Binding of Nitric Oxide in CDGSH-type [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2

Abstract

Iron-sulfur proteins are among the primary targets of nitric oxide in cells. Previous studies have shown that iron-sulfur clusters hosted by cysteine residues in proteins are readily disrupted by nitric oxide forming a protein-bound dinitrosyl iron complex, thiolate-bridged di-iron tetranitrosyl complex, or octanitrosyl cluster. Here we report that human mitochondrial protein Miner2 [2Fe-2S] clusters can bind nitric oxide without disruption of the clusters. Miner2 is a member of a new CDGSH iron-sulfur protein family that also includes two mitochondrial proteins: the type II diabetes-related mitoNEET and the Wolfram syndrome 2-linked Miner1. Miner2 contains two CDGSH motifs, and each CDGSH motif hosts a [2Fe-2S] cluster via three cysteine and one histidine residues. Binding of nitric oxide in the reduced Miner2 [2Fe-2S] clusters produces a major absorption peak at 422 nm without releasing iron or sulfide from the clusters. The EPR measurements and mass spectrometry analyses further reveal that nitric oxide binds to the reduced [2Fe-2S] clusters in Miner2, with each cluster binding one nitric oxide. Although the [2Fe-2S] cluster in purified human mitoNEET and Miner1 fails to bind nitric oxide, a single mutation of Asp-96 to Val in mitoNEET or Asp-123 to Val in Miner1 facilitates nitric oxide binding in the [2Fe-2S] cluster, indicating that a subtle change of protein structure may switch mitoNEET and Miner1 to bind nitric oxide. The results suggest that binding of nitric oxide in the CDGSH-type [2Fe-2S] clusters in mitochondrial protein Miner2 may represent a new nitric oxide signaling mode in cells.

Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells

“…Indeed, MiNT is identified as an essential gene in high-resolution genetic vulnerability analyses in a recent study (30). Also, whereas most [2Fe-2S] clusters are destroyed by binding of nitric oxide (NO), MiNT is reported to stably bind NO (31). This feature of MiNT could suggest that it has a role in NO signaling.…”

EPR spectroscopy of complex biological iron–sulfur systems

“…Recombinant expression is typically without this sequence, e.g., human mitoNEET (i.e., mitochondrial NEET) is used in the biochemical literature as a label for a heterologously expressed protein (actually three paralogs: mitoNEET, Miner1, and Miner2, in which Miner is an abbreviation of mitoNEET related [28, 29]) that misses the first 32 amino acids. The remainder sequence contains an unusual binding motif with 3 Cys and 1 His to bind a single [2Fe–2S] (2+;+) cluster.…”

Mass Spectrometric Identification of [4Fe–4S](NO)x Intermediates of Nitric Oxide Sensing by Regulatory Iron–Sulfur Cluster Proteins

“…ESI‐MS utilising solution and ionization conditions under which proteins remain folded enables accurate mass detection of intact proteins and protein complexes, and has been used extensively to study protein–protein interactions, interactions of proteins with drug molecules, oligonucleotides, carbohydrates, and lipids, as well as protein structural changes . ESI‐MS of iron–sulfur cluster proteins, where the cluster remains bound following vapourisation/ionisation, has also been shown to be a valuable technique, and recently provided detailed mechanistic information of the O 2 ‐sensing reaction of the [4Fe–4S] cluster binding FNR regulator and evidence of NO‐binding to the [2Fe‐2S] clusters of human Miner2 and variant forms of Miner1 and MitoNEET …”

“…[21] ESI-MS of iron-sulfur clusterp roteins, where the cluster remains bound following vapourisation/ionisation, has also been shown to be avaluable technique, [22] and recently provided detailed mechanistic information of the O 2 -sensing reactiono f the [4Fe-4S]c lusterb inding FNR regulator [23] and evidenceo f NO-binding to the [2Fe-2S] clusters of human Miner2 and variant forms of Miner1 and MitoNEET. [24] Here, we report the application of ESI-MS under non-denaturing conditions to studies of the reactions of the [4Fe-4S] clusters of NsrR and WhiD with NO.T he data reveal, in remarkable detail, intermediates and products of the reaction of the clusters with NO and enablet he nitrosylation mechanismso f the two regulators to be distinguished. .…”