Conformational dynamics of the cytochrome P450 BM3/N-palmitoylglycine complex: the proposed "proximal-distal" transition probed by temperature-jump spectroscopy |
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Authors: | Brenner Sibylle Hay Sam Girvan Hazel M Munro Andrew W Scrutton Nigel S |
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Institution: | Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom. |
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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 approximately 800 s(-1) in the wild-type, and approximately 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 A 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. |
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