Conformational Dynamics of the Cytochrome P450 BM3/N-Palmitoylglycine Complex: The Proposed “Proximal-Distal” Transition Probed by Temperature-Jump Spectroscopy

Sibylle Brenner, Sam Hay, Hazel M. Girvan, Andrew W. Munro, Nigel S. Scrutton

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)7879-7886
Number of pages8
JournalJournal of Physical Chemistry B
Volume111
Issue number27
Early online date16 Jun 2007
DOIs
Publication statusPublished - 1 Jul 2007
Externally publishedYes

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