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

Abstract

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.

Mass Spectrometric Identification of [4Fe–4S](NO)x Intermediates of Nitric Oxide Sensing by Regulatory Iron–Sulfur Cluster Proteins

“…In the expression, the x + charge of the iron–sulfur cluster offsets the number of protons required to achieve the observed charge state ( n +) . Predicted masses are given as the isotope average of the neutral protein or protein complex, in which cluster‐ or Fe/S/NO‐binding is expected to be charge‐compensated …”

“…ESI‐MS utilising solution and ionization conditions under which proteins remain folded enables accurate mass detection of intact proteins and protein complexes, and has been used extensively to study protein–protein interactions, interactions of proteins with drug molecules, oligonucleotides, carbohydrates, and lipids, as well as protein structural changes . ESI‐MS of iron–sulfur cluster proteins, where the cluster remains bound following vapourisation/ionisation, has also been shown to be a valuable technique, and recently provided detailed mechanistic information of the O 2 ‐sensing reaction of the [4Fe–4S] cluster binding FNR regulator and evidence of NO‐binding to the [2Fe‐2S] clusters of human Miner2 and variant forms of Miner1 and MitoNEET …”

Origin and phylogenetic relationships of [4Fe–4S]‐containing O2 sensors of bacteria

“…). It is not known whether the similarity extends to details such as the formation of an [4Fe–3S] intermediate and of the Cys persulfides as described for FNR Ec (Crack et al ., ).…”

“…The FNR Ec , FNR Bs , WhiB3/D and NreB proteins use [4Fe–4S] 2+ clusters for O 2 sensing (Khoroshilova et al ., ; Jakimowicz et al ., ; Reents et al ., ; Singh et al ., ; Crack et al ., ; ; Mullner et al ., ; Zhang et al ., ). For FNR Ec , NreB and to some extent also for WhiB3/D, the response to O 2 appears to be similar by the conversion to a [2Fe–2S] 2+ cluster, despite the use of unrelated protein types and Cys clusters for ligation that differ in spacing of the Cys residues and sequence (Fig.…”

“…Step 2 apparently involves a second oxidation by O 2 (Crack et al ., ; Zhang et al ., ). The two sulfide ions in step 2 are not released into aqueous solution but oxidized to sulfane (S 0 ) and form a persulfide with two of the Cys ligands (RS − ) of the cluster.…”