Structural relaxation of protoporphyrin-IX-substituted cytochrome b562 and its mutants at low temperatures studied with persistent spectral hole burning

Cytochrome b562 and three of its mutants in which only one amino acid is substituted were studied with persistent spectral hole burning. Structural relaxation at low temperatures was evaluated by performing temperature cycling experiments from 22 up to 100 K. There are remarkable differences in spectral diffusion behavior between the wild type and the mutants (N6D at 35 K and M7C above 60 K). The relaxation of the –OH group in N6D (asparagine 6 is replaced by aspartic acid) which occurs at 35 K at the site 10 Å vertically apart from the protoporphyrin ring is reflected by an increase in holewidth of 0.3 cm−1. The relaxation of the –SCH3 groups of methionine 7 in wild type cytochrome b562 was found to occur at 60 K by comparing the results with those of M7C (methionine is replaced by cysteine).