Functional Differentiation and Regulatory Mechanisms of Ferrochelatases HemH1 and HemH2 in Bacillus thuringiensis Under Iron and Oxidative Stress

Ferrochelatase is the terminal enzyme in heme biosynthesis. <i>Bacillus thuringiensis</i> (Bt) 97-27 contains two ferrochelatases, HemH1 and HemH2, but their regulatory mechanisms and functional differences under virous environmental stimuli remain unclear. This study confirmed that the iron uptake regulator protein (Fur) bound to the promoters of <i>hemH1</i> and <i>hemH2</i>, with Fe²⁺ or Fe³⁺ enhancing this binding. Heterologous expression of HemH1 and HemH2 in <i>Escherichia coli</i> showed that pEH2/BL grew better than pEH1/BL under different 2,2'-Bipyridyl, Fe²⁺, and Fe³⁺ concentrations. Under iron limitation, the heme precursor ALA production decreased significantly in both strains. The heme production of pEH2/BL decreased sharply under iron-limited conditions, while that of pEH1/BL decreased significantly under iron-rich conditions. The H₂O₂ sensitivity experiment revealed that <i>E. coli</i> pEH1/BL was more tolerant to H₂O₂ than pEH2/BL. In Bt, Δ<i>hemH2</i> was most sensitive to H₂O₂ stress, but complementation of <i>hemH1</i> or <i>hemH2</i> partially restored H₂O₂ resistance, with the overexpressed strain pHH2/Bt being most tolerant. β-galactosidase assays indicated that Fur positively regulated <i>hemH1</i> and negatively regulated <i>hemH2</i> under normal conditions, but this regulation reversed with 2.5 mM Fe³⁺. qRT-PCR showed upregulation of genes related to heme synthesis, oxidative stress, and ferrous iron transport. This study reveals the functional differentiation of HemH1 and HemH2 under the joint regulation of Fur and environmental factors, highlighting their synergistic roles in heme synthesis, heavy metal detoxification, and oxidative stress resistance to maintain bacterial physiological homeostasis.