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

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P204. NMR Study of Heme Binding to HmuY Protein
from Porphyromonas gingivalis
J. Wojaczyński, a H. Połata, b T. Olczak, b and L. Latos-Grażyński a
a
Department of Chemistry, University of Wrocław, 14 F. Joliot-Curie St., 50 383 Wrocław, Poland
e-mail: jw@wchuwr.pl
b
Faculty of Biotechnology, University of Wrocław, Tamka 2, 50 138 Wrocław, Poland
Porphyromonas gingivalis, a 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.
The aim of this study was to characterize the nature of heme binding to HmuY using 1 H NMR spectroscopy. The
protein was expressed, purified and detailed magnetic resonance investigations were performed. We found that
the heme complexed to HmuY is in a low-spin Fe(III) hexa-coordinate environment. The nature of coordinating
ligands and the possibility of additional heme binding by HmuY was thoroughly explored.
heme methyl signals
30 20 10 0 -10
δ [ppm]
D 2 O, 323 K
Using site-directed mutagenesis, several single and double HmuY mutants were constructed with the methionine,
histidine, cysteine, and tyrosine residues replaced by an alanine residue. The ability of the mutated proteins to
bind heme was reflected by their 1 H NMR spectra which gave a closer insight into the heme-protein interactions.
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