhuR, a dual function binding and signaling outer membrane receptor which is encoded by the hemin utilization ( hurIR-bhuRSTUV ) locus of B. bronchiseptica, B. pertussis, and B. parapertussis. It was speculated that BhuR mediated hemin uptake occurs through one of the following mechanisms: (1) BhuR binds hemoproteins to remove heme through a destructive process, (2) the cell produces proteases to degrade hemoproteins, freeing heme for binding by BhuR, (3) the cell produces hemophores, which are peptides capable of removing heme from hemoproteins which BhuR then binds or, (4) BhuR binds and internalizes hemoproteins and degrades them within the cell to liberate heme. The precise mechanism of hemin uptake has not been established. To that end, in vitro growth experiments using a panel of hemoproteins, wild type (wt) B. bronchiseptica , and a B. bronchiseptica mutant in which BhuR was functionally inactivated established that certain hemoproteins prone to spontaneous heme release supported growth of the bacterium. To establish that BhuR recognizes hemin that is released from hemoproteins, B. bronchiseptica was grown in Fe depleted culture medium supplemented with hemoproteins sequestered within dialysis tubing. These experiments demonstrated that direct contact between bacteria and hemoprotein was not required for hemin acquisition. These observations also revealed that hemophores were not required for hemin acquisition. To extend and confirm these findings, bacterial growth was monitored by growing bacteria in Fe depleted broth supplemented with mutant sperm whale myoglobins, each of which exhibited different affinities for heme. These observations demonstrated that growth rates of the bacterium correlated with the rate of heme loss from sperm whale myoglobin. To establish that BhuR is a hemin receptor, plasmid-encoded bhuR was expressed in Escherichia coli and the cells assayed for the capacity to absorb hemin from solution. In an effort to determine which BhuR amino acids bind hemin, molecular modeling was performed to delineate possible targets for site-specific, oligonucleotide-directed mutagenesis to substitute histidine or putatively surface exposed tyrosine with alanine. Single-point substitution mutant BhuR polypeptides were measured for ability to absorb hemin from solution. Results from these experiments suggested a novel heme binding motif in BhuR in which histidine and several putatively surface-exposed tyrosines were not required for hemin capture. Collectively, these experiments indicated a novel hemin acquisition mechanism which many pathogenic microorganisms may employ to acquire Fe from heme.