Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3

Tobias W B Ost, Caroline S Miles, Andrew W Munro, Jane Murdoch, Graeme A Reid, Stephen K Chapman

    Research output: Contribution to journalArticle

    Abstract

    Site-directed mutants of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. While the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT > F393Y much greater than F393H > F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H much greater than F393Y approximate to wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] and substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous-form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, so explaining lower overall turnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen.

    Original languageEnglish
    Pages (from-to)13421-13429
    Number of pages9
    JournalBiochemistry
    Volume40
    Issue number45
    DOIs
    Publication statusPublished - 13 Nov 2001

    Keywords

    • GENE
    • ESCHERICHIA-COLI
    • P-450 BM3
    • ELECTRON-TRANSFER
    • NITRIC-OXIDE SYNTHASE
    • CYTOCHROME-P-450
    • MOLECULAR-CLONING
    • EXPRESSION
    • BACILLUS-MEGATERIUM
    • CRYSTAL-STRUCTURE

    Cite this

    Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., & Chapman, S. K. (2001). Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3. Biochemistry, 40(45), 13421-13429. https://doi.org/10.1021/bi010716m
    Ost, Tobias W B ; Miles, Caroline S ; Munro, Andrew W ; Murdoch, Jane ; Reid, Graeme A ; Chapman, Stephen K. / Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3. In: Biochemistry. 2001 ; Vol. 40, No. 45. pp. 13421-13429.
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    abstract = "Site-directed mutants of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. While the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT > F393Y much greater than F393H > F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H much greater than F393Y approximate to wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] and substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous-form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, so explaining lower overall turnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen.",
    keywords = "GENE, ESCHERICHIA-COLI, P-450 BM3, ELECTRON-TRANSFER, NITRIC-OXIDE SYNTHASE, CYTOCHROME-P-450, MOLECULAR-CLONING, EXPRESSION, BACILLUS-MEGATERIUM, CRYSTAL-STRUCTURE",
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    Ost, TWB, Miles, CS, Munro, AW, Murdoch, J, Reid, GA & Chapman, SK 2001, 'Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3', Biochemistry, vol. 40, no. 45, pp. 13421-13429. https://doi.org/10.1021/bi010716m

    Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3. / Ost, Tobias W B ; Miles, Caroline S; Munro, Andrew W; Murdoch, Jane; Reid, Graeme A; Chapman, Stephen K.

    In: Biochemistry, Vol. 40, No. 45, 13.11.2001, p. 13421-13429.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3

    AU - Ost, Tobias W B

    AU - Miles, Caroline S

    AU - Munro, Andrew W

    AU - Murdoch, Jane

    AU - Reid, Graeme A

    AU - Chapman, Stephen K

    PY - 2001/11/13

    Y1 - 2001/11/13

    N2 - Site-directed mutants of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. While the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT > F393Y much greater than F393H > F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H much greater than F393Y approximate to wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] and substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous-form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, so explaining lower overall turnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen.

    AB - Site-directed mutants of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. While the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT > F393Y much greater than F393H > F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H much greater than F393Y approximate to wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] and substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous-form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, so explaining lower overall turnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen.

    KW - GENE

    KW - ESCHERICHIA-COLI

    KW - P-450 BM3

    KW - ELECTRON-TRANSFER

    KW - NITRIC-OXIDE SYNTHASE

    KW - CYTOCHROME-P-450

    KW - MOLECULAR-CLONING

    KW - EXPRESSION

    KW - BACILLUS-MEGATERIUM

    KW - CRYSTAL-STRUCTURE

    U2 - 10.1021/bi010716m

    DO - 10.1021/bi010716m

    M3 - Article

    VL - 40

    SP - 13421

    EP - 13429

    JO - Biochemistry

    JF - Biochemistry

    SN - 0006-2960

    IS - 45

    ER -

    Ost TWB, Miles CS, Munro AW, Murdoch J, Reid GA, Chapman SK. Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3. Biochemistry. 2001 Nov 13;40(45):13421-13429. https://doi.org/10.1021/bi010716m