HutZ from <i>Aliivibrio fischeri</i> Inhibits HutW-Mediated Anaerobilin Formation by Sequestering Heme

Anaerobilin synthase catalyzes the decyclization of the heme protoporphyrin ring, an O₂-independent reaction that liberates iron and produces the linear tetrapyrrole, anaerobilin. The marine bacterium <i>Aliivibrio fischeri</i>, the enteric pathogen <i><i>Escherichia coli</i></i> O157:H7, and the opportunistic oral pathogen <i>Fusobacterium nucleatum</i> encode anaerobilin synthase as part of their heme uptake/utilization operons, designated <i>chu</i> (<i>E. coli</i> O157:H7), <i>hmu</i> (<i>F. nucleatum</i>), and <i>hut</i> (<i>A. fischeri</i>). <i>F. nucleatum</i> and <i>E. coli</i> O157:H7 contain accessory proteins (ChuS, ChuY, and HmuF) encoded in their respective operons that mitigate against the cytotoxicity of labile heme and anaerobilin by functioning in heme trafficking and anaerobilin reduction. However, the <i>hut</i> operon of <i>A. fischeri</i> and other members of the <i>Vibrionaceae</i> family including the enteric pathogen <i>Vibrio cholerae</i> do not contain homologues to these accessory proteins, raising questions as to how members of this family mitigate against anaerobilin and heme toxicity. Herein, we show that HutW (anaerobilin synthase) from <i>A. fischeri</i> produces anaerobilin, but that HutX and HutZ, encoded downstream of HutW, do not catalyze anaerobilin reduction in the presence of excess NAD(P)H, FAD, and FMN. However, we show that HutZ prevents labile heme and anaerobilin cytotoxicity by binding tightly to heme, sequestering it from HutW, and preventing anaerobilin formation. Thus, <i>A. fischeri</i> is seemingly unable to extract iron from heme using the <i>hutWXZ</i> gene products. Our results further suggest that the structurally distinct <i>chu, hmu,</i> and <i>hut</i> operons have functionally converged to protect the cell from anaerobilin accumulation and heme cytotoxicity.