Heme d formation in a Shewanella benthica hemoglobin

In our continued investigations of microbial globins, we solved the structure of a truncated hemoglobin from Shewanella benthica, an obligate psychropiezophilic bacterium. The distal side of the heme active site is lined mostly with hydrophobic residues, with the exception of a tyrosine, Tyr34 (CD1) and a histidine, His24 (B13). We found that purified SbHbN, when crystallized in the ferric form with polyethylene glycol as precipitant, turned into a green color over weeks. The electron density obtained from the green crystals accommodated a trans heme d, a chlorin-type derivative featuring a γ-spirolactone and a vicinal hydroxyl group on a pyrroline ring. In solution, exposure of the protein to one equivalent of hydrogen peroxide resulted in a similar green color change, but caused by the formation of multiple products. These were oxidation species released on protein denaturation, likely including heme d, and a species with heme covalently attached to the polypeptide. The Tyr34Phe replacement prevented the formation of both heme d and the covalent linkage. The ready modification of heme b by SbHbN expands the range of chemistries supported by the globin fold and offers a route to a novel heme cofactor. The ferric group 1 truncated hemoglobin from the extremophile Shewanella benthica KT99 reacts with peroxides. In solution, several products are obtained including a heme–protein covalent adduct. In the crystal, the product is solely trans heme d. The results add to the repertory of hemoglobin chemistry. [Display omitted] •The group 1 truncated hemoglobin of Shewanella benthica reacts with peroxides.•In the crystal (reagent polyethylene glycol), the product is heme d.•In solution (reagent H2O2), products include a species with heme-protein cross-link.•Tyr34 on the distal side of the heme is implicated in the reactions.•Cyanide bound to the heme iron inhibits the reactions.