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

Anaerobilin synthase catalyzes the decyclization of the\nheme protoporphyrin\nring, an O<sub>2</sub>-independent reaction that liberates iron and\nproduces the linear tetrapyrrole, anaerobilin. The marine bacterium Aliivibrio fischeri, the enteric pathogen <i>Escherichia coli</i> O157:H7,\nand the opportunistic oral pathogen Fusobacterium nucleatum encode anaerobilin synthase as part of their heme uptake/utilization\noperons, designated <i>chu</i> (E. coli O157:H7), <i>hmu</i> (F. nucleatum), and <i>hut</i> (A. fischeri). F. nucleatum and E. coli O157:H7 contain accessory proteins (ChuS,\nChuY, and HmuF) encoded in their respective operons that mitigate\nagainst the cytotoxicity of labile heme and anaerobilin by functioning\nin heme trafficking and anaerobilin reduction. However, the <i>hut</i> operon of A. fischeri and other members of the <i>Vibrionaceae</i> family including\nthe enteric pathogen Vibrio cholerae do not contain homologues to these accessory proteins, raising questions\nas to how members of this family mitigate against anaerobilin and\nheme toxicity. Herein, we show that HutW (anaerobilin synthase) from A. fischeri produces anaerobilin, but that HutX and\nHutZ, encoded downstream of HutW, do not catalyze anaerobilin reduction\nin the presence of excess NAD(P)H, FAD, and FMN. However, we show\nthat HutZ prevents labile heme and anaerobilin cytotoxicity by binding\ntightly to heme, sequestering it from HutW, and preventing anaerobilin\nformation. Thus, A. fischeri is seemingly\nunable to extract iron from heme using the <i>hutWXZ</i> gene products. Our results further suggest that the structurally\ndistinct <i>chu, hmu,</i> and <i>hut</i> operons\nhave functionally converged to protect the cell from anaerobilin accumulation\nand heme cytotoxicity.