Regulation of FNR Dimerization by Subunit Charge Repulsion (2006)

Dimerization of the global anaerobic transcription factor FNR is essential for FNR activity. Under aerobic conditions FNR is an inactive monomeric species because it lacks the oxygen labile [4Fe-4S] cluster required for dimerization. In this study, we investigated the protein side chains that inhibit FNR dimerization under aerobic conditions. Substitution of Asp154 within the predicted dimerization helix with residues containing neutral or positively charged side chains increased FNR activity under aerobic conditions, whereas replacement of Asp154 with Glu inhibited FNR activity similar to WT-FNR. Similar results were obtained when making analogous substitutions of Glu150. In vitro analysis of representative FNR mutant proteins indicated that their increased activity under aerobic conditions resulted an [4Fe-4S] of dimerization. In of residues with of FNR activity under aerobic conditions. that side chains is to inhibit dimerization under aerobic conditions. that under anaerobic conditions. of the of indicated that whereas that the FNR the to for dimerization Dimerization of the global anaerobic transcription factor FNR is essential for FNR activity. Under aerobic conditions FNR is an inactive monomeric species because it lacks the oxygen labile [4Fe-4S] cluster required for dimerization. In this study, we investigated the protein side chains that inhibit FNR dimerization under aerobic conditions. Substitution of Asp154 within the predicted dimerization helix with residues containing neutral or positively charged side chains increased FNR activity under aerobic conditions, whereas replacement of Asp154 with Glu inhibited FNR activity similar to WT-FNR. Similar results were obtained when making analogous substitutions of Glu150. In vitro analysis of representative FNR mutant proteins indicated that their increased activity under aerobic conditions resulted an [4Fe-4S] of dimerization. In of residues with of FNR activity under aerobic conditions. that side chains is to inhibit dimerization under aerobic conditions. that under anaerobic conditions. of the of indicated that whereas that the FNR the to for dimerization is the transcription factor transcription of under anaerobic conditions of for In the of FNR is with containing [4Fe-4S] cluster Dimerization is required for FNR because the to the of to or transcription Under aerobic conditions, FNR because FNR is to monomeric is inactive for of the monomeric species results the of the [4Fe-4S] cluster to cluster the cluster is the of FNR to is the [4Fe-4S] cluster the of FNR to to the of transcription of predicted to similar their for of this the transcription factor the the transcription factor when the of their of is helix helix an an the or dimerization that of FNR that the of FNR of predicted that the residues within this is the charged is that this an dimerization because to dimerization under aerobic conditions, protein when the [4Fe-4S] cluster is that to results an transcription factor that residues dimerization of FNR the of the [4Fe-4S] cluster of FNR is predicted to the dimerization helix to the that of the [4Fe-4S] cluster is to that the of Asp154 to this we vitro to the the of the residues the of dimerization of the aerobic of to the of the the this substitutions of FNR were of mutant proteins containing the or mutant proteins containing the or were the or the to it when with with with the were were the were the of to the of the the for of were of were the were for or FNR protein this or of to of with of to of activity of FNR mutant activity to the were with were to an of whereas anaerobic were to an of to or with to an of with for under of the [4Fe-4S] cluster the analysis of of to for the of the FNR protein that the [4Fe-4S] cluster for the to FNR to the cluster of cluster containing or to an of to with or with FNR with for the were with protein that to of the of or the an to that anaerobic of of of FNR or mutant were the the of the to with were the or were to under anaerobic conditions anaerobic of FNR protein with of to for were with the of protein were of or cluster protein with of the protein with the were the an with an analysis were were of to an anaerobic of FNR with of the [4Fe-4S] cluster the to for of to of under the conditions that of to the of the with the of Asp154 for of that Asp154 to resulted FNR protein that under aerobic conditions the of [4Fe-4S] cluster the this dimerization we the with side chains of to FNR activity dimerization under aerobic conditions. FNR activity the of activity containing under of the or mutant the were for activity under aerobic conditions the FNR mutant with Glu inactive WT-FNR. In of the charged side with or residues this FNR proteins that were under aerobic conditions. the mutant protein under aerobic conditions the charged of analysis that the FNR mutant proteins were similar to that of that the activity is to the protein Under anaerobic conditions, the the of activity that of the mutant proteins were to of the [4Fe-4S] of substitutions Asp154 FNR activity. activity of FNR containing substitutions the of under aerobic conditions or anaerobic conditions with activity is of the activity of the containing under anaerobic conditions of FNR is indicated the the the increased activity of under aerobic conditions with an dimerization with of monomeric were of species the that it is under