Substitution of a haem-iron axial ligand in flavocytochrome b2

Caroline S Miles, Forbes D C Manson, Graeme A Reid, Stephen K Chapman

    Research output: Contribution to journalArticlepeer-review

    11 Citations (Scopus)

    Abstract

    The importance of haem-iron axial coordination in flavocytochrome b2 (L-lactate: cytochrome-c oxidoreductase) has been examined by replacing one of the ligating histidines, His-43, with methionine. The His-43 --> Met mutation (H43M) results in a distinct colour change from red in the wild-type enzyme to green in the mutant enzyme. The electronic absorption spectrum indicates that only approx. 5% of the haem binding sites are occupied. There is no evidence of any absorption band at 695 nm (characteristic of methionine ligation) suggesting that methionine does not act as an axial ligand in the mutant enzyme. The H43M-mutant enzyme shows a band around 640-650 nm which is usually associated with high-spin ferric-haem proteins, either five coordinate or with a weak-field ligand in the sixth position. The EPR spectrum of the H43M-enzyme at 7 K shows a g-value near 6.0, indicating that the haem-iron is high-spin in contrast to its low-spin state in the wild-type enzyme. The His-43 --> Met mutation has only a small effect on the lactate dehydrogenase activity of the enzyme as measured with ferricyanide as external electron acceptor, but greatly reduces its cytochrome-c reductase activity.

    Original languageEnglish
    Pages (from-to)82-86
    Number of pages5
    JournalBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
    Volume1202
    Issue number1
    DOIs
    Publication statusPublished - 3 Sept 1993

    Keywords

    • BINDING
    • B2
    • FLAVODEHYDROGENASE
    • HEME
    • FLAVOCYTOCHROME-B(2)
    • CYTOCHROME
    • SITE-DIRECTED MUTAGENESIS
    • L-LACTATE
    • BAKERS-YEAST
    • AXIAL LIGAND

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