The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

Patricia White, Forbes D C Manson, Claire E Brunt, Stephen K Chapman, Graeme A Reid

    Research output: Contribution to journalArticle

    Abstract

    The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this 'hinge-swap' enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; k(cat), for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25-degrees-C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the k(cat) for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-H-2]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

    Original languageEnglish
    Pages (from-to)89-94
    Number of pages6
    JournalBiochemical Journal
    Volume291
    Issue number1
    Publication statusPublished - 1 Apr 1993

    Keywords

    • SITE
    • HANSENULA-ANOMALA
    • BAKERS-YEAST
    • MUTAGENESIS
    • SIMULATION

    Cite this

    White, P., Manson, F. D. C., Brunt, C. E., Chapman, S. K., & Reid, G. A. (1993). The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2. Biochemical Journal, 291(1), 89-94.
    White, Patricia ; Manson, Forbes D C ; Brunt, Claire E ; Chapman, Stephen K ; Reid, Graeme A. / The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2. In: Biochemical Journal. 1993 ; Vol. 291, No. 1. pp. 89-94.
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    abstract = "The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this 'hinge-swap' enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; k(cat), for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25-degrees-C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the k(cat) for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-H-2]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.",
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    White, P, Manson, FDC, Brunt, CE, Chapman, SK & Reid, GA 1993, 'The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2', Biochemical Journal, vol. 291, no. 1, pp. 89-94.

    The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2. / White, Patricia; Manson, Forbes D C; Brunt, Claire E; Chapman, Stephen K; Reid, Graeme A.

    In: Biochemical Journal, Vol. 291, No. 1, 01.04.1993, p. 89-94.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

    AU - White, Patricia

    AU - Manson, Forbes D C

    AU - Brunt, Claire E

    AU - Chapman, Stephen K

    AU - Reid, Graeme A

    PY - 1993/4/1

    Y1 - 1993/4/1

    N2 - The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this 'hinge-swap' enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; k(cat), for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25-degrees-C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the k(cat) for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-H-2]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

    AB - The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this 'hinge-swap' enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; k(cat), for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25-degrees-C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the k(cat) for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-H-2]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

    KW - SITE

    KW - HANSENULA-ANOMALA

    KW - BAKERS-YEAST

    KW - MUTAGENESIS

    KW - SIMULATION

    M3 - Article

    VL - 291

    SP - 89

    EP - 94

    JO - Biochemical Journal

    JF - Biochemical Journal

    SN - 0264-6021

    IS - 1

    ER -

    White P, Manson FDC, Brunt CE, Chapman SK, Reid GA. The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2. Biochemical Journal. 1993 Apr 1;291(1):89-94.