Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Abstract

Biogenesis of iron-sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron-sulfur protein assembly machinery, two human key proteins-NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsinform a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron-sulfur cluster proteins. The Ndor1-anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.Fe/S protein maturation | CIAPIN1 domain | diflavin reductase I ron-sulfur clusters (ISCs) are ancient inorganic cofactors that are crucial for many protein functions in eukaryotic and bacterial cells (1-3). The clusters are composed of inorganic sulfide and ferric/ ferrous iron atoms, the latter being preferentially coordinated by cysteinyl residues (4-6). Because inorganic sulfide and ferrous/ferric iron atoms are toxic in vivo, biosynthesis of ISC proteins within cells is a highly regulated process that requires complex protein machineries for the mobilization of Fe and S atoms from appropriate sources, for their assembly into ISC forms and their final delivery to the recipient proteins (7-9). Three distinct protein machineries are operative and essential in the (nonplant) eukaryotic cells for the biogenesis of ISC proteins: (i) the ISC assembly machinery in the mitochondrial matrix, (ii) the ISC export machinery located in the mitochondrial intermembrane space, and (iii) the cytosolic iron-sulfur protein assembly (CIA) machinery.The CIA machinery comprises several proteins (10, 11), among which one named Dre2 has been recently identified in yeast (12). The C-terminal domain of Dre2 (residues 228-348) is able to bind two ISCs, a [2Fe-2S] and a [4Fe-4S] (12, 13). The [2Fe-2S] cluster of Dre2 receives electrons from a cytosolic diflavin reductase, termed Tah18 (13), which contains a FAD and a FMN prosthetic group, respectively, bound in two distinct structural domains, to accept electrons from NADPH (14). Dre2 and Tah18 are protein partners forming a stable complex in vivo (13, 15). The C-terminal region of Dre2 (residues 173-348) is fundamenta...

Human anamorsin binds [2Fe–2S] clusters with unique electronic properties

“…The acidic iron(III) solution was prepared by dissolving 57 Fe 2 O 3 in 12 M HCl to a final iron concentration of 125 mM. FMN-Ndor1 (residues 1-174 of the full-length sequence) was expressed and purified as previously reported [6].…”

“…Since electrospray ionization mass spectrometry data indicate the presence of only one [2Fe-2S] unit per protein molecule bound to the CIAPIN1 domain [6], binding of a [2Fe-2S] cluster in one of the two motifs disfavors the binding of a second [2Fe-2S] cluster in the other motif. Consistently, it is not possible to chemically reconstitute the FL protein with both [2Fe-2S] clusters bound at stoichiometric levels; the protein-to-Fe-to-S ratio in reconstituted samples never exceeded 1:2:2 [9].…”

“…For [2Fe-2S] ferredoxins of known structure and presumably others as well, one iron atom is closer to the surface (approximately 5 Å ), and it has been established by NMR spectroscopy that the added electron resides on this iron [4]. Here, we focused our attention on the human Fe/S binding protein anamorsin, which has recently been proposed to be part of an electron transfer chain functioning in the early steps of cytosolic Fe/S protein biogenesis [5,6]. The electron transfer process requires the formation of a stable complex with the diflavin reductase Ndor1 [6].…”

“…Here, we focused our attention on the human Fe/S binding protein anamorsin, which has recently been proposed to be part of an electron transfer chain functioning in the early steps of cytosolic Fe/S protein biogenesis [5,6]. The electron transfer process requires the formation of a stable complex with the diflavin reductase Ndor1 [6]. Ndor1 contains a FAD and a FMN prosthetic group and receives electrons from NADPH [7].…”

“…This protein consists of two structural domains: the first binds FMN, and the second binds FAD and NADPH [8]. The FMN binding domain (FMN-Ndor1 hereafter) is the part of Ndor1 directly interacting with anamorsin which receives one electron from FMN [6]. Human anamorsin coordinates a [2Fe-2S] cluster through four cysteines arranged in a CX 8 CX 2 CXC (M1) motif (where X indicates any amino acid) [9].…”