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Updated: Dec 29, 2025

Analyzing Large Protein Complexes by Structural Mass Spectrometry
Published on: June 19, 2010
Characterization of [2Fe-2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass
Mengxuan Jia1,2, Sambuddha Sen1, Christine Wachnowsky1
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 43210, USA.
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.
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