The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo

Hannah Rosenbach,George E Cutsail,Serena Debeer,Marina Pascual-Ortiz,James A Birrell,Ingrid Span,Ursula Fleig,Eva Walla

doi:10.1007/s00775-020-01840-w

Abstract

The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365−920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371−920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe–S cluster in Asp1365−920 inside the cell. However, we show that the Fe–S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe–S cluster in vivo that is not involved in its pyrophosphatase activity.

Highlights

Iron–sulfur (Fe–S) clusters are ancient and versatile cofactors that are ubiquitously found in all organisms
Our work reveals that Asp1 contains an Fe–S cluster in vivo that is not involved in its pyrophosphatase activity
We show that the C-terminal pyrophosphatase domain of S. pombe Asp1 (Asp1365−920) isolated from E. coli BL21(DE3) ∆iscR or S. pombe cells under strictly anaerobic conditions contains a [2Fe-2S] cluster

Summary

Introduction

Iron–sulfur (Fe–S) clusters are ancient and versatile cofactors that are ubiquitously found in all organisms. Fe–S clusters associated with proteins are essential for numerous biological processes, including electron transfer, substrate binding and activation, redox catalysis, sensing of iron and oxygen, DNA replication and repair, regulation of gene expression, tRNA modification, and genome instability [1–4]. Most Fe–S clusters are bound to the protein backbone by cysteine residues. In many Fe–S proteins, the cysteine ligands that bind the cofactor are arranged in characteristic cysteine pattern [5]. Despite the constant discovery of novel ligation patterns, it remains challenging to identify Fe–S-containing proteins purely based on sequence analysis. Another challenge in identifying protein-bound Fe–S cofactors is their sensitivity to oxygen [6–9].

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Conclusion