conditions. that the of the is to dimerization of FNR under aerobic of of the is for protein for the of that charged residues inhibited dimerization under aerobic conditions that charged within the dimerization helix of dimerization. this we the activity of of of the predicted FNR dimerization to under aerobic conditions. the dimerization helix that results an protein under aerobic conditions, to is In with increased FNR activity under aerobic conditions with the replacement of the charged with positively charged resulted the activity of the substitutions the of activity for similar of the of the dimerization helix it that residues the of the helix FNR activity. mutant containing of to Glu activity under aerobic conditions that the of the predicted for dimerization under aerobic under aerobic of FNR proteins with within the dimerization activity under aerobic conditions of containing of to activity of activity under aerobic conditions of the with of to proteins were containing the activity is of the activity of the containing FNR activity under aerobic conditions of containing of to FNR activity under aerobic conditions of the with of to proteins were containing the activity is of the activity of the containing of substitutions FNR activity. activity of FNR containing substitutions the of under aerobic conditions or anaerobic conditions with activity is of the activity of the containing under anaerobic conditions of FNR is indicated the the similar to the of substitutions were their activity mutant containing the of the of the side activity under aerobic conditions the that the of Glu is dimerization the that side with replacement of with with an the activity the mutant that positively or charged is dimerization of of substitutions Asp154 Glu150. FNR activity of containing substitutions the of under aerobic conditions or anaerobic conditions with activity is of the activity of the containing under anaerobic conditions of FNR is indicated the the to positively charged residues resulted FNR activity under aerobic conditions proteins neutral charged the the of positively charged residues is FNR activity the the this that of residues is of FNR an inactive the activity of the mutant under aerobic conditions with of the the of the protein that of the protein the that is that protein is the of the protein is the to of the that the of this mutant protein is activity that the mutant of the of the side the Glu side chains the side the of to the the Glu side chains WT-FNR. results the that the FNR is for of to the of residues within the predicted to dimerization when is the activity of the proteins with substitutions to were under aerobic conditions In this of substitutions that an or of FNR of the of of of the substitutions of that the of the helix is similar to for whereas the the of the helix is of dimerization the activity of In to with with that dimerization when the Asp154 is substitutions increased activity of Substitution of to increased activity under aerobic anaerobic conditions the activity of similar In substitutions of or the of the predicted increased the activity of under aerobic conditions substitutions activity or activity the residues to activity of to the of the helix that is required to the of the the of were FNR residues to the to the within the dimerization helix the predicted the that or with under anaerobic conditions, of is FNR of within of Similar results were obtained of containing of cluster or an FNR [4Fe-4S] cluster that is to or In with to that of FNR to were of of FNR predicted for of of of with for the of of the for indicated for species were or whereas the of to it that of the cluster the of the that of FNR of an to the cluster containing of of whereas the of the of the that were similar for the for the of is under conditions is to the monomeric results that the of the residues that of the [4Fe-4S] of of [4Fe-4S] of the is of dimerization to the FNR activity is of FNR that to or transcription or vitro the of FNR the the of that required for FNR dimerization. In the that the of [4Fe-4S] the of the dimerization for the aerobic of the protein for of FNR of dimerization to the that dimerization residues Asp154 the dimerization the of of with residues the of helix the that is an or of the dimerization inhibit dimerization that the side chains of or the the monomeric the of dimerization Asp154 the Glu150. of of an dimerization when FNR because is the for to In the that of the predicted to dimerization is predicted to the of substitutions dimerization of the or the [4Fe-4S] because replacement of residues with dimerization of the of FNR that lacks analogous to the of that to of FNR the of the [4Fe-4S] the of of residues to that dimerization is inhibited the of this is for the of that the that predicted cluster of FNR with the that of containing cluster with of the analogous to of of that this cluster charged an the analogous whereas an the analogous of residues the FNR that of dimerization the of of of with of FNR to the of FNR that to an of the is an of the that to the of the FNR proteins the of the it that to the that is of the to the of the dimerization helix of for of the dimerization helix of the to the that we the for dimerization FNR the of the the dimerization helix similar to to FNR to dimerization with to the of of their the of their In of FNR the cluster of the cluster that of [4Fe-4S] FNR when FNR is the monomeric the of the the inactive