A novel Rieske iron-sulfur protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum: sequencing of the gene, expression in E. coli and characterization of the protein.
The crenarchaeon Pyrobaculum aerophilum is with an optimal growth temperature of 100 degrees C one of the most thermophilic organisms known to possess an aerobic respiratory chain. The analysis of DNA sequences from the Pyrobaculum genome project lead to the identification of an open reading frame potentially coding for a Rieske iron-sulfur protein. The complete gene (named 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 of 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 delta1-23 truncated holo-protein is redox active. It displays the typical spectroscopic properties of a Rieske protein. 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 equilibria with pKa values of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parR protein clusters together with the two other available archaeal Rieske sequences from Sulfolobus on a separate branch of the phylogenetic tree apart from the proteins from thermophilic bacteria like Aquifex and Thermus.
Henninger
T
T
Institut für Biochemie, Medizinische Universität zu Lübeck, Germany.
Anemüller
S
S
Fitz-Gibbon
S
S
Miller
J H
JH
Schäfer
G
G
Schmidt
C L
CL
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D004926:Escherichia coli; D019143:Evolution, Molecular; D019766:Genes, Archaeal; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019624:Thermoproteaceae / Q000235:genetics* / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III