The ferric spin state equilibrium of the heme iron was analyzed in wild-type cytochrome P450 BM3 and its F87G mutant by using temperature (T)-jump relaxation spectroscopy in combination with static equilibrium experiments. No relaxation process was measurable in the substrate-free enzyme indicating a relaxation process with a rate constant ≫ 10 000 s-1. In contrast, a slow spin state transition process was observed in the N-palmitoylglycine (NPG)-bound enzyme species. This transition occurred with an observed rate constant (298 K) of ∼800 s-1 in the wild-type, and ∼2500 s-1 in the F87G mutant, suggesting a significant contribution of the phenylalanine side chain to a reaction step rate limiting the actual spin state transition. These findings are discussed in terms of an equilibrium between different binding modes of the substrate, including a position 7.5 Å away from the heme iron ("distal") and the catalytically relevant "proximal" binding site, and are in accordance with results from X-ray crystallography, NMR studies, and molecular dynamics simulations.
|Number of pages||8|
|Journal||Journal of Physical Chemistry B|
|Early online date||16 Jun 2007|
|Publication status||Published - 1 Jul 2007|