of FNR this were that when the [4Fe-4S] cluster is to the or is In the results that of FNR because the to the is similar to with indicated that is to the of when the the the [4Fe-4S] the of the of the of the cluster the is the increased of the is to because of the to the residues of In the of the of for the of with cluster to FNR when it that to FNR the of is for FNR to inactive under aerobic conditions that the FNR of the the to FNR activity to to the FNR dimerization is is the transcription factor transcription of under anaerobic conditions of for In the of FNR is with containing [4Fe-4S] cluster Dimerization is required for FNR because the to the of to or transcription Under aerobic conditions, FNR because FNR is to monomeric is inactive for of the monomeric species results the of the [4Fe-4S] cluster to cluster the cluster is the of FNR to is the [4Fe-4S] cluster the of FNR to dimerization. FNR to the of transcription of predicted to similar their for of this the transcription factor the the transcription factor when the of their of is helix helix an an the or dimerization that of FNR that the of FNR of predicted that the residues within this is the charged is that this an dimerization because to dimerization under aerobic conditions, protein when the [4Fe-4S] cluster is that to results an transcription factor that residues dimerization of FNR the of the [4Fe-4S] cluster of FNR is predicted to the dimerization helix to the that of the [4Fe-4S] cluster is to that the of Asp154 to dimerization. In this we vitro to the the of the residues the of dimerization of the aerobic of to the of the the this substitutions of FNR were of mutant proteins containing the or mutant proteins containing the or were the or the to it when with with with the were were the were the of to the of the the for of were of were the were for or FNR protein this or of to of with of to of activity of FNR mutant activity to the were with were to an of whereas anaerobic were to an of to or with to an of with for under of the [4Fe-4S] cluster the analysis of of to for the of the FNR protein that the [4Fe-4S] cluster for the to FNR to the cluster of cluster containing or to an of to with or with FNR with for the were with protein that to of the of or the an to that anaerobic of of of FNR or mutant were the the of the to with were the or were to under anaerobic conditions anaerobic of FNR protein with of to for were with the of protein were of or cluster protein with of the protein with the were the an with an analysis were were of to an anaerobic of FNR with of the [4Fe-4S] cluster the to for of to of under the conditions that of to the of the with the this substitutions of FNR were of mutant proteins containing the or mutant proteins containing the or were the or the to it when with with with the were were the were the of to the of the the for of were of were the were for or FNR protein activity of FNR mutant activity to the were with were to an of whereas anaerobic were to an of to or with to an of with for under of the [4Fe-4S] cluster the analysis of of to for the of the FNR protein that the [4Fe-4S] cluster for the to FNR to the cluster of cluster containing or to an of to with or with FNR with for the were with protein that to of the of or the an to that anaerobic of of of FNR or mutant were the the of the to with were the or were to under anaerobic conditions anaerobic of FNR protein with of to for were with the of protein were of or cluster protein with of the protein with the were the an with an analysis were were of to an anaerobic of FNR with of the [4Fe-4S] cluster the to for of to of under the conditions that of to the of the with the of Asp154 for of that Asp154 to resulted FNR protein that under aerobic conditions the of [4Fe-4S] cluster the this dimerization we the with side chains of to FNR activity dimerization under aerobic conditions. FNR activity the of activity containing under of the or mutant the were for activity under aerobic conditions the FNR mutant with Glu inactive WT-FNR. In of the charged side with or residues this FNR proteins that were under aerobic conditions. the mutant protein under aerobic conditions the charged of analysis that the FNR mutant proteins were similar to that of that the activity is to the protein Under anaerobic conditions, the the of activity that of the mutant proteins were to of the [4Fe-4S] the increased activity of under aerobic conditions with an dimerization with of monomeric were of species the that it is under conditions. that the of the is to dimerization of FNR under aerobic of of the is for protein for the of that charged residues inhibited dimerization under aerobic conditions that charged within the dimerization helix of dimerization. this we the activity of of of the predicted FNR dimerization to under aerobic conditions. the dimerization helix that results an protein under aerobic conditions, to is In with increased FNR activity under aerobic conditions with the replacement of the charged with positively charged resulted the activity of the substitutions the of activity for similar of the of the dimerization helix it that residues the of the helix FNR activity. mutant containing of to Glu activity under aerobic conditions that the of the predicted for dimerization under aerobic under aerobic of FNR proteins with within the dimerization activity under aerobic conditions of containing of to activity of activity under aerobic