A novel Rieske iron-sulfur protein from the hyperthermophilic crenarchaeonPyrobaculum aerophilum: Sequencing of the gene, expression in E-coli and characterization of the protein

The crenarchaeon Pyrobaculum aerophilum is with an optimal growth temperatu re of 100 degrees C one of the most thermophilic organisms known to possess an aerobic respiratory chain. The analysis of DNA sequences from the Pyrob aculum genome project lead to the identification of an open reading frame p otentially coding for a Rieske iron-sulfur protein. The complete gene (name d parR) was cloned and sequenced. The deduced amino acid sequence displays unusual amino acid exchanges and a so far unknown sequence insertion. The N -terminus shows similarities to bacterial signal sequences. Several forms o f the gene were expressed in E. coli in order to verify the classification as a Rieske protein and to facilitate biophysical studies. Soluble, thermo- stable proteins with correctly inserted iron-sulfur clusters were expressed from two versions of the gene. The Delta 1-23 truncated hole-protein is re dox active. It displays the typical spectroscopic properties of a Rieske pr otein. The redox potential was determined to be +215 mV at pH 6.5 and is pH dependent above pH 7.5 revealing the influence of two protonation equilibr ia with pKa values of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parR protein clusters together with the two other available archaeal Ri eske sequences from Sulfolobus on a separate branch of the phylogenetic tre e apart from the proteins from thermophilic bacteria like Aquifex and Therm us.