title: Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae.
abstract: HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV-visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤5%) in comparison to the wild-type protein (~20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8M GdnHCl at 37°C, the half-life was 5h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30302-3965, United States.
authors.forename: Rizvan C
authors.initials: RC
authors.lastname: Uluisik
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 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, ND 58108-6050, United States.
authors.forename: Gudrun S
authors.initials: GS
authors.lastname: Lukat-Rodgers
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108-6050, United States.
authors.forename: Seth A
authors.initials: SA
authors.lastname: Adrian
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, MD 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, MD 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, ND 58108-6050, United States. Electronic address: Kent.Rodgers@ndsu.edu.
authors.forename: Kenton R
authors.initials: KR
authors.lastname: Rodgers
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30302-3965, United States. Electronic address: ddixon@gsu.edu.
authors.forename: Dabney W
authors.initials: DW
authors.lastname: Dixon
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003353:Corynebacterium diphtheriae / Q000737:chemistry* / Q000235:genetics; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D000072417:Protein Domains; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins; D006418:Heme
keywords: Corynebacterium; Guanidinium; Heme; Hemin; Hemoglobin; Raman