ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P156. Heme Coordination in Porphyromonas gingivalis HmuY Protein
H. Połata , M. Olczak , T. Olczak
Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137, Wrocław, Poland
e-mail: hpolata@tlen.pl
Periodontitis is an infectious disease to which genetic, microbial, immunological, and environmental factors
combine to influence disease risk and progression, resulting in the destruction of tooth-supporting tissues.
Porphyromonas gingivalis, Gram-negative, anaerobic bacterium implicated in the development and progression
of chronic periodontitis, requires iron and heme for growth. One of the mechanisms of heme uptake in this
bacterium comprises the outer-membrane heme transporter HmuR and a putative heme-binding lipoprotein
HmuY. We propose that heme derived from host sources binds to HmuY and is then delivered to HmuR, which
further transports the heme molecule into the bacterial cell. The aim of this study was to characterize the nature
of heme binding to HmuY. The protein was expressed, purified and detailed spectroscopic investigations (UV-
Vis absorption spectroscopy, spectrofluorimetry, CD, and MCD) were performed. The spectrophotometric
titrations showed a 1:1 molar ratio of heme binding to HmuY with a Kd of ~3 uM. Ferric heme bound to HmuY
may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. The heme complexed to HmuY
is in a low-spin Fe(III) hexa-coordinate environment and the heme binding does not change the protein's
structure. Using site-directed mutagenesis, several single and double HmuY mutants were constructed and the
ability of the mutated proteins to bind heme was analyzed. This analysis identified histidines 134 and 166 as
potential heme ligands. It is also likely that a reversible coordination by a histidine chain may occur, allowing
heme transfer from hemoglobin to HmuY and subsequently to HmuR.
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