title: Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization.
abstract: The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV-visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on π donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT-CO.
authors: Department of Chemistry, Georgia State University, Atlanta, GA, 30302-3965, USA.
authors: Elizabeth B
authors: EB
authors: Draganova
authors: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108-6050, USA.
authors: Seth A
authors: SA
authors: Adrian
authors: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108-6050, USA.
authors: Gudrun S
authors: GS
authors: Lukat-Rodgers
authors: Department of Chemistry, Georgia State University, Atlanta, GA, 30302-3965, USA.
authors: Cyrianne S
authors: CS
authors: Keutcha
authors: 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, USA.
authors: Michael P
authors: MP
authors: Schmitt
authors: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108-6050, USA.
authors: Kenton R
authors: KR
authors: Rodgers
authors: Department of Chemistry, Georgia State University, Atlanta, GA, 30302-3965, USA. ddixon@gsu.edu.
authors: Dabney W
authors: DW
authors: Dixon
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D006418:Heme; D006860:Hydrogen Bonding; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; C408133:HmuT protein, Corynebacterium diphtheriae; D008074:Lipoproteins; D006418:Heme
keywords: Electrospray; Heme binding; Heme uptake; Resonance Raman; Thermal unfolding