Conformational dynamics of the cytochrome P450 BM3/N-palmitoylglycine complex: the proposed "proximal-distal" transition probed by temperature-jump spectroscopy.
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.
Brenner
Sibylle
S
Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Hay
Sam
S
Girvan
Hazel M
HM
Munro
Andrew W
AW
Scrutton
Nigel S
NS
D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase; D010169:Palmitic Acids / Q000737:chemistry*; D010649:Phenylalanine; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D010169:Palmitic Acids; C095773:N-palmitoylglycine; D006418:Heme; D010649:Phenylalanine; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D005998:Glycine