title: Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.
abstract: The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.
authors.affiliation: Department of Chemistry, Georgia State University , Atlanta, Georgia 30302-3965, United States.
authors.forename: Elizabeth B
authors.initials: EB
authors.lastname: Draganova
authors.affiliation: Department of Chemistry, Georgia State University , Atlanta, Georgia 30302-3965, United States.
authors.forename: Neval
authors.initials: N
authors.lastname: Akbas
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University , Fargo, North Dakota 58108-6050, United States.
authors.forename: Seth A
authors.initials: SA
authors.lastname: Adrian
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University , Fargo, North Dakota 58108-6050, United States.
authors.forename: Gudrun S
authors.initials: GS
authors.lastname: Lukat-Rodgers
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina , Columbia, South Carolina 29208, United States.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Collins
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina , Columbia, South Carolina 29208, United States.
authors.forename: John H
authors.initials: JH
authors.lastname: Dawson
authors.affiliation: Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation, and Research, Food and Drug Administration , Silver Spring, Maryland 20993, United States.
authors.forename: Courtni E
authors.initials: CE
authors.lastname: Allen
authors.affiliation: Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation, and Research, Food and Drug Administration , Silver Spring, Maryland 20993, United States.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Schmitt
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University , Fargo, North Dakota 58108-6050, United States.
authors.forename: Kenton R
authors.initials: KR
authors.lastname: Rodgers
authors.affiliation: Department of Chemistry, Georgia State University , Atlanta, Georgia 30302-3965, United States.
authors.forename: Dabney W
authors.initials: DW
authors.lastname: Dixon
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D003353:Corynebacterium diphtheriae / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D008024:Ligands; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C408133:HmuT protein, Corynebacterium diphtheriae; D008024:Ligands; D008074:Lipoproteins; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D006639:Histidine