conditions of the with of to proteins were containing the activity is of the activity of the containing FNR activity under aerobic conditions of containing of to FNR activity under aerobic conditions of the with of to proteins were containing the activity is of the activity of the containing of substitutions FNR activity. activity of FNR containing substitutions the of under aerobic conditions or anaerobic conditions with activity is of the activity of the containing under anaerobic conditions of FNR is indicated the the similar to the of substitutions were their activity mutant containing the of the of the side activity under aerobic conditions the that the of Glu is dimerization the that side with replacement of with with an the activity the mutant that positively or charged is dimerization of of substitutions Asp154 Glu150. FNR activity of containing substitutions the of under aerobic conditions or anaerobic conditions with activity is of the activity of the containing under anaerobic conditions of FNR is indicated the the to positively charged residues resulted FNR activity under aerobic conditions proteins neutral charged the the of positively charged residues is FNR activity the the this that of residues is of FNR an inactive the activity of the mutant under aerobic conditions with of the the of the protein that of the protein the that is that protein is the of the protein is the to of the that the of this mutant protein is activity that the mutant of the of the side the Glu side chains the side the of to the the Glu side chains WT-FNR. results the that the FNR is for of to the of residues within the predicted to dimerization when is the activity of the proteins with substitutions to were under aerobic conditions In this of substitutions that an or of FNR of the of of of the substitutions of that the of the helix is similar to for whereas the the of the helix is of dimerization the activity of In to with with that dimerization when the Asp154 is substitutions increased activity of Substitution of to increased activity under aerobic anaerobic conditions the activity of similar In substitutions of or the of the predicted increased the activity of under aerobic conditions substitutions activity or activity the residues to activity of to the of the helix that is required to the of the the of were FNR residues to the to the within the dimerization helix the predicted the that or with under anaerobic conditions, of is FNR of within of Similar results were obtained of containing of cluster or an FNR [4Fe-4S] cluster that is to or In with to that of FNR to were of of FNR predicted for of of of with for the of of the for indicated for species were or whereas the of to it that of the cluster the of the that of FNR of an to the cluster containing of of whereas the of the of the that were similar for the for the of is under conditions is to the monomeric results that the of the residues that of the [4Fe-4S] of of [4Fe-4S] of the is of Asp154 for of that Asp154 to resulted FNR protein that under aerobic conditions the of [4Fe-4S] cluster the this dimerization we the with side chains of to FNR activity dimerization under aerobic conditions. FNR activity the of activity containing under of the or mutant the were for activity under aerobic conditions the FNR mutant with Glu inactive WT-FNR. In of the charged side with or residues this FNR proteins that were under aerobic conditions. the mutant protein under aerobic conditions the charged of analysis that the FNR mutant proteins were similar to that of that the activity is to the protein Under anaerobic conditions, the the of activity that of the mutant proteins were to of the [4Fe-4S] the increased activity of under aerobic conditions with an dimerization with of monomeric were of species the that it is under conditions. that the of the is to dimerization of FNR under aerobic conditions. for the of that charged residues inhibited dimerization under aerobic conditions that charged within the dimerization helix of dimerization. this we the activity of of of the predicted FNR dimerization to under aerobic conditions. the dimerization helix that results an protein under aerobic conditions, to is In with increased FNR activity under aerobic conditions with the replacement of the charged with positively charged resulted the activity of the substitutions the of activity for similar of the of the dimerization helix it that residues the of the helix FNR activity. mutant containing of to Glu activity under aerobic conditions that the of the predicted for dimerization under aerobic conditions. similar to the of substitutions were their activity mutant containing the of the of the side activity under aerobic conditions the that the of Glu is dimerization the that side with replacement of with with an the activity the mutant that positively or charged is dimerization of to positively charged residues resulted FNR activity under aerobic conditions proteins neutral charged the the of positively charged residues is FNR activity the the this that of residues is of FNR an inactive the activity of the mutant under aerobic conditions with of the the of the protein that of the protein the that is that protein is the of the protein is the to of the that the of this mutant protein is activity that the mutant of the of the side the Glu side chains the side the of to the the Glu side chains WT-FNR. results the that the FNR is for dimerization. of