Analyses by Fourier transform infrared spectroscopies of protein structures of soluble NADH-cytochrome b5 reductases prepared by site-directed mutagenesis: Comparison with ferredoxin-NADP+ reductase
✍ Scribed by Satoshi Yoshida; Toshitsugu Yubisui; Komei Shirabe; Masazumi Takeshita
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 222 KB
- Volume
- 3
- Category
- Article
- ISSN
- 1075-4261
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✦ Synopsis
Fourier-transform infrared (FTIR) spectroscopy was used to study the change of protein structure of NADH-cytochrome b 5 reductase in a soluble form. Recombinant mutant cytochrome b 5 reductases, serine 127 to proline (S127P), and alanine (S127A) were investigated, where the mutation on Ser-127 to proline is a case found in patients of type II methemoglobinemia. The secondary structure of cytochrome b 5 reductase was revealed tentatively by FTIR using resolution enhancement and bandfitting techniques, providing the contents of a-helix (22%), b-sheet (30%), random coil (27%), and b-turn (22%) for the wild-type cytochrome b 5 reductase. The mutant enzyme, S127P, was more sensitive to the thermal denaturation than the wild type with increasing b-sheet structures observed at 1624 and 1672 cm 01 during the heat treatment and relatively decreasing in intensities of bands around 1640-1660 cm 01 during heat treatment. The secondary structure of ferredoxin-NADP / reductase, one of the same family as cytochrome b 5 reductase, predicted from FTIR data was similar to that of the wild-type cytochrome b 5 reductase but significantly different in the content of bsheet and was consistent with the X-ray crystallographic data of ferredoxin-NADP / reductase. The mutation on Ser-127 to proline or alanine in cytochrome b 5 reductase caused only a small change (3 or 9%, respectively) in total of a-helix, random coil, and b-turn contents and almost no change in the b-sheet content. These results suggest that the lability of the mutated cytochrome b 5 reductases might not result simply from the secondary structural change but from possibly the tertiary structural change, including the peptide side chain positional and the protein-protein interactional changes.