The active form of the oxygen sensor fumarate nitrate reductase regulator (FNR) of Escherichia coli contains a [4Fe-4S] cluster which is converted to a [2Fe-2S] cluster after reaction with air, resulting in inactivation of FNR. Reaction of reconstituted [4Fe-4S].FNR with air resulted within 5 min in conversion to apoFNR. The rate was comparable to the rate known for [4Fe-4S].FNR/[2Fe-2S].FNR cluster conversion, suggesting that apoFNR is a product of [2Fe-2S].FNR decomposition and a final form of air-inactivated FNR in vitro. Formation of apoFNR and the redox state of the cysteinyl residues were determined in vitro by alkylation. FNR contains five cysteinyl residues, four of which (Cys20, Cys23, Cys29 and Cys122) ligate the FeS clusters. Alkylated FNR and proteolytic fragments thereof were analyzed by MALDI-TOF. ApoFNR formed by air inactivation of [4Fe-4S].FNR in vitro contained one or two disulfides. Only disulfide pairs Cys16/20 and Cys23/29 were formed; Cys122 was never part of a disulfide. The same type of disulfide was found in apoFNR obtained during isolation of FNR, suggesting that cysteine disulfide formation follows a fixed pattern. ApoFNR, including the form with two disulfides, can be reconstituted to [4Fe-4S].FNR after disulfide reduction. The experiments suggest that apoFNR is a major form of FNR under oxic conditions.