Mass spectrometric identification of intermediates in the O 2 -driven [4Fe-4S] to [2Fe-2S] cluster conversion in FNR

• Published in: Proceedings of the National Academy of Sciences - PNAS 2017-04, Vol.114 (16), p.E3215
• Main Authors: Crack, Jason C, Thomson, Andrew J, Le Brun, Nick E
• Format: Article
• Language: English
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1620987114

The iron-sulfur cluster containing protein Fumarate and Nitrate Reduction (FNR) is the master regulator for the switch between anaerobic and aerobic respiration in and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O that leads to conversion to a [2Fe-2S] form with loss of high-affinity DNA binding. Here, we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a [3Fe-3S] cluster and persulfide-coordinated [2Fe-2S] clusters [[2Fe-2S](S) , where = 1 or 2]. Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion and not as a subsequent, secondary reaction to generate [2Fe-2S](S) species. This methodology shows great potential for broad application to studies of protein cofactor-small molecule interactions.