The Radical SAM Heme Synthase AhbD from Methanosarcina barkeri Contains Two Auxiliary [4Fe-4S] Clusters

Abstract

In archaea and sulfate-reducing bacteria, heme is synthesized via the siroheme-dependent pathway. The last step of this route is catalyzed by the Radical SAM enzyme AhbD and consists of the conversion of iron-coproporphyrin III into heme. AhbD belongs to the subfamily of Radical SAM enzymes containing a SPASM/Twitch domain carrying either one or two auxiliary iron–sulfur clusters in addition to the characteristic Radical SAM cluster. In previous studies, AhbD was reported to contain one auxiliary [4Fe-4S] cluster. In this study, the amino acid sequence motifs containing conserved cysteine residues in AhbD proteins from different archaea and sulfate-reducing bacteria were reanalyzed. Amino acid sequence alignments and computational structural models of AhbD suggested that a subset of AhbD proteins possesses the full SPASM motif and might contain two auxiliary iron–sulfur clusters (AuxI and AuxII). Therefore, the cluster content of AhbD from Methanosarcina barkeri was studied using enzyme variants lacking individual clusters. The purified enzymes were analyzed using UV/Visible absorption and EPR spectroscopy as well as iron/sulfide determinations showing that AhbD from M. barkeri contains two auxiliary [4Fe-4S] clusters. Heme synthase activity assays suggested that the AuxI cluster might be involved in binding the reaction intermediate and both clusters potentially participate in electron transfer.