AnkB, an ankyrin repeat protein form Vibrio cholerae, functions as a heme chaperone for catalase maturation

AnkB is an ankyrin repeat-containing protein from Vibrio cholerae. In this study, we examined its role in facilitating heme incorporation into catalase, an enzyme that converts hydrogen peroxide into molecular oxygen and water. To obtain soluble protein, we expressed and purified a truncated construct, ΔAnkB, lacking the predicted transmembrane region. Spectroscopic heme titration experiments revealed that ΔAnkB binds heme with an apparent dissociation constant (K_d) of 0.17 μM, which is comparable to that of catalase (KatB, 0.38 μM). Although mutagenesis studies did not identify a definitive heme ligand residue, absorption spectra of heme-reconstituted mutants indicate that Tyr95 and Tyr87 contribute to heme coordination. Blue Native-PAGE and size-exclusion chromatography did not detect a stable ΔAnkB-KatB complex; however, kinetic analyses demonstrated efficient heme transfer from ΔAnkB to apo-KatB, displaying biphasic kinetics with a fast phase (k ≈ 1.35 s^-1) and a slow phase (k ≈ 0.013 s^-1). Notably, the fast phase was absent when apo-myoglobin was used as the acceptor, a representative hemoprotein, suggesting that ΔAnkB interacts transiently and specifically with KatB to facilitate direct heme transfer rather than simple dissociation into bulk solvent. The genomic proximity of ankB to katB further supports its physiological involvement in KatB maturation. These findings suggest that AnkB contributes to the antioxidant defense in V. cholerae by facilitating heme incorporation during KatB maturation.