The hybrid-cluster protein ('prismane protein') from Escherichia coli - Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-20] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S]

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-admired S = 3/2 ground-state paramagnet ism 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 prot eins are present in a range of bacterial and archaeal species. In this pape r we describe the isolation and spectroscopic characterization of the hybri d-cluster protein from Escherichia coli. EPR spectroscopy shows the presenc e: of a hybrid cluster in the E. coli protein with characteristics similar to those in file proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the pre sence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene enc oding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and arch aea, 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-clu ster protein willi NADH as an electron donor. Midpoint potentials (25 degre es C, pH 7.5) for the Fe/S clusters in both: proteins indicate that electro ns derived from the oxidation of NADH(E-m NADH/NAD(+) couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (E-m = -2 20 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 tra nsitions). The physiological function of the hybrid-cluster protein has not yet been e lucidated. The protein is only detected in the facultative anaerobes E, col i and Morganella morganii after cultivation under anaerobic conditions in t he presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitr ite respiration.