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

Abstract

Fourier-transform infrared (FTIR) spectroscopy was used to study the change of protein structure of NADH-cytochrome b₅ reductase in a soluble form. Recombinant mutant cytochrome b₅ 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 b₅ reductase was revealed tentatively by FTIR using resolution enhancement and band-fitting techniques, providing the contents of α-helix (22%), β-sheet (30%), random coil (27%), and β-turn (22%) for the wild-type cytochrome b₅ reductase. The mutant enzyme, S127P, was more sensitive to the thermal denaturation than the wild type with increasing β-sheet structures observed at 1624 and 1672 cm⁻¹ during the heat treatment and relatively decreasing in intensities of bands around 1640–1660 cm⁻¹ during heat treatment. The secondary structure of ferredoxin-NADP⁺ reductase, one of the same family as cytochrome b₅ reductase, predicted from FTIR data was similar to that of the wild-type cytochrome b₅ reductase but significantly different in the content of β-sheet and was consistent with the X-ray crystallographic data of ferredoxin-NADP⁺ reductase. The mutation on Ser-127 to proline or alanine in cytochrome b₅ reductase caused only a small change (3 or 9%, respectively) in total of α-helix, random coil, and β-turn contents and almost no change in the β-sheet content. These results suggest that the lability of the mutated cytochrome b₅ 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. © 1997 John Wiley & Sons, Inc. Biospect 3: 215–223, 1997