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

Significance The transcriptional regulator Fumarate and Nitrate Reduction is the master switch for the transition between anaerobic and aerobic respiration in many other bacteria. It fulfills this role by controlling the expression of >300 genes in response to O 2 . It senses O 2 through a [4Fe-4S] cluster cofactor, which undergoes conversion to a [2Fe-2S] cluster on reaction with O 2 , leading to loss of DNA binding. By using time-resolved electrospray ionization mass spectrometry, we gained insight into the reaction through detection of cluster conversion intermediates and products, including a [3Fe-3S] cluster. The data also show that sulfide released from the cluster is oxidized during the conversion reaction and not after it. Our methodology has great potential for broad application to studies of cofactor reactivities.