Isolation and characterization of mutated FhlA proteins which activate transcription of thehycoperon (formate hydrogenlyase) ofEscherichia coliin the absence of molybdate

Escherichia coli growing under anaerobic conditions produces H(2) and CO(2) by the enzymatic cleavage of formate catalyzed by formate hydrogenlyase (FHL) consisting of a molybdoenzyme formate dehydrogenase H (fdhF), hydrogenase 3 (hyc), and intermediate electron carriers (hyc). Transcription of both the fdhF and hyc operons requires the activator, FhlA protein, as well as formate and molybdate. Several fhlA mutants with an altered response to the required effector molybdate were isolated and these FhlA mutated proteins activated hyc transcription in the absence of molybdate, but only in the presence of formate. Mutated protein FhlA126 carries a single mutation (R495C) in the conserved central domain of the modular, sigma(54)-dependent, enhancer-binding protein. FhlA57 contains two mutations; one in the unique N-terminal domain (E205K) and a second in the central domain (P442S). Both mutations in FhlA132 are located in the N-terminal domain (A42T and E363K). Both FhlA126 and FhlA132 proteins activated the hyc operon even in the absence of ModE and MoeA, two components of Mo-metabolism which are required for hyc-lac expression in wild-type E. coli. Based on these results, a model is proposed in which the native FhlA protein interacts with a unique form of Mo (MoeA product?) as a second effector for optimum expression of the hyc operon in E. coli.