Changing the heme ligation in flavocytochrome b(2): substitution of histidine-66 by cysteine

Christopher G Mowat, Caroline S Miles, Andrew W Munro, Myles R Cheesman, Luca G Quaroni, Graeme A Reid, Stephen K Chapman

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    Abstract

    Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b(2) (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b(2)) which remains a competent L-lactate dehydrogenase (k(cat) 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degreesC, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+/-5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate, Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a v(4) band at 1345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b(2) heme is P450-like, Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

    Original languageEnglish
    Pages (from-to)584-592
    Number of pages9
    JournalJournal of Biological Inorganic Chemistry
    Volume5
    Issue number5
    DOIs
    Publication statusPublished - 1 Oct 2000

    Keywords

    • ESCHERICHIA-COLI
    • heme-iron ligation
    • site-directed mutagenesis
    • MAGNETIC CIRCULAR-DICHROISM
    • CYTOCHROME-C
    • INTRAMOLECULAR ELECTRON-TRANSFER
    • CATALYTIC CYCLE
    • PARAMAGNETIC-RESONANCE SPECTROSCOPY
    • FLAVODEHYDROGENASE
    • HEMOPROTEINS
    • flavocytochrome
    • LIGAND-BINDING
    • BAKERS-YEAST
    • L-lactate dehydrogenase

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