ABSTRACT
The TT1485 gene from Thermus thermophilus HB8 encodes a hypothetical protein of unknown function with about 20 sequence homologs of bacterial or archaeal origin. Together they form a family of uncharacterized proteins, the cluster of orthologous group COG3253. Using a combination of amino acid sequence analysis, three-dimensional structural studies and biochemical assays, we identified TT1485 as a novel heme-binding protein. The crystal structure reveals that this protein is a pentamer and each monomer exhibits a beta-barrel fold. TT1485 is structurally similar to muconolactone isomerase, but this provided no functional clues. Amino acid sequence analysis revealed remote homology to a heme enzyme, chlorite dismutase. Strikingly, amino acid residues that are highly conserved in the homologous hypothetical proteins and chlorite dismutase cluster around a deep cavity on the surface of each monomer. Molecular modeling shows that the cavity can accommodate a heme group with a strictly conserved His as a heme ligand. TT1485 reconstituted with iron protoporphyrin IX chloride gave a low chlorite dismutase activity, indicating that TT1485 catalyzes a reaction other than chlorite degradation. The presence of a possible Fe-His-Asp triad in the heme proximal site suggests that TT1485 functions as a novel heme peroxidase to detoxify hydrogen peroxide within the cell.
Subject(s)
Carrier Proteins/chemistry , Hemeproteins/chemistry , Peroxidases/chemistry , Protein Structure, Secondary , Thermus thermophilus/chemistry , Amino Acid Sequence , Carrier Proteins/metabolism , Carrier Proteins/physiology , Crystallography, X-Ray , Evolution, Molecular , Genes, Archaeal , Genes, Bacterial , Heme-Binding Proteins , Hemeproteins/metabolism , Hemeproteins/physiology , Kinetics , Models, Molecular , Molecular Sequence Data , Oxidoreductases/chemistry , Oxidoreductases/genetics , Peroxidases/metabolism , Peroxidases/physiology , Sequence Homology , Thermus thermophilus/metabolismABSTRACT
Recombinant holo Thermus thermophilus [7Fe-8S] ferredoxin was synthesized by cloning from Thermus thermophilus HB8 gene. A specific sequence (Pro-His-Val-Ile) at the N-terminus of the recombinant ferredoxin was determined by a rapid and highly sensitive mass spectral method using a novel Ru(II) Edman reagent, [(tpy)Ru(tpy-C6H4-NCS)](PF6)2 (tpy=terpyridine). The formation of the recombinant holoTtFd was established by the characteristic absorptions and CD extrema as [7Fe-8S] ferredoxin. The catalytic electron-transfer reactivity of the [7Fe-8S] ferredoxin between ferredoxin-NADP+ reductase and cytochrome c was recognized.
