A-Type Carrier Proteins Are Involved in [4Fe-4S] Cluster Insertion into the Radical S -Adenosylmethionine Protein MoaA for the Synthesis of Active Molybdoenzymes

Abstract

Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them were characterized in detail in Escherichia coli, namely IscA, SufA and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA involved in [4Fe-4S] cluster insertion into the S-adenosyl-methionine dependent radical SAM protein MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth of nitrate respiration, based on low gene expression levels. IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics and gene regulation. Still remaining critical gaps in our knowledge are how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SusA and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.

Iron limitation indirectly reduces the Escherichia coli torCAD operon expression by a reduction of molybdenum cofactor availability

“…The ferric uptake repressor (Fur) regulates the expression of iron homeostasis genes in response to intracellular iron levels. Since the first step of Moco biosynthesis depends on the presence of [4Fe-4S] clusters in MoaA ( 50 ), Moco biosynthesis subsequently depends on the presence of iron and Fe-S clusters and consequently on the regulators Fur, FNR, and IscR, as reported previously ( 21 , 32 ).…”

Multimodal Spectroscopic Analysis of the Fe–S Clusters of the as-Isolated Escherichia coli SufBC2D Complex

“…Our results reveal that the as-isolated SufBC 2 D complex, containing a [2Fe-2S] cluster, can transfer its cluster to apoferredoxin, in agreement with its scaffold function. The current accepted view is that bacterial IscU and SufBC 2 D behave like scaffolds, assembling a [2Fe-2S] cluster that is subsequently transferred to A-type proteins, where it can be converted to [4Fe-4S] (via a reductive coupling) for targeting to apo-targets. − It should be noted, however, that A-type proteins are also instrumental in the maturation of [2Fe-2S]-containing proteins such as IscR or SoxR in E. coli. , The observation of a [2Fe-2S] cluster on as-purified SufBC 2 D is in agreement with this view.…”

A multidisciplinary analysis of the as-isolated Escherichia coli SufBC2D complex reveals the presence of two new iron-sulfur clusters

“…The current admitted view is that bacterial IscU and SufBC2D act as scaffolds assembling a [2Fe-2S] cluster that is subsequently transferred to A-type proteins, which are thought to convert them to [4Fe-4S] (via a reductive coupling) for targeting to apo-targets. [41][42][43][44][45][46][47] Note however that Atype are also instrumental in maturation of [2Fe-2S] containing proteins such as IscR or SoxR in E. coli. 48,49 The observation of a [2Fe-2S] cluster on as-purified SufBC2D is in agreement with this view.…”