Direct iron-protein coordination is dispensable for heme trafficking and the tetrapyrrole reductase activity of HmuF from Fusobacterium nucleatum.

HmuF from Fusobacterium nucleatum is a new member of the flavodoxin superfamily that traffics heme to anaerobilin synthase for decyclization of the porphyrin ring. HmuF then catalyzes the reduction of the linearized tetrapyrrole, termed anaerobilin. Sequence alignment of HmuF homologs of other gram-negative anaerobes reveals that the iron-coordinating histidine residue (H134) is not conserved in HmuF homologs of Leptotrichia and Campylobacter. Given this lack of sequence conservation, we created H134F, H134Y, H134A, H134C and H134M variants of HmuF to explore the importance of H134 on HmuF function. All variants retained the FMN cofactor and were purified with sub-stoichiometric amounts of heme bound to the protein. The electronic absorption spectra of the Fe3+-heme complexes suggest that the ferric heme is high-spin, and among the variants, only H134Y directly coordinates with the iron. Difference spectroscopy showed that each variant binds one equivalent of heme, and competition experiments with apomyoglobin reveal the heme dissociation rate constants (koff) for HmuF H134Y and H134C are comparable to wild-type HmuF, while HmuF H134A, HmuF H134M, and HmuF H134F exhibit 8-, 15- and 200-fold faster koff values, respectively. All variants can traffic heme to anaerobilin synthase for radical-mediated decyclization of the heme and catalyze the subsequent reduction of the linearized tetrapyrrole. Combined, these studies demonstrate that van der Waals interactions between the heme macrocycle and the protein, as well as the FMN cofactor, regulate heme binding affinity. Direct protein-iron coordination is also not essential for heme trafficking and anaerobilin reductase activities of HmuF.