The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.
A cytochrome c‐552 gene from a thermophilic hydrogen‐oxidizing bacterium, Hydrogenobacter thermophilus, was cloned by using two oligonucleotide probes, which had been synthesized based on the known amino acid sequence of the protein. A 780‐bp PstI –SphI fragment of the cloned DNA was sequenced and found to contain the entire structural gene coding for cytochrome c‐552 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. Cytochrome c‐552 is synthesized in vivo as a precursor having an N‐terminal signal sequence consisting of 18 amino acid residues. The cloned cytochrome c‐552 gene without its own signal sequence was introduced into the pKK223‐3 vector and expressed in E. coli upon induction with isopropyl β‐d‐thiogalactoside. An expressed cytochrome c‐552 protein had a methionine residue at the N‐terminus since an initiation signal was introduced before the first amino acid residue of the mature cytochrome c‐552. The heme c was attached to apo‐type cytochrome c‐552 in the cytoplasm of E. coli and the holoprotein had spectral properties, similar to the authentic cytochrome c‐552 from H. thermophilus.
The denaturation of the c-type cytochrome of the thermophilic bacterium Hydrogenobacter thermophilus cytochrome c-552 by heat and guanidine hydrochloride was studied by measuring the change in circular dichroic spectra. The melting temperature (T1/2) of cytochrome c-552 in the presence of 1.5 M guanidine hydrochloride was 34 degrees C higher than that of the c-type cytochrome of Pseudomonas aeruginosa cytochrome c-551. Hydrogenobacter cytochrome c-552 is a much more stable protein than cytochrome c-551 of the mesophilic bacterium P. aeruginosa, even though their amino acid sequences are 56% identical and they have numerous other similarities. However, notwithstanding these similarities between the sequences of the cytochromes c-552 and c-551 that were compared, it is very likely that these differences in stability could be due to some heretofore undefined differences in their spatial structures. It has been suggested that alpha-helix structure and electrostatic interaction could be the source of the stable spatial structure of cytochrome c-552.
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