Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.
Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.
Sook
Brian R
BR
Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.
Block
Darci R
DR
Sumithran
Suganya
S
Montañez
Griselle E
GE
Rodgers
Kenton R
KR
Dawson
John H
JH
Eichenbaum
Zehava
Z
Dixon
Dabney W
DW
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry; D002942:Circular Dichroism; D004563:Electrochemistry; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D058085:Iron Compounds / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013297:Streptococcus pyogenes / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D058085:Iron Compounds; C523920:SiaA protein, Streptococcus pyogenes