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| | {{STRUCTURE_1p0x| PDB=1p0x | SCENE= }} | | {{STRUCTURE_1p0x| PDB=1p0x | SCENE= }} |
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| - | '''F393Y mutant heme domain of flavocytochrome P450 BM3'''
| + | ===F393Y mutant heme domain of flavocytochrome P450 BM3=== |
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| - | ==Overview==
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| - | In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s(-)(1). The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_14653735}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 14653735 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_14653735}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Fatty acid hydroxylase]] | | [[Category: Fatty acid hydroxylase]] |
| | [[Category: Monooxygenase]] | | [[Category: Monooxygenase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 04:32:35 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 04:19:05 2008'' |
Revision as of 01:19, 29 July 2008
Template:STRUCTURE 1p0x
F393Y mutant heme domain of flavocytochrome P450 BM3
Template:ABSTRACT PUBMED 14653735
About this Structure
1P0X is a Single protein structure of sequence from Bacillus megaterium. Full crystallographic information is available from OCA.
Reference
Oxygen activation and electron transfer in flavocytochrome P450 BM3., Ost TW, Clark J, Mowat CG, Miles CS, Walkinshaw MD, Reid GA, Chapman SK, Daff S, J Am Chem Soc. 2003 Dec 10;125(49):15010-20. PMID:14653735
Page seeded by OCA on Tue Jul 29 04:19:05 2008
