Transcriptional regulation of a hybrid cluster (prismane) protein

HCP (hybrid-cluster protein) contains two Fe/S clusters, one of which is a hybrid [4Fe-2S-2O] cluster. Despite intensive study, its physiological function has not been reported. The Escherichia coli hcp gene is located in a two-gene operon with hcr , which encodes an NADH-dependent HCP reductase. E. coli HCP is detected after anaerobic growth with nitrate or nitrite: possible roles for it in hydroxylamine or nitric oxide reduction have been proposed. To study the regulation and role of HCP, an hcp::lacZ fusion was constructed and transformed into fnr , arcA and norR mutant strains of E. coli . Transcription from the hcp promoter was induced during anaerobic growth. Only the fnr mutant was defective in hcp expression. Nitrate- and nitrite-induced transcription from the hcp promoter was activated by the response regulator proteins NarL and NarP. Gel retardation assays were used to show that FNR (fumarate-nitrate regulation) and NarL form a complex with the hcp promoter. Transcription of the hcp-hcr operon initiates at a thymine nucleotide located 31 bp upstream of the translation-initiation codon. HCP has been overexpressed from a recombinant plasmid for physiological studies.