Identification of two heme-binding sites in the cytoplasmic heme-trafficking protein PhuS from Pseudomonas aeruginosa and their relevance to function.
PhuS is a cytoplasmic, 39 kDa heme-binding protein from Pseudomonas aeruginosa. It has previously been shown to transfer heme to its cognate heme oxygenase. It is expressed from the phu operon, which encodes a group of proteins known to actively internalize and transport heme from host organisms. This study combines the spectral resolution of resonance Raman spectroscopy with site-directed mutagenesis to identify and characterize the heme-bound states of holo-PhuS. This combined approach has identified a site in monomeric PhuS having alternate His ligands at positions 209 and 212. A second distinct binding site is present in dimeric PhuS. This site supports six-coordinate, low-spin heme, even when both His209 and His212 are mutated to Ala. The presence of conserved His and Tyr residues in all of the homologs characterized to date suggest that the dimer could be of the domain-swapped type in which two protein molecules are cross-linked by bound heme. The multiple heme-bound states and their sensitivity to pH suggest the possibility that these cytoplasmic heme-binding proteins have multiple functions that are toggled by variations in intracellular conditions.
Block
Darci R
DR
Department of Chemistry, Biochemistry and Molecular Biology, North Dakota State University, 1231 Albrecht Avenue, Fargo, North Dakota 58105, USA.
Lukat-Rodgers
Gudrun S
GS
Rodgers
Kenton R
KR
Wilks
Angela
A
Bhakta
Mehul N
MN
Lansky
Ila B
IB
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D014443:Tyrosine; D006418:Heme; D006639:Histidine