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 band-fitting techniques, providing the contents of alpha-helix (22%), beta-sheet (30%), random coil (27%), and beta-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 beta-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 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 beta-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 b(5) reductase caused only a small change (3 or 9%, respectively) in total of alpha-helix, random coil, and beta-turn contents and almost no change in the beta-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. (C) 1997 John Wiley & Sons, Inc.