Anaerobic acquisition of [4Fe 4S] clusters by the inactive FNR(C20S) variant and restoration of activity by second-site amino acid substitutions

The FNR protein of Escherichia coli controls the transcription of target ge nes in response to anoxia. The anaerobic incorporation of oxygen-sensitive [4Fe 4S] clusters promotes dimerization, which in turn enhances DNA binding . Four potential iron ligands (C20, C23, C29 and C122) are essential for no rmal FNR activity in vivo. Three FNR variants (C20S, C23G and C29G) retaine d the ability to incorporate oxygen-sensitive [4Fe 4S] clusters and to bind target DNA with essentially unimpaired affinity, suggesting that their fai lure to function normally in vivo resides at a later stage in the signal tr ansduction pathway. The C122 variant failed to assemble iron-sulphur cluste rs and to bind DNA. Second-site substitutions that partially restore activi ty to FNR(C20S) were generated by error-prone polymerase chain reaction and were located in the dimer interface, in the activating regions (AR1, 2 or 3) or close to C122. Substitutions at E47, R48, E123, I124, E127 or T128 al lowed the extent of the FNR AR2 surface to be defined. Only one revertant, FNR(C20S Y69F G149S), specifically corrected the C20S defect. It was conclu ded that [4Fe 4S] cluster acquisition, dimerization and DNA binding are not sufficient to confer transcription regulatory activity on FNR: the iron-su lphur cluster must also be correctly liganded in order to establish effecti ve activating contacts between FNR and RNA polymerase.