Allosteric mechanisms in P450eryF probed with 1-pyrenebutanol, a novel fluorescent substrate.

1-Pyrenebutanol (1-PB) has been used as a new fluorescent substrate for P450eryF to explore the molecular mechanisms of cooperativity. Hydroxylation of 1-PB by P450eryF was detected by both fluorometric and chromatographic assays. Binding was monitored by a substrate-induced low-to-high spin shift, as well as by fluorescence resonance energy transfer (FRET) from 1-PB to the heme. Spectrophotometric titration showed that P450eryF has high affinity for 1-PB with distinct positive cooperativity (S(50) = 12.4 +/- 2.2 microM, n = 2.3 +/- 0.6), as also revealed in activity measurements. FRET analysis showed a different binding process obeying a simple bimolecular mechanism with a K(D) = 2.15 +/- 0.8 microM that suggests the presence of the higher affinity binding site. 1-PB binding at this site appears not to modulate the spin state directly but rather to facilitate the spin shift caused by the interactions of P450eryF with the other substrate molecule.