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.

Pushing the limits of native MS: Online SEC-native MS for structural biology applications

“…Benefits of such systems is that using individual nozzles for each ligand/mixture of ligand prevents from cross contamination of capillaries or columns. SEC-nMS can also be of interest to investigate protein-metal interactions, as reported by Jia et al for [2Fe-2S] cluster-bridged complexes, for which offline nanoESI-nMS failed to detect cluster-bound species 44 . As iron-sulfur clusters are sensitive to oxygen, SEC-nMS helps to minimize the possibility to oxidize unstable analytes due to reduced sample preparation and shorter time of analysis.…”