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

• Published in: Biochemistry (Easton) 2016-02, Vol.55 (6), p.884-893
• Main Authors: Sekine, Yukari, Tanzawa, Takehito, Tanaka, Yoshikazu, Ishimori, Koichiro, Uchida, Takeshi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cytoplasm - metabolism; Heme - chemistry; Heme - genetics; Heme - metabolism; Hemeproteins - chemistry; Hemeproteins - genetics; Hemeproteins - metabolism; Intracellular Fluid - metabolism; Molecular Sequence Data; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Transport - physiology; Vibrio cholerae - genetics; Vibrio cholerae - metabolism; X-Ray Diffraction
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.5b01273

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.