HutZ, one of the crucial proteins of the iron uptake system in Vibrio cholerae, was purified, which binds to heme at a stoichiometry of 1 : 1. In the presence of ascorbic acid, the HutZ-bound heme degrades via the same intermediates observed in heme oxygenase, suggesting that HutZ works as a heme degradation enzyme.

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.

Heme degradation reaction of HutZ from Vibrio cholerae is regulated by the subunit–subunit interactions.

HutZ is a heme-degrading enzyme in Vibrio cholerae. It converts heme to biliverdin via verdoheme, suggesting that it follows the same reaction mechanism as that of mammalian heme oxygenase. However, none of the key intermediates have been identified. In this study, we applied steady-state and time-resolved UV-vis absorption and resonance Raman spectroscopy to study the reaction of heme-HutZ complex with H2O2 or ascorbic acid. We characterized three intermediates: oxyferrous heme, meso-hydroxyheme, and verdoheme complexes. Our data suport the view that HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence and structural homology.