The hybrid‐cluster protein (‘prismane protein’) from Escherichia coli

Abstract

Hybrid-cluster proteins (`prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe±4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe±2S±2O] cluster, which can attain four redox states.Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe±2S] cluster instead of a [4Fe±4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe±2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 8C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (E m NADH/NAD + couple: 2320 mV) are transferred along the [2Fe±2S] cluster of the NADH oxidoreductase (E m = ±220 mV) and the [2Fe±2S] cluster of the hybrid-cluster protein (E m = ±35 mV) to the hybrid cluster (E m = ±50, +85 and +365 mV for the three redox transitions).The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.Keywords: EPR; hybrid-cluster protein (`prismane protein'); NAD(H) oxidoreductase; nitrate regulation; redox titration.The`hybrid-cluster protein' (HCP, formerly named`prismane protein') was initially purified from species of the strictly anaerobic sulfate-reducing bacterial genus Desulfovibrio: D. vulgaris (Hildenborough) [1,2] and D. desulfuricans ATCC 27774 [3,4]. This soluble cytoplasmic protein has been extensively studied because of the unusual properties of its redox-active iron clusters. Initial characterization with EPR and Mo Èssbauer spectroscopy suggested the presence of an Fe/S cluster with magnetic properties similar to those of synthetic [6Fe±6S] model compounds (`prismane' clusters; hence the name`prismane protein' was initially proposed for this protein) [1,5].Recently, the three-dimensional structure of the D. vulgaris HCP' at 1.7 A Ê resolution was obtained by X-ray crystallography [6]. The structure reveal...

Hydroxylamine Assimilation by Rhodobacter capsulatus E1F1

“…2A). Similarity to class 2 HCPs of E. coli, Salmonella, and other facultative anaerobic Gram-negative bacteria (50% identity and 63% similarity), all of them including the N-terminal C-X 2 -C-X 11 -C-X 6 -C motif for binding a [2Fe-2S] cluster, was higher than to the class 1 HCPs present in Desulfovibrio and other strictly anaerobic bacteria (43% identity and 55% similarity), which have a different sequence C-X 2 -C-X 7-8 -C-X 5 -C for the coordination of the N-terminal [4Fe-4S] cluster (18,19 protein is homologous to the Nitrosomonas europaea nsrR gene product (39% identity and 55% similarity, Fig. 2B), which is a nitrite-sensitive negative regulator of the expression of nirK, the gene encoding the copper-nitrite reductase (39).…”

“…As the E. coli and Morganella morganii HCPs are only detected in cells grown anaerobically with nitrate or nitrite, it has been suggested that HCP have a role in nitrate and/or nitrite respiration (18). A similar increase in the expression of hcp gene was observed during anaerobic growth with nitrate in Shewanella oneidensis (22), and a nitrite-inducible Fnr-dependent promoter was identified upstream of nipAB, the hcp-hcr homologues of S. enterica (21).…”

“…Activity-The physiological function of the HCP proteins is still unknown, but it has been recently demonstrated that E. coli HCP has hydroxylamine reductase activity (24) and is only expressed during anaerobic growth with nitrate (18). A nitriteinducible HCP has also been described in Salmonella (21).…”

“…An unusual feature of the hybrid center is that it has four different oxidation states within the biologically relevant range of redox potentials. EPR spectra of the reduced HCP from Escherichia coli indicate the presence of an N-terminal [2Fe-2S] center instead of a [4Fe-4S] cluster (18). The Cys motif for binding this cluster 1 in Desulfovibrio and other strictly anaerobic bacteria has the C-X 2 -C-X 7-8 -C-X 5 -C spacing, whereas in E. coli and other facultative anaerobic bacteria has the sequence C-X 2 -C-X 11 -C-X 6 -C (18,19).…”

“…However, hcp genes are not present in the completely sequenced genomes of some prokaryotes. In E. coli (18), Thiobacillus ferrooxidans (20). and Salmonella enterica (21), a gene coding for a putative NADH oxidoreductase (hcr) is located immediately downstream of the hcp gene, and the HCR protein of E. coli catalyzes reduction of HCP in the presence of NADH (18).…”