Premium
Characterization of [2Fe–2S]‐Cluster‐Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods
Author(s) -
Jia Mengxuan,
Sen Sambuddha,
Wachnowsky Christine,
Fidai Insiya,
Cowan James A.,
Wysocki Vicki H.
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201915615
Subject(s) - chemistry , cluster (spacecraft) , iron–sulfur cluster , ferredoxin , sulfur , mass spectrometry , characterization (materials science) , combinatorial chemistry , crystallography , nanotechnology , biochemistry , organic chemistry , chromatography , materials science , enzyme , computer science , programming language
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.