Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ

Yukari Sekine, Takehito Tanzawa, Yoshikazu Tanaka, Koichiro Ishimori, Takeshi Uchida

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)884-893
Number of pages10
JournalBiochemistry
Volume55
Issue number6
DOIs
StatePublished - Feb 16 2016
Externally publishedYes

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Hemeproteins
Vibrio cholerae
Heme
Carrier Proteins
Enzymes
Proteins
Escherichia coli O157
Surface Plasmon Resonance
Operon
heme-binding protein
Surface plasmon resonance
Escherichia coli
Ligands
Stoichiometry
Spectrum analysis
Raman scattering
Crystal structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ. / Sekine, Yukari; Tanzawa, Takehito; Tanaka, Yoshikazu; Ishimori, Koichiro; Uchida, Takeshi.

In: Biochemistry, Vol. 55, No. 6, 16.02.2016, p. 884-893.

Research output: Contribution to journalArticle

Sekine, Yukari ; Tanzawa, Takehito ; Tanaka, Yoshikazu ; Ishimori, Koichiro ; Uchida, Takeshi. / Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ. In: Biochemistry. 2016 ; Vol. 55, No. 6. pp. 884-893.
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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.",
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