title: Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ.
abstract: HutZ is a cytoplasmic heme-binding protein from Vibrio cholerae. Although we have previously identified HutZ as a heme-degrading enzyme [Uchida, T., et al. (2012) Chem. Commun. 48, 6741-6743], the heme transport protein for HutZ remained unknown. To identify the heme transport protein for HutZ, we focused on the heme utilization operon, hutWXZ. To this end, we constructed an expression system for HutX in Escherichia coli and purified it to homogeneity. An absorption spectral analysis demonstrated that HutX binds heme with a 1:1 stoichiometry and a dissociation constant of 7.4 nM. The crystal structure of HutX displays a fold similar to that of the homologous protein, ChuX, from E. coli O157:H7. A structural comparison of HutX and ChuX, and resonance Raman spectra of heme-HutX, suggest that the axial ligand of the ferric heme is Tyr90. The heme bound to HutX is transferred to HutZ with biphasic dissociation kinetics of 8.3 × 10(-2) and 1.5 × 10(-2) s(-1), values distinctly larger than those for transfer from HutX to apomyoglobin. Surface plasmon resonance experiments confirmed that HutX interacts with HutZ with a dissociation constant of ∼400 μM. These results suggest that heme is transferred from HutX to HutZ via a specific protein-protein interaction. Therefore, we can conclude that HutX is a cytoplasmic heme transport protein for HutZ.
authors.affiliation: Graduate School of Chemical Sciences and Engineering, Hokkaido University , Sapporo 060-0810, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Sekine
authors.affiliation: Graduate School of Life Science, Hokkaido University , Sapporo 060-0810, Japan.
authors.forename: Takehito
authors.initials: T
authors.lastname: Tanzawa
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University , Sapporo 060-0810, Japan.|PRESTO, Japan Science and Technology Agency , Sapporo 060-0810, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Graduate School of Chemical Sciences and Engineering, Hokkaido University , Sapporo 060-0810, Japan.|Department of Chemistry, Faculty of Science, Hokkaido University , Sapporo 060-0810, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ishimori
authors.affiliation: Graduate School of Chemical Sciences and Engineering, Hokkaido University , Sapporo 060-0810, Japan.|Department of Chemistry, Faculty of Science, Hokkaido University , Sapporo 060-0810, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D006418:Heme