TYR-143 facilitates interdomain electron transfer in flavocytochrome B2

Caroline S Miles, Nathalie Rouvière-Fourmy, Florence Lederer, F Scott Mathews, Graeme A Reid, Michael T Black, Stephen K Chapman

    Research output: Contribution to journalArticle

    Abstract

    The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143 --> Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the H-2 kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25-degrees-C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-H-2]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

    Original languageEnglish
    Pages (from-to)187-192
    Number of pages6
    JournalBiochemical Journal
    Volume285
    Issue number1
    Publication statusPublished - 1 Jul 1992

    Keywords

    • PROSTHETIC GROUPS
    • COMPUTER-SIMULATION
    • KINETIC SCHEME
    • PROTEINS
    • L-LACTATE
    • BAKERS-YEAST

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