The importance of threonine-301 from cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase) in substrate binding as demonstrated by site-directed mutagenesis.

Threonine-301 from rabbit liver cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase) has been replaced by histidine via site-directed mutagenesis. In the oxidized state the mutant P-450s exhibited typical low-spin type absorption spectra of P-450 and their reduced CO complexes showed a Soret peak at 450 nm. However, no spectral change was induced on addition of substrates for their wild-type counterparts. The mutant P-450s were also completely devoid of the hydroxylase activity. These findings suggest that threonine-301, which is highly conserved in P-450s and located at the distal heme surface, plays an important role in substrate binding.