The radical <scp>SAM</scp> enzyme <scp>N</scp>ir<scp>J</scp> cleaves off two propionate side chains with the release of acrylate during heme <i>d</i>₁ biosynthesis

Heme d₁ is an iron-containing, modified tetrapyrrole that serves as an essential prosthetic group in cytochrome cd₁ nitrite reductases. The biosynthesis of heme d₁ from the precursor siroheme requires three or four enzymatic steps, including the removal of two propionate side chains, the latter being catalyzed by the radical SAM enzyme NirJ. Although the removal of the propionate side chains by NirJ has been shown previously, several aspects of NirJ catalysis remained elusive, including the type of its auxiliary iron-sulfur cluster as well as the identity of the cleavage byproduct and the actual product of the NirJ reaction. Here, we demonstrate by Mössbauer spectroscopy that NirJ contains a [4Fe-4S] cluster ligated by cysteine residues as its auxiliary cluster. We show that acrylate is released during the NirJ reaction as the cleavage byproduct, as observed by HPLC-UV and HPLC-MS analysis of enzyme activity assay mixtures after derivatization. Finally, we provide strong evidence from HPLC-UV/Vis and HPLC-MS analysis that the NirJ reaction product contains methylene groups at positions C3 and C8 of the tetrapyrrole macrocycle. Based on these results, we propose a revised version of the NirJ reaction mechanism, including a potential role of the auxiliary iron-sulfur cluster as an electron donor for radical quenching.