to the of residues within the predicted to dimerization when is the activity of the proteins with substitutions to were under aerobic conditions In this of substitutions that an or of FNR of the of of of the substitutions of that the of the helix is similar to for whereas the the of the helix is of dimerization the activity of In to with with that dimerization when the Asp154 is substitutions increased activity of Substitution of to increased activity under aerobic anaerobic conditions the activity of similar In substitutions of or the of the predicted increased the activity of under aerobic conditions substitutions activity or activity the residues to activity of to the of the helix that is required to the of the the of were FNR residues to the to the within the dimerization helix the predicted the that or with under anaerobic conditions, of is FNR of within of Similar results were obtained of containing of cluster or an FNR [4Fe-4S] cluster that is to or In with to that of FNR to were of of FNR predicted for of the for indicated for species were or whereas the of to it that of the cluster the of the that of FNR of an to the cluster containing of of whereas the of the of the that were similar for the for the of is under conditions is to the monomeric results that the of the residues that of the [4Fe-4S] of dimerization to the FNR activity is of FNR that to or transcription or vitro the of FNR the the of that required for FNR dimerization. In the that the of [4Fe-4S] the of the dimerization for the aerobic of the protein for of FNR of dimerization to the that dimerization residues Asp154 the dimerization the of of with residues the of helix the that is an or of the dimerization inhibit dimerization that the side chains of or the the monomeric the of dimerization Asp154 the Glu150. of of an dimerization when FNR because is the for to In the that of the predicted to dimerization is predicted to the of substitutions dimerization of the or the [4Fe-4S] because replacement of residues with dimerization of the of FNR that lacks analogous to the of that to of FNR the of the [4Fe-4S] the of of residues to that dimerization is inhibited the of this is for the of that the that predicted cluster of FNR with the that of containing cluster with of the analogous to of of that this cluster charged an the analogous whereas an the analogous of residues the FNR that of dimerization the of of of with of FNR to the of FNR that to an of the is an of the that to the of the FNR proteins the of the it that to the that is of the to the of the dimerization helix of for of the dimerization helix of the to the that we the for dimerization FNR the of the the dimerization helix similar to to FNR to dimerization with to the of of their the of their In of FNR the cluster of the cluster that of [4Fe-4S] FNR when FNR is the monomeric the of the the inactive of FNR this were that when the [4Fe-4S] cluster is to the or is In the results that of FNR because the to the is similar to with indicated that is to the of when the the the [4Fe-4S] the of the of the of the cluster the is the increased of the is to because of the to the residues of In the of the of for the of with cluster to FNR when it that to FNR the of is for FNR to inactive under aerobic conditions that the FNR of the the to FNR activity to to the FNR dimerization is of dimerization to the FNR activity is of FNR that to or transcription or vitro the of FNR the the of that required for FNR dimerization. In the that the of [4Fe-4S] the of the dimerization for the aerobic of the protein for of FNR of dimerization to the that dimerization residues Asp154 the dimerization the of of with residues the of helix the that is an or of the dimerization inhibit dimerization that the side chains of or the the monomeric the of dimerization Asp154 the Glu150. of of an dimerization when FNR because is the for to In the that of the predicted to dimerization is predicted to the of substitutions dimerization of the or the [4Fe-4S] because replacement of residues with dimerization of the of FNR that lacks analogous to the of that to of FNR the of the [4Fe-4S] the of of residues to that dimerization is inhibited the of this is for the of that the that predicted cluster of FNR with the that of containing cluster with of the analogous to of of that this cluster charged an the analogous whereas an the analogous of residues the FNR that of dimerization the of of of with of FNR to the of FNR that to an of the is an of the that to the of the FNR proteins the of the it that to the that is of the to the of the dimerization helix of for of the dimerization helix of the to the that we the for dimerization FNR the of the the dimerization helix similar to to FNR to dimerization with to the of of their the of their In of FNR the cluster of the cluster that of [4Fe-4S] FNR when FNR is the monomeric the of the the inactive of FNR this were that when the [4Fe-4S] cluster is to the or is In the results that of FNR because the to the is similar to with indicated that is to the of when the the the [4Fe-4S] the of the of the of the cluster the is the increased of the is to because of the to the residues of In the of the of for the of with cluster to FNR when In it that to FNR the of is for FNR to inactive under aerobic conditions that the FNR of the the to FNR activity to to the FNR dimerization is for the of the of the for of the protein for the the of the the of with the of