Isolation and characterization of the cytochrome domain of flavocytochrome b2 expressed independently in Escherichia coli

Claire E Brunt, Mark C Cox, Andrew G P Thurgood, Geoffrey R Moore, Graeme A Reid, Stephen K Chapman

    Research output: Contribution to journalArticle

    Abstract

    The cytochrome domain of flavocytochrome b2 (L-lactate dehydrogenase) was expressed in the bacterium Escherichia coli and a purification procedure was developed. When expressed in E. coli, the b2-cytochrome domain contains protohaem IX and has an electronic absorption spectrum identical with that of the cytochrome b2 'core' produced by proteolytic cleavage of the enzyme isolated from yeast. The b2-cytochrome domain isolated from E. coli has an M(r) of 10 500 and a redox potential of -31 +/- 2 mV. High-field n.m.r. studies indicate pK(a) values for the haem propionate groups to be 4.8 and 4.6, consistent with these groups being exposed to solvent rather than buried inside the protein. Using n.m.r. spectroscopy, we have determined an electron self-exchange rate constant for the b2-cytochrome domain of 2.3 x 10(6) M-1.s-1, which is more than two orders of magnitude larger than the value obtained for microsomal cytochrome b5, a homologue of b2-cytochrome domain.

    Original languageEnglish
    Pages (from-to)87-90
    Number of pages4
    JournalBiochemical Journal
    Volume283
    Issue number1
    Publication statusPublished - 1 Apr 1992

    Keywords

    • B5
    • B-2
    • PROTEINS
    • DEHYDROGENASE
    • CORE

    Cite this