Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in Bacillus subtilis

Abstract

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.

Metal homeostasis and resistance in bacteria

“…One active area of research is to define the mechanisms of iron allocation to satisfy these needs, which likely involves accessory proteins such as frataxin. 85 …”

The Listeria monocytogenes Fur‐regulated virulence protein FrvA is an Fe(II) efflux P1B4‐type ATPase

“…Precipitate was removed by centrifugation and the supernatant was analyzed by fluorescence spectroscopy. The fluorescence emission of PPIX was scanned from 500 to 680 nm with excitation at 410 nm as described (Mielcarek et al, 2015). Fluorescence intensity at 596 nm (peak) was normalized and plotted.…”

SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY

“…Although in vitro results, specially when protein-protein interactions and protein oligomerization are concerned, are not always easy to verify directly in living cells, it still should be possible to link the findings to processes observed in vivo . As noted in the introduction, one of the primary functions of CyaY is related to its involvement in the ISC assembly machinery [ 19 , 61 ], but also in heme synthesis [ 20 ], and presumably in the regulation of ISC assembly [ 21 ]. In addition, heme biding appears to induce oligomerization of the protein, although the type of the oligomers is not known.…”

“…coli frataxin CyaY has been obtained [ 19 ]. CyaY from Bacilus subtilis has also been shown to interact with ferrochelatase, the terminal enzyme of heme synthesis that catalyzes iron insertion into protoporphyrin IX [ 20 ]. In addition, it was shown that Vibrio cholerae CyaY binds heme with dissociation constant in the nano-molar range [ 21 ], and that heme biding induced oligomerization of the protein.…”