Functional spectrum and specificity of mitochondrial ferredoxins FDX1 and FDX2.

Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of [2Fe-2S] ferredoxins, FDX1 (aka adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity.

Mitochondrial [2Fe-2S] ferredoxins: new functions for old dogs.

Ferredoxins (FDXs) comprise a large family of iron-sulfur proteins that shuttle electrons from NADPH and FDX reductases into diverse biological processes. This review focuses on the structure, function and specificity of mitochondrial [2Fe-2S] FDXs that are related to bacterial FDXs due to their endosymbiotic inheritance. Their classical function in cytochrome P450-dependent steroid transformations was identified around 1960, and is exemplified by mammalian FDX1 (aka adrenodoxin). Thirty years later the essential function in cellular Fe/S protein biogenesis was discovered for the yeast mitochondrial FDX Yah1 that is additionally crucial for the formation of haem a and ubiquinone CoQ6 . In mammals, Fe/S protein biogenesis is exclusively performed by the FDX1 paralog FDX2, despite the high structural similarity of both proteins. Recently, additional and specific roles of human FDX1 in haem a and lipoyl cofactor biosyntheses were described. For lipoyl synthesis, FDX1 transfers electrons to the radical S-adenosyl methionine-dependent lipoyl synthase to kickstart its radical chain reaction. The high target specificity of the two mammalian FDXs is contained within small conserved sequence motifs, that upon swapping change the target selection of these electron donors.

The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d1 biosynthesis.

Heme d1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd1 nitrite reductase of many denitrifying bacteria. In the course of heme d1 biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18-didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d1 molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S-adenosyl-l-methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron-sulfur clusters. While the N-terminal [4Fe-4S] cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18-didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.

The PqqD homologous domain of the radical SAM enzyme ThnB is required for thioether bond formation during thurincin H maturation.

Thurincin H is a 31-residue, ribosomally synthesized bacteriocin originating from the thn operon of Bacillus thuringiensis SF361. It is the only known sactipeptide carrying four thioether bridges between four cysteines and the α-carbons of a serine, an asparagine and two threonine residues. By analysis of the thn operon and use of in vitro studies we now reveal that ThnB is a radical S-adenosylmethionine (SAM) enzyme containing two [4Fe-4S] clusters. Furthermore, we confirm the involvement of ThnB in the formation of the thioether bonds present within the structure of thurincin H. Finally, we show that the PqqD homologous N-terminal domain of ThnB is essential for maturation of the thurincin H precursor peptide, but not for the SAM cleavage activity of ThnB.