Characterization of [2Fe–2S]‐Cluster‐Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods

Abstract

Many iron–sulfur proteins involved in cluster trafficking form [2Fe–2S]‐cluster‐bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe–2S]‐cluster‐bridged proteins and their transient cluster‐transfer intermediates. The use of this mechanistic and protein‐characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA‐like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster‐transfer reactions between the holo‐dimers and apo‐ferredoxin (FDX2) are monitored, and intermediate [2Fe–2S] species, such as (FDX2:GLRX5:[2Fe–2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe–2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron–sulfur‐cluster‐bridged protein complexes and transient iron–sulfur‐cluster transfer intermediates.

Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 2 Caused by CYS59TYR BOLA3 Mutation

“…BOLA3 is known to form a hetero-dimeric complex with apo GLRX5 with an affinity constant of 1.2 × 10 5 M −1 [22][23][24][25]. To test whether the Cys59Tyr mutation affected the formation of the apo BOLA3-GLRX5 complex, we stepwise added apo GLRX5 to 15 Nlabelled C59Y BOLA3, or vice versa adding C59Y BOLA3 to apo 15 N-labelled GLRX5, and the interaction was followed through 1 H− 15 N HSQC NMR experiments (see the Materials and Methods section for details).…”

“…BOLA3 is known to form a hetero-dimeric complex with apo GL constant of 1.2 × 10 5 M −1 [22][23][24][25]. To test whether the Cys59Tyr muta mation of the apo BOLA3-GLRX5 complex, we stepwise added apo G C59Y BOLA3, or vice versa adding C59Y BOLA3 to apo 15 N-labelled teraction was followed through 1 H− 15 N HSQC NMR experiments (s As following step, chemical shift perturbations and line broadening analyses were performed by comparing the 1 H− 15 N HSQC spectra of 15 N-labelled BOLA3 and 15 Nlabelled C59Y BOLA3, respectively, with those recorded in presence of equimolar amounts of apo GLRX5, at two different starting concentrations of 15 N-labelled BOLA3 and 15 Nlabelled C59Y BOLA3 (100 and 800 µM).…”

“…A combination of in vitro and in vivo studies support that BOLA3 complexed with GLRX5 could function in [2Fe–2S] cluster trafficking and/or insertion reactions in the mitochondrial iron–sulfur protein biogenesis [ 23 ]. Recently, the BOLA3–GLRX5 complex in its [2Fe–2S] 2+ bound state was shown to transfer the cluster to both apo human ferredoxins FDX1 and FDX2 with rate constants comparable to other cluster donors to FDX proteins [ 24 , 25 ], as well as to transfer its cluster to apo NFU1 to form a [4Fe–4S] 2+ cluster-bound NFU1 dimer [ 26 ]. However, considering that the yeast homologue of human BOLA3 was shown not to be required for the maturation of mitochondrial [2Fe–2S] proteins [ 23 ], the cluster transfer to FDXs is very likely not physiologically relevant.…”

Cluster exchange reactivity of [2Fe‐2S]‐bridged heterodimeric BOLA1‐GLRX5

“…We have previously demonstrated that [2Fe-2S]bound BOLA3-GLRX5 heterodimeric complex is indeed well-suited for cluster trafficking, with cluster uptake from donors such as ISCU or [2Fe-2S](GS) 4 resulting in the [2Fe-2S]-bridged BOLA3-GLRX5 complex, and the [2Fe-2S] in BOLA3-GLRX5 readily transferrable to downstream cluster acceptors [9,16], but not to ISCA1 or ISCA2 that has been implicated in [4Fe-4S] cluster trafficking [9].…”

Sensing mechanisms of iron–sulfur cluster regulatory proteins elucidated using native mass spectrometry

“…[38][39][40][41] Recently, native MS has been applied to a much broader range of Fe-S proteins, enabling the determination of cluster types, and, remarkably, the nature of chemistry taking place at clusters, in some cases in real time. [42][43][44][45][46][47][48][49][50][51][52] Aspects of these advances are the focus of this